U.S. patent application number 13/710276 was filed with the patent office on 2013-07-18 for peptides that home to tumor lymphatic vasculature and methods of using same.
This patent application is currently assigned to SANFORD-BURNHAM MEDICAL RESEARCH INSTITUTE. The applicant listed for this patent is Sanford-Burnham Medical Research Institute. Invention is credited to Jason A. Hoffman, Pirjo Laakkonen, Kimmo Porkka, Erkki Ruoslahti.
Application Number | 20130183241 13/710276 |
Document ID | / |
Family ID | 32176139 |
Filed Date | 2013-07-18 |
United States Patent
Application |
20130183241 |
Kind Code |
A1 |
Laakkonen; Pirjo ; et
al. |
July 18, 2013 |
PEPTIDES THAT HOME TO TUMOR LYMPHATIC VASCULATURE AND METHODS OF
USING SAME
Abstract
The present invention provides a conjugate containing a moiety
linked to a homing molecule that selectively homes to tumor
lymphatic vasculature. The invention also provides a method of
directing a moiety to tumor lymphatic vasculature in a subject by
administering to the subject a conjugate containing a moiety linked
to a homing molecule that selectively homes to tumor lymphatic
vasculature.
Inventors: |
Laakkonen; Pirjo; (Helsinki,
FI) ; Porkka; Kimmo; (Helsinki, FI) ; Hoffman;
Jason A.; (La Jolla, CA) ; Ruoslahti; Erkki;
(Rancho Santa Fe, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanford-Burnham Medical Research Institute; |
La Jolla |
CA |
US |
|
|
Assignee: |
SANFORD-BURNHAM MEDICAL RESEARCH
INSTITUTE
La Jolla
CA
|
Family ID: |
32176139 |
Appl. No.: |
13/710276 |
Filed: |
December 10, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12657529 |
Jan 22, 2010 |
8329859 |
|
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13710276 |
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|
11712043 |
Feb 27, 2007 |
7671021 |
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12657529 |
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10290385 |
Nov 5, 2002 |
7192921 |
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11712043 |
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60421145 |
Nov 8, 2001 |
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Current U.S.
Class: |
424/9.1 ;
514/19.3 |
Current CPC
Class: |
C07K 7/08 20130101; C07K
7/06 20130101; A61K 38/00 20130101; A61K 38/08 20130101; C07K
14/001 20130101; C07K 2319/00 20130101; A61P 35/00 20180101; A61P
43/00 20180101 |
Class at
Publication: |
424/9.1 ;
514/19.3 |
International
Class: |
A61K 38/08 20060101
A61K038/08 |
Goverment Interests
[0002] This invention was made with government support under CA
74238, CA 82715 and Cancer Center Support Grant CA 30199 awarded by
the National Cancer Institute. The government has certain rights in
this invention.
Claims
1-39. (canceled)
40. A method of directing a moiety to tumor lymphatic vasculature
in a subject, comprising administering to the subject a conjugate
which comprises a moiety linked to a homing molecule that
selectively homes to tumor lymphatic vasculature, thereby directing
the moiety to tumor lymphatic vasculature.
41. The method of claim 40, wherein said homing molecule is
cyclic.
42. The method of claim 40, wherein said homing molecule is a
peptide or peptidomimetic.
43. The method of claim 40, wherein said homing molecule comprises
the amino acid sequence GNKRTRG (SEQ ID NO: 2) or a conservative
variant or peptidomimetic thereof.
44. The method of claim 43, wherein said homing molecule comprises
the amino acid sequence GNKRTRG (SEQ ID NO: 2).
45. The method of claim 43, wherein said homing molecule comprises
the amino acid sequence CGNKRTRGC (SEQ ID NO: 1) or a conservative
variant or peptidomimetic thereof.
46. The method of claim 45, wherein said homing molecule comprises
the amino acid sequence CGNKRTRGC (SEQ ID NO: 1).
47. The method of claim 40, wherein said moiety is a therapeutic
agent.
48. The method of claim 40, wherein said moiety is a cancer
chemotherapeutic agent.
49. The method of claim 40, wherein said moiety is a cytotoxic
agent.
50. The method of claim 40, wherein said moiety is a anti
lymphangiogenic agent.
51. The method of claim 40, wherein said moiety is a detectable
label.
52. The method of claim 40, wherein said moiety is a phage.
53. A method of imaging tumor lymphatic vasculature in a subject,
comprising (a) administering to the subject a conjugate comprising
a detectable label linked to a homing molecule that selectively
homes to tumor lymphatic vasculature; and (b) detecting said
conjugate, thereby imaging said tumor lymphatic vasculature.
54. The method of claim 53, wherein said homing molecule is
cyclic.
55. The method of claim 53, wherein said homing molecule is a
peptide or peptidomimetic.
56. The method of claim 53, wherein said homing molecule comprises
the amino acid sequence GNKRTRG (SEQ ID NO: 2) or a conservative
variant or peptidomimetic thereof.
57-59. (canceled)
60. The method of claim 53, wherein said detectable label is a
radionuclide.
61. The method of claim 60, wherein said radionuclide is selected
from the group consisting of indium 111, technetium 99, carbon 11,
and carbon 13.
62-71. (canceled)
72. A method of reducing the number of tumor lymphatic vessels in a
subject, comprising administering to the subject a conjugate which
comprises a moiety linked to a homing molecule that selectively
homes to tumor lymphatic vasculature, thereby reducing the number
of tumor lymphatic vessels in said subject.
73-91. (canceled)
Description
[0001] This application is a continuation of U.S. Ser. No.
12/657,529, filed Jan. 22, 2010, which is a continuation of U.S.
Ser. No. 11/712,043, filed Feb. 27, 2007, now U.S. Pat. No.
7,671,021, which is a continuation of U.S. Ser. No. 10/290,385,
filed Nov. 5, 2002, now U.S. Pat. No. 7,192,921, which is based on,
and claims the benefit of U.S. Provisional Application No.
60/421,145, filed Nov. 8, 2001, which was converted from U.S. Ser.
No. 10/007,792, each of which the entire contents are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the fields of
molecular medicine and drug delivery and, more specifically, to
molecules that selectively home to the lymphatic vasculature of
specific tumors.
[0005] 2. Background Information
[0006] Metastasis, the spreading of cancer from a primary site to a
secondary site often in another organ, contributes significantly to
cancer patient mortality. Metastasis occurs commonly, for example,
in breast cancer and in the middle and later stages of bone cancer.
Most often, systemic chemotherapy is necessary to manage cancer
metastasis or to diminish the likelihood that metastasis will
occur. However, undesirable side effects such as severe nausea,
vomiting, neuropathy, hair loss and drop in blood cell count can
occur upon systemic treatment with a chemotherapeutic agent and
significantly impact the quality of patient life. In addition, such
undesirable side effects often limit the amount of a treatment that
can be safely administered, thereby reducing cancer patient
survival rates.
[0007] Cancers metastasize through tumor vasculature, which is
diverse in both its cellular and molecular compositions, exhibiting
variation in the type of cells that line the vessels and their
complement of cell-surface receptors. Blood vessels are one type of
tumor vasculature, and archetypal blood vessels are entirely lined
with endothelial cells. Tumor blood vessels also can be mosaic or
lined by both endothelial and tumor cells, while other vessels are
formed entirely from tumor cells. Lymphatic vessels, which also
occur within several tumor types, are a second type of tumor
vasculature. The lymphatic vasculature is an important route for
the spreading of cancer, and animal experiments have shown a
positive correlation between metastasis and the number of lymphatic
vessels in and around a tumor.
[0008] In view of the undesirable side effects that limit
conventional systemic chemotherapy designed to reduce or prevent
metastasis, there is a need for molecules which selectively home to
tumor lymphatic vasculature and which are suitable, for example,
for selectively targeting agents to ablate tumor lymphatic
vasculature, thereby reducing the risk of tumor metastasis. The
present invention satisfies this need by providing molecules that
selectively home to tumor lymphatic vasculature, for example, to
breast cancer and osteosarcoma lymphatic vasculature. Related
advantages also are provided.
SUMMARY OF THE INVENTION
[0009] The present invention provides an isolated peptide or
peptidomimetic containing the amino acid sequence GNKRTRG (SEQ ID
NO: 2), or a peptidomimetic thereof. The invention further provides
an isolated peptide or peptidomimetic containing the amino acid
sequence CGNKRTRGC (SEQ ID NO: 1) or a peptidomimetic thereof. A
peptide or peptidomimetic of the invention can be, for example,
cyclic or otherwise conformationally constrained and can have a
variety of lengths, for example, a length of less than 100
residues, a length of less than 50 residues, a length less than 20
residues, or a length of less than 15 residues.
[0010] The present invention further provides a conjugate
containing a moiety linked to a homing molecule that selectively
homes to tumor lymphatic vasculature. In one embodiment, the
conjugate contains a homing molecule which selectively homes to
tumor lymphatic vasculature other than melanoma vasculature. In
another embodiment, the conjugate contains a homing molecule which
selectively homes to tumor lymphatic vasculature and which is not
an anti-VEGFR-3 or anti-LYVE-1 antibody or antigen-binding fragment
thereof. In a further embodiment, the conjugate contains a homing
molecule which selectively homes to tumor lymphatic vasculature and
which is not an antibody or antigen-binding fragment thereof.
[0011] In a conjugate of the invention, the homing molecule that
selectively homes to tumor lymphatic vasculature can be, for
example, a peptide or peptidomimetic. In one embodiment, the
peptide or peptidomimetic portion of the conjugate has a length of
at most 200 residues. In another embodiment, the peptide or
peptidomimetic portion of the conjugate has a length of at most 50
residues. In further embodiments, the conjugate contains a cyclic
or otherwise conformationally constrained homing molecule, such as
a peptide or peptidomimetic, that selectively homes to tumor
lymphatic vasculature.
[0012] A homing molecule useful in a conjugate of the invention can
be, for example, a homing peptide or peptidomimetic containing the
amino acid sequence GNKRTRG (SEQ ID NO: 2), or a conservative
variant or peptidomimetic thereof. If desired, such a peptide or
peptidomimetic can be cyclic or otherwise conformationally
constrained. A homing molecule useful in a conjugate of the
invention also can be, for example, a homing peptide or
peptidomimetic containing the amino acid sequence CGNKRTRGC (SEQ ID
NO: 1), or a conservative variant or peptidomimetic thereof. In
specific embodiments, such a homing peptide or peptidomimetic is
cyclic or otherwise conformationally constrained. A variety of
moieties are useful in a conjugate of the invention including,
without limitation, therapeutic agents, cancer chemotherapeutic
agents, cytotoxic agents, anti-lymphangiogenic agents, detectable
labels and phage.
[0013] If desired, a conjugate of the invention can contain
multiple homing molecules which each selectively homes to tumor
lymphatic vasculature. In one embodiment, a conjugate of the
invention contains at least two homing molecules which each
selectively homes to tumor lymphatic vasculature. In further
embodiments, a conjugate of the invention contains at least 10
homing molecules, or at least 100 homing molecules, which each
selectively homes to tumor lymphatic vasculature. In another
embodiment, the invention provides a conjugate containing a phage
linked to at least 100 homing molecules which each selectively
homes to tumor lymphatic vasculature. In a further embodiment, the
invention provides a conjugate in which a phage or other particle
that is linked to at least 100, 200, 300, 400 or 500 identical or
non-identical homing molecules which each selectively homes to
tumor lymphatic vasculature
[0014] A conjugate of the invention can contain, for example, a
moiety linked to at least two homing molecules which each
selectively homes to tumor lymphatic vasculature and which each
independently includes the amino acid sequence GNKRTRG (SEQ ID NO:
2), or a conservative variant or peptidomimetic thereof. In a
further embodiment, the invention provides a conjugate containing a
moiety linked to at least ten homing molecules which each
selectively homes to tumor lymphatic vasculature and which each
independently includes the amino acid sequence GNKRTRG (SEQ ID NO:
2) or a conservative variant or peptidomimetic thereof. In yet
another embodiment, the invention provides a conjugate containing a
moiety linked to at least 100 homing molecules which each
selectively homes to tumor lymphatic vasculature and which each
independently includes the amino acid sequence GNKRTRG (SEQ ID NO:
2) or a conservative variant or peptidomimetic thereof. Moieties
useful in a conjugate of the invention containing multiple homing
molecules include, but are not limited to, phage moieties.
[0015] The present invention also provides a method of directing a
moiety to tumor lymphatic vasculature in a subject by administering
to the subject a conjugate which contains a moiety linked to a
homing molecule that selectively homes to tumor lymphatic
vasculature, thereby directing the moiety to tumor lymphatic
vasculature. In a method of the invention, the homing molecule can
be, for example, cyclic or otherwise conformationally constrained
and further can be, for example, a peptide or peptidomimetic. In
one embodiment, the homing molecule is a peptide containing the
amino acid sequence GNKRTRG (SEQ ID NO: 2), or a conservative
variant or peptidomimetic thereof. In another embodiment, the
homing molecule is a peptide that contains the amino acid sequence
CGNKRTRGC (SEQ ID NO: 1), or a conservative variant or
peptidomimetic thereof. A variety of moieties are useful in a
method of the invention including, for example, therapeutic agents,
cancer chemotherapeutic agents, cytotoxic agents,
anti-lymphangiogenic agents, detectable labels and phage.
[0016] The present invention also provides a method of imaging
tumor lymphatic vasculature in a subject by administering to the
subject a conjugate which contains a detectable label linked to a
homing molecule that selectively homes to tumor lymphatic
vasculature, and detecting the conjugate, thereby imaging the tumor
lymphatic vasculature. In a method of the invention for imaging
tumor lymphatic vasculature, the homing peptide can be, for
example, cyclic or otherwise conformationally constrained and
further can be, for example, a peptide or peptidomimetic. In one
embodiment, the homing molecule is a peptide containing the amino
acid sequence GNKRTRG (SEQ ID NO: 2), or a conservative variant or
peptidomimetic thereof. In another embodiment, the homing molecule
is a peptide containing the amino acid sequence CGNKRTRGC (SEQ ID
NO: 1), or a conservative variant or peptidomimetic thereof. A
detectable label useful in an imaging method of the invention can
be, for example, a radionuclide or a fluorescent molecule. Examples
of radionuclides useful as detectable labels include, but are not
limited to, indium-111, technetium-99, carbon-11, and
carbon-13.
[0017] Further provided by the invention is a method of reducing or
inhibiting tumor metastasis in a subject by administering to the
subject a conjugate which contains a moiety linked to a homing
molecule that selectively homes to tumor lymphatic vasculature,
thereby reducing or inhibiting tumor metastasis. In a method of the
invention for reducing or inhibiting tumor metastasis, the homing
molecule can be, for example, cyclic or otherwise conformationally
constrained and further can be, for example, a peptide or
peptidomimetic. In one embodiment, the homing molecule is a peptide
containing the amino acid sequence GNKRTRG (SEQ ID NO: 2), or a
conservative variant or peptidomimetic thereof. In another
embodiment, the homing molecule is a peptide containing the amino
acid sequence CGNKRTRGC (SEQ ID NO: 1), or a conservative variant
or peptidomimetic thereof. A variety of moieties are useful in a
method of the invention for reducing or inhibiting tumor
metastasis. Such a moieties include, without limitation, cancer
chemotherapeutic agents, cytotoxic agents and anti-lymphangiogenic
agents.
[0018] The present invention further provides a method of reducing
the number of tumor lymphatic vessels in a subject by administering
to the subject a conjugate which contains a moiety linked to a
homing molecule that selectively homes to tumor lymphatic
vasculature, thereby reducing the number of tumor lymphatic vessels
in the subject. In a method of the invention, the homing molecule
can be, for example, cyclic or otherwise conformationally
constrained and further can be, for example, a peptide or
peptidomimetic. In one embodiment, the homing molecule is a peptide
containing the amino acid sequence GNKRTRG (SEQ ID NO: 2), or a
conservative variant or peptidomimetic thereof. In another
embodiment, the homing molecule is a peptide containing the amino
acid sequence CGNKRTRGC (SEQ ID NO: 1), or a conservative variant
or peptidomimetic thereof. A variety of moieties can be useful in a
method of the invention for reducing the number of tumor lymphatic
vessels including, without limitation, cancer chemotherapeutic
agents, cytotoxic agents and anti-lymphangiogenic agents.
[0019] The present invention additionally provides a method of
treating cancer in a subject by administering to the subject a
conjugate which contains a moiety linked to a homing molecule that
selectively homes to tumor lymphatic vasculature. In a method of
the invention, the homing molecule can be, for example, cyclic or
otherwise conformationally constrained and further can be, for
example, a peptide or peptidomimetic. In one embodiment, the homing
molecule is a peptide containing the amino acid sequence GNKRTRG
(SEQ ID NO: 2), or a conservative variant or peptidomimetic
thereof. In another embodiment, the homing molecule is a peptide
containing the amino acid sequence CGNKRTRGC (SEQ ID NO: 1), or a
conservative variant or peptidomimetic thereof. A variety of
moieties can be useful in a method of the invention for treating
cancer in a subject including, but not limited to, cancer
chemotherapeutic agents, cytotoxic agents and anti-lymphangiogenic
agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] This patent or application file contains at least one color
photograph. Copies of this patent or patent application publication
with the color photographs will be provided by the Patent &
Trademark Office upon request and payment of the necessary fee.
[0021] FIGS. 1A-1E show ex vivo binding and in vivo homing of phage
displaying CGNKRTRGC (SEQ ID NO: 1). FIG. 1A shows Binding of
recombinant T7 phage displaying CGNKRTRGC (SEQ ID NO: 1), CGEKRTRGC
(SEQ ID NO: 3) or CGNKRTRGV (SEQ ID NO: 4) to primary MDA-MB-435
breast carcinoma tumor cell suspensions prepared from 435 breast
carcinoma xenografts. FIG. 1B shows Correlation of ex vivo binding
of CGNKRTRGC (SEQ ID NO: 1)-displaying phage with peptide copy
number displayed. FIG. 1C shows In vivo homing of CGNKRTRGC (SEQ ID
NO: 1) phage to MDA-MB-435 breast carcinoma and KRIB osteosarcoma
xenografts. FIG. 1D shows In vivo homing to normal tissues (normal
kidney, lung, spleen, skin or breast tissue). FIG. 1E shows
Internalization of CGNKRTRGC (SEQ ID NO: 1) phage by MDA-MB-435
breast carcinoma tumor cells.
[0022] FIGS. 2A-2B show in vitro internalization and nuclear
localization of fluorescein-conjugated peptide CGNKRTRGC (SEQ ID
NO: 1) in 435 cells. FIG. 2A shows Fluorescein-conjugated CGNKRTRGC
(SEQ ID NO: 1) peptide (green). Nuclei are visualized by DAPI
staining (blue). FIG. 2B shows Fluorescein-conjugated control
peptide.
[0023] FIGS. 3A-3I show localization of fluorescent peptide
CGNKRTRGC (SEQ ID NO: 1) in tumors following intravenous injection.
FIGS. 3A-3C show Fluorescent peptide CGNKRTRGC (SEQ ID NO: 1)
staining (green). FIGS. 3D-3F show Tomato lectin staining detected
with streptavidin-conjugated Alexa 594 (red). FIGS. 3G-3I show
Peptide and blood vessel staining present in the same microscopic
field.
[0024] FIGS. 4A-4C show distinct localization of lymphatic vessel
markers and blood vessel markers. Lymphatic vessels were visualized
in 435 tumor sections using rabbit anti-LYVE1 and goat anti-rabbit
Alexa 594 and appear red in the photomicrographs (FIGS. 4A and 4C).
Blood vessels were labeled with fluorescein-conjugated tomato
lectin and appear green in photomicrographs (FIGS. 4B and 4C).
[0025] FIGS. 5A-5H show co-localization of fluorescein-conjugated
peptide CGNKRTRGC (SEQ ID NO: 1) with the lymphatic markers VEGFR-3
and LYVE-1. FIGS. 5A, 5C and 5E show Fluorescein-conjugated
CGNKRTRGC (SEQ ID NO: 1; green) and lymphatic marker VEGFR-3 (red).
FIG. 5G shows Fluorescein-conjugated CGNKRTRGC (SEQ ID NO: 1;
green) and lymphatic marker LYVE-1 (red). FIGS. 5B, 5D, 5F and 5H
show Fluorescein-conjugated SEQ ID NO: 1 (green) and DAPI nuclear
staining (blue).
[0026] FIG. 6 shows homing of CGNKRTRGC (SEQ ID NO: 1) phage to
MDA-MB-435 xenograft tumors or brain following intravenous or
subcutaneous injection.
[0027] FIG. 7 shows MDA-MB-435 cells incubated with CGNKRTRGC (SEQ
ID NO: 1) or with a control peptide (CGEKRTRGC; SEQ ID NO: 3).
After staining with trypan blue, the total number of cells were
counted, and the percentage of cells stained with trypan blue was
determined.
[0028] FIG. 8 shows the effect of injection of peptide CGNKRTRGC
(SEQ ID NO: 1) on the growth of MDA-MB-435 human breast carcinoma
xenografts in vivo.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention is directed, in part, to the discovery
of homing molecules which selectively home to tumor lymphatic
vasculature, for example, the lymphatic vasculature of breast
cancer tumors and osteosarcomas, in preference to normal lymphatic
vasculature. A homing molecule of the invention also can
selectively home, for example, to the lymphatic vasculature of
squamous carcinomas. As disclosed herein, peptide CGNKRTRGC (SEQ ID
NO: 1) was identified by a combination of ex vivo and in vivo
selection as selectively homing to the lymphatic vasculature of
several tumors in preference to the lymphatic vasculature of a
variety of normal tissues. As shown in FIG. 1A, about 5000 times
more CGNKRTRGC (SEQ ID NO: 1)-displaying phage than nonrecombinant
control T7 phage bound to tumor cell suspensions prepared from
MDA-MB-435 breast tumor xenografts. The CGNKRTRGC (SEQ ID NO: 1)
phage also bound to tumor cell suspensions prepared from KRIB human
osteosarcoma xenografts but not to C8161 melanoma or HL-60 human
leukemia cells (see Example 1).
[0030] As further disclosed herein in Example 2, the CGNKRTRGC (SEQ
ID NO: 1) phage selectively homed to MDA-MB-435 breast tumor and
KRIB osteosarcomas in vivo. As shown in FIG. 1C, the mean phage
titer of CGNKRTRGC (SEQ ID NO: 1)-displaying phage was about
60-fold greater than that of nonrecombinant phage recovered from
435 breast tumors and was 15-fold greater than that of
nonrecombinant control T7 phage recovered from KRIB osteosarcomas.
Furthermore, the CGNKRTRGC (SEQ ID NO: 1)-displaying phage did not
home to tumors obtained with a melanoma (C8161) or leukemia (HL-60)
cell line, or to a variety of normal tissues including kidney,
lung, spleen, and skin, and displayed minimal affinity for normal
breast tissue (see FIGS. 1C and 1D).
[0031] As demonstrated in Example 3, the homing peptide, SEQ ID NO:
1, was internalized by cells. In particular, following injection of
SEQ ID NO: 1 phage into the tail vein of a mouse bearing a
MDA-MB-435 breast tumor xenograft, ten to twenty times more
CGNKRTRGC (SEQ ID NO: 1)-displaying phage were recovered from tumor
samples treated with NP-40 detergent, which lyses the cells, than
from untreated samples, indicating that the SEQ ID NO: 1-bearing
phage had been internalized (see FIG. 1E). Internalization of
CGNKRTRGC (SEQ ID NO: 1)-displaying phage was confirmed by
internalization of fluorescein-labeled peptide SEQ ID NO: 1 in
cultured 435 cells, indicating that the tumor cells share the
receptor for peptide SEQ ID NO: 1 with the lymphatic vessel cells.
As shown in FIGS. 2A and B, fluorescein-conjugated SEQ ID NO: 1 was
internalized and translocated to the nucleus of 435 cells, while
there was no detectable internalization of a control peptide,
which, like SEQ ID NO: 1, contained three basic residues.
Furthermore, fluorescein-conjugated SEQ ID NO:1 also was
internalized and transported into nuclei in vivo as shown in FIG.
5.
[0032] As further disclosed herein, upon injection into the tail
vein of nude mice carrying an MDA-MB-435 breast tumor, both
CGNKRTRGC (SEQ ID NO: 1)-displaying phage and fluorescein
conjugated peptide SEQ ID NO: 1 localized to vessel-like structures
and some individual cells within the breast cancer xenograft (see
Example 4). The vessels in which the phage or
fluorescein-conjugated peptide localized were negative for markers
CD31 and Meca-32, which are expressed by blood vessel endothelial
cells in preference to lymphatic endothelial cells, indicating that
the vessel-like structures targeted by peptide CGNKRTRGC (SEQ ID
NO: 1) were not blood vessels. As further disclosed herein in FIG.
5, fluorescein-conjugated peptide CGNKRTRGC (SEQ ID NO: 1)
co-localized with the lymphatic markers VEGFR-3 and LYVE-1 in 435
tumor tissue. However, the SEQ ID NO: 1-bearing phage did not home
to the C8161 melanoma xenografts, even though these xenografts
contained as many VEGFR-3/LYVE-1-positive/tomato lectin negative
vessels as the 435 tumors. Together, these results indicate that
peptide SEQ ID NO: 1 selectively homes to VEGFR-3 and LYVE-1
positive lymphatic vessels of several different tumors. Combined
with data showing that cultured 435 and KRIB cells bound SEQ ID NO:
1-bearing phae and internalized fluorescein-labeled peptide SEQ ID
NO: 1, these results indicate that the CGNKRTRGC peptide (SEQ ID
NO: 1) recognizes a lymphatic vessel target molecule present on
tumor cells as well as lymphatic vessel cells of the same tumors.
As disclosed herein, this target molecule is not significantly
expressed in the lymphatic vessels of normal tissues.
[0033] Results disclosed herein further indicate that CGNKRTRGC
peptide (SEQ ID NO: 1) has cytotoxic activity in cell culture and
in vivo. As shown in FIG. 7, significantly enhanced cytotoxicity
was observed in cultured MDA-MB-435 cells incubated with peptide
SEQ ID NO: 1 as compared to cells incubated with control peptide.
Furthermore, when MDA-MB-435 human breast carcinoma cell xenografts
were treated by intravenous injection of CGNKRTRGC peptide (SEQ ID
NO: 1) twice a week, tumor volumes decreased. As shown in FIG. 8,
tumor growth was slowed significantly in mice treated with peptide
SEQ ID NO: 1, whereas the tumor volume continued to increase
rapidly in mice not treated with the peptide. These results
demonstrate that peptide CGNKRTRGC (SEQ ID NO: 1) has cytotoxic
activity both in cell culture and in vivo and further indicate that
this peptide as well as structurally related peptides and
peptidomimetics, and molecules that bind the same receptor can be
useful for slowing or preventing tumor growth in vivo.
[0034] Based on these findings, the present invention provides
homing molecules and conjugates useful, for example, for reducing
or preventing tumor metastasis in cancer patients having a primary
tumor. The conjugates of the invention can be administered, for
example, to a subject having pre-metastatic breast or bone cancer
or to a subject having early or late stage metastatic breast or
bone cancer. Conjugates of the invention also can be useful, for
example, for imaging tumor lymphatic vasculature, such as breast
cancer or osteosarcoma lymphatic vasculature.
[0035] Thus, the present invention provides an isolated peptide or
peptidomimetic containing the amino acid sequence GNKRTRG (SEQ ID
NO: 2), or a peptidomimetic thereof. The invention further provides
an isolated peptide or peptidomimetic containing the amino acid
sequence CGNKRTRGC (SEQ ID NO: 1) or a peptidomimetic thereof. A
peptide or peptidomimetic of the invention can be, for example,
cyclic or otherwise conformationally constrained and can have a
variety of lengths, for example, a length of less than 100
residues, a length of less than 50 residues, a length less than 20
residues, or a length of less than 15 residues. In one embodiment,
a peptide or peptidomimetic of the invention which contains the
amino acid sequence GNKRTRG (SEQ ID NO: 2) or CGNKRTRGC (SEQ ID NO:
1), or a peptidomimetic of one of these sequences, has cytotoxic
activity. It is understood that a peptide containing, for example,
the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2 includes the
specified amino acids as a contiguous sequence in which the
specified amino acids are not separated by other amino acids.
[0036] The peptides and peptidomimetics of the invention are
provided in isolated form. As used herein in reference to a peptide
or peptidomimetic of the invention, the term "isolated" means a
peptide or peptidomimetic that is in a form that is relatively free
from material such as contaminating polypeptides, lipids, nucleic
acids and other cellular material that normally is associated with
the peptide or peptidomimetic in a cell or that is associated with
the peptide or peptidomimetic in a library or in a crude
preparation.
[0037] The peptides and peptidomimetics of the invention, including
the bifunctional, multivalent and homing peptides and
peptidomimetics discussed below, can have a variety of lengths. A
peptide or peptidomimetic of the invention can have, for example, a
relatively short length of less than eight, nine, ten, 12, 15, 20,
25, 30, 35 or 40 residues. A peptide or peptidomimetic of the
invention also can be useful in the context of a significantly
longer sequence. For example, as disclosed herein, peptide
CGNKRTRGC (SEQ ID NO: 1) maintained the ability to home when fused
to a phage coat protein, confirming that a peptide of the invention
can have selective homing activity when embedded in larger protein
sequence. Thus, a peptide or peptidomimetic of the invention can
have, for example, a length of up to 50, 100, 150 or 200 residues.
As used herein, the term "residue" refers to amino acids or analogs
thereof.
[0038] The present invention also provides an isolated peptide or
peptidomimetic containing the amino acid sequence GNKRTRG (SEQ ID
NO: 2) or CGNKRTRGC (SEQ ID NO: 1), or a conservative variant or
peptidomimetic of one of these sequences. 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.
[0039] The invention further provides a chimeric protein containing
a peptide or peptidomimetic of the invention, or a homing peptide
or peptidomimetic of the invention, fused to a heterologous
protein. In one embodiment, the invention provides a chimeric
protein containing a homing peptide or peptidomimetic that
selectively homes to tumor lymphatic vasculature fused to a
heterologous protein. In one embodiment, the homing peptide or
peptidomimetic that selectively homes to tumor lymphatic
vasculature has cytotoxic activity. In another embodiment, the
heterologous protein has a therapeutic activity. In a further
embodiment, the heterologous protein is an antibody or
antigen-binding fragment thereof. In other embodiments, the
invention provides a chimeric protein in which a peptide or
peptidomimetic containing the amino acid sequence CGNKRTRGC (SEQ ID
NO: 1) or GNKRTRG (SEQ ID NO: 2), or a conservative variant or
peptidomimetic of one of these sequences, is fused to a
heterologous protein. 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 peptide of the invention or upon which the
peptidomimetic is derived. A chimeric protein of the invention can
have a variety of lengths, for example, up to 100, 200, 300, 400,
500 or 800 residues.
[0040] The invention also provides a bifunctional peptide which
contains a homing peptide that selectively homes to tumor lymphatic
vasculature, such as the lymphatic vasculature of breast cancers or
osteosarcomas, 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-lymphangiogenic activity or pro-apoptotic
activity in addition to selective homing activity. As exemplary
bifunctional peptides, the invention provides
CGNKRTRGC-GG-.sub.D(KLAKLAK).sub.2 and
GNKRTRG-GG-.sub.D(KLAKLAK).sub.2. In such peptides, the CGNKRTRGC
(SEQ ID NO: 1) portion exhibits selective homing activity and
cytotoxic activity, while the .sub.D(KLAKLAK).sub.2 portion
exhibits pro-apoptotic activity.
[0041] The present invention further provides an isolated
multivalent peptide or peptidomimetic that includes at least two
motifs each independently containing the amino acid sequence
GNKRTRG (SEQ ID NO: 2), or a conservative variant or peptidomimetic
thereof. The multivalent peptide or peptidomimetic can have, for
example, at least three, at least five or at least ten of such
motifs each independently containing the amino acid sequence
GNKRTRG (SEQ ID NO: 2), or a conservative variant or peptidomimetic
thereof. In particular embodiments, the multivalent peptide or
peptidomimetic has two, three, four, five, six, seven, eight, nine,
ten, fifteen or twenty identical or non-identical motifs of the
amino acid sequence GNKRTRG (SEQ ID NO: 2), or a conservative
variant or peptidomimetic thereof. In another embodiment, the
multivalent peptide or peptidomimetic contains identical motifs,
which consist of the amino acid sequence SEQ ID NO: 2, or
conservative variants or peptidomimetics thereof. In a further
embodiment, the multivalent peptide or peptidomimetic contains
contiguous motifs, which are not separated by any intervening amino
acids. In yet further embodiments, the multivalent peptide or
peptidomimetic is cyclic or otherwise conformationally constrained,
or has cytotoxic activity.
[0042] In an isolated multivalent peptide or peptidomimetic of the
invention, at least one motif can be, if desired, CGNKRTRGC (SEQ ID
NO: 1), or a conservative variant or peptidomimetic thereof. In
particular embodiments, a multivalent peptide or peptidomimetic of
the invention has two, three, four, five, six, seven, eight, nine,
ten, fifteen or twenty identical or non-identical motifs of the
amino acid sequence CGNKRTRGC (SEQ ID NO: 1), or a conservative
variant or peptidomimetic thereof. Such multivalent peptides or
peptidomimetics can be, if desired, contiguous and further can be,
if desired, cyclic or otherwise conformationally constrained.
[0043] Thus, the invention provides peptides and peptidomimetics,
including bifunctional and multivalent peptides and
peptidomimetics, and homing peptides and peptidomimetics discussed
further below. 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
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).
[0044] A variety of peptidomimetics are known in the art including,
for example, peptide-like molecules which 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, for example, 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 which mimics peptide secondary structure can
contain, for example, 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. A peptidomimetic
also can be a peptide-like molecule which contains, for example, an
amide bond isostere such as a retro-inverso modification; reduced
amide bond; methylenethioether or methylenesulfoxide 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.
[0045] 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 or a target molecule that binds the peptide 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 tumor lymphatic vasculature.
[0046] An isolated peptide or peptidomimetic of the invention, or a
homing molecule of the invention as discussed further below, can be
cyclic, or otherwise conformationally constrained. As used herein,
a "conformationally constrained" molecule, such as a peptide or
peptidomimetic, is one in which the three-dimensional structure is
maintained substantially in one spatial arrangement over time.
Conformationally constrained molecules can have improved properties
such as increased affinity, metabolic stability, membrane
permeability or solubility. Methods of conformational constraint
are well known in the art and include cyclization.
[0047] In one embodiment, a peptide or peptidomimetic of the
invention, or a homing molecule such as a homing peptide or
peptidomimetic, is cyclic. As used herein, the term "cyclic" refers
to a molecule having non-adjacent components linked to one another
through a covalent or ionic bond or through an equivalent
interaction such that a rigid or semi-rigid three dimensional
structure of the molecule is maintained.
[0048] As used herein in reference to a peptide or peptidomimetic,
the term cyclic refers to a structure including an intramolecular
bond between two non-adjacent amino acids or amino acid analogues.
The cyclization can be effected through a covalent or non-covalent
bond. Intramolecular bonds include, but are not limited to,
backbone to backbone, side-chain to backbone and side-chain to
side-chain bonds. A preferred method of cyclization is through
formation of a disulfide bond between the side-chains of
non-adjacent amino acids or amino acid analogs. Residues capable of
forming a disulfide bond include, for example, cysteine (Cys),
penicillamine (Pen), .beta.,.beta.-pentamethylene cysteine (Pmc),
.beta.,.beta.-pentamethylene-.beta.-mercaptopropionic acid (Pmp)
and functional equivalents thereof (see, also, Table 1).
TABLE-US-00001 TABLE 1 AMINO ACIDS AND AMINO ACID ANALOGS USEFUL
FOR CYCLIZATION THREE LETTER AMINO ACID* CODE TYPE OF BOND
.gamma.-amino-adipic acid Adp Lactam Aspartic acid Asp Lactam
Cysteine Cys Disulfide Glutamic acid Glu Lactam Leucine Leu
Lysinonorleucine Lysine Lys Lactam and Lysinonorleucine
-(aminomethyl) benzoic acid Mamb Lactam Ornithine Orn Lactam
Penicillamine Pen Disulfide .alpha.,.beta.-diaminopropionic --
Lactam acid .beta.,.beta.-pentamethylene cysteine Pmc Disulfide
.beta.,.beta.-pentamethylene-.beta.- Pmp Disulfide
mercaptopropionic acid Tyrosine Tyr Dityrosine -- includes amino
acids and analogs thereof.
[0049] A peptide or peptidomimetic also can cyclize, for example,
via a lactam bond, which can utilize a side-chain group of one
amino acid or analog thereof to form a covalent attachment to the
N-terminal amine of the amino-terminal residue. Residues capable of
forming a lactam bond include aspartic acid (Asp), glutamic acid
(Glu), lysine (Lys), ornithine (Orn),
.alpha.,.beta.-diaminopropionic acid, .gamma.-amino-adipic acid
(Adp) and M-(aminomethyl)benzoic acid (Mamb). Cyclization
additionally can be effected, for example, through the formation of
a lysinonorleucine bond between lysine (Lys) and leucine (Leu)
residues or a dityrosine bond between two tyrosine (Tyr)
residues.
[0050] The present invention also provides an isolated homing
peptide or peptidomimetic that selectively homes to tumor lymphatic
vasculature. In one embodiment, the isolated homing peptide or
peptidomimetic selectively homes to tumor lymphatic vasculature
other than melanoma vasculature. In another embodiment, the
isolated homing peptide or peptidomimetic is not an anti-VEGFR-3 or
anti-LYVE-1 antibody or antigen-binding fragment thereof. In a
further embodiment, the isolated homing peptide or peptidomimetic
is not an antibody or antigen-binding fragment thereof. An isolated
homing peptide or peptidomimetic of the invention can include, for
example, the amino acid sequence GNKRTRG (SEQ ID NO: 2) or the
amino acid sequence CGNKRTRGC (SEQ ID NO: 1), or a conservative
variant or peptidomimetic of one of these sequences.
[0051] The present invention further provides a conjugate which
contains a moiety linked to a homing molecule that selectively
homes to tumor lymphatic vasculature. In one embodiment, the
conjugate contains a homing molecule which selectively homes to
tumor lymphatic vasculature other than melanoma vasculature. In
another embodiment, the conjugate contains a homing molecule which
selectively homes to tumor lymphatic vasculature and which is not
an anti-VEGFR-3 or anti-LYVE-1 antibody or antigen-binding fragment
thereof. In a further embodiment, the conjugate contains a homing
molecule which selectively homes to tumor lymphatic vasculature and
which is not an antibody or antigen-binding fragment thereof. In
yet a further embodiment, the conjugate contains a homing molecule
which selectively homes to tumor lymphatic vasculature and which
has cytotoxic activity.
[0052] In a conjugate of the invention, the homing molecule that
selectively homes to tumor lymphatic vasculature can be, for
example, a peptide or peptidomimetic. In one embodiment, the
peptide or peptidomimetic portion of the conjugate has a length of
at most 200 residues. In another embodiment, the peptide or
peptidomimetic portion of the conjugate has a length of at most 50
residues. In further embodiments, the conjugate contains a cyclic
or otherwise conformationally constrained homing molecule, such as
a peptide or peptidomimetic, that selectively homes to tumor
lymphatic vasculature. In yet further embodiments, the peptide or
peptidomimetic portion of the conjugate has cytotoxic activity.
[0053] A homing molecule useful in a conjugate of the invention can
be, for example, a homing peptide or peptidomimetic containing the
amino acid sequence GNKRTRG (SEQ ID NO: 2), or a conservative
variant or peptidomimetic thereof. If desired, such a peptide or
peptidomimetic can be cyclic or otherwise conformationally
constrained. A homing molecule useful in a conjugate of the
invention also can be, for example, a homing peptide or
peptidomimetic containing the amino acid sequence CGNKRTRGC (SEQ ID
NO: 1), or a conservative variant or peptidomimetic thereof. In
specific embodiments, such a homing peptide or peptidomimetic is
cyclic or otherwise conformationally constrained. A variety of
moieties are useful in a conjugate of the invention including,
without limitation, therapeutic agents, cancer chemotherapeutic
agents, cytotoxic agents, anti-lymphangiogenic agents, detectable
labels and phage.
[0054] As disclosed herein, peptide SEQ ID NO: 1 recognizes a
lymphatic vessel target "receptor" which is expressed on some tumor
cells as well as lymphatic vessel cells of the same tumors but
which is not significantly expressed in the lymphatic vessels of
normal tissues. The binding of SEQ ID NO: 1 to this target receptor
forms the basis for the selective homing activity of peptide SEQ ID
NO: 1 and related peptides and peptidomimetics. Based on this
discovery, it is clear that molecules structurally unrelated to SEQ
ID NO: 1 but which bind the same target receptor also have the same
characteristic of selective homing to tumor lymphatic vasculature.
Such molecules can be identified by the ability to specifically
bind to, or compete for binding to, the target receptor bound by
SEQ ID NO: 1. Thus, the invention provides a molecule that
specifically binds the receptor bound by peptide SEQ ID NO: 1; such
a molecule also is characterized by the ability to selectively home
to tumor lymphatic vasculature. In one embodiment, the molecule is
a peptide or peptidomimetic, which can have, for example, a length
of at most 20, 50 or 200 residues.
[0055] The invention also provides a conjugate which contains a
moiety linked to a molecule that specifically binds the receptor
bound by peptide SEQ ID NO: 1 and that selectively homes to tumor
lymphatic vasculature. In such a conjugate, the molecule can be,
for example, a peptide or peptidomimetic, and the moiety can be any
of the moieties disclosed herein as useful in the conjugates of the
invention.
[0056] The present invention also provides a method of directing a
moiety to tumor lymphatic vasculature in a subject by administering
to the subject a conjugate which contains a moiety linked to a
homing molecule that selectively homes to tumor lymphatic
vasculature, thereby directing the moiety to tumor lymphatic
vasculature. In one embodiment, a method of directing a moiety to
tumor lymphatic vasculature is practiced with a conjugate
containing a homing molecule which selectively homes to tumor
lymphatic vasculature other than melanoma vasculature. In another
embodiment, a method of the invention is practiced with a conjugate
containing a homing molecule which selectively homes to tumor
lymphatic vasculature and which is not an anti-VEGFR-3 or
anti-LYVE-1 antibody or antigen-binding fragment thereof. In a
further embodiment, a method of the invention is practiced with a
conjugate containing a homing molecule which selectively homes to
tumor lymphatic vasculature and which is not an antibody or
antigen-binding fragment thereof. In yet a further embodiment, a
method of the invention is practiced with a conjugate that contains
a homing molecule which selectively homes to tumor lymphatic
vasculature and which has cytotoxic activity.
[0057] In a method of the invention, the homing molecule can be,
for example, cyclic or otherwise conformationally constrained and
further can be, for example, a peptide or peptidomimetic. In one
embodiment, the homing molecule is a peptide containing the amino
acid sequence GNKRTRG (SEQ ID NO: 2), or a conservative variant or
peptidomimetic thereof. In another embodiment, the homing molecule
is a peptide that contains the amino acid sequence CGNKRTRGC (SEQ
ID NO: 1), or a conservative variant or peptidomimetic thereof. A
variety of moieties are useful in a method of the invention
including, for example, therapeutic agents, cancer chemotherapeutic
agents, cytotoxic agents, anti-lymphangiogenic agents, detectable
labels and phage.
[0058] It is understood that a variety of routes of administration
are useful in the methods of the invention. Such routes include
both systemic and local administration, including, without
limitation, oral administration, intravenous injection,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, transdermal diffusion or electrophoresis, local
injection; extended release delivery devices including locally
implanted extended release devices including bioerodible and
reservoir-based implants.
[0059] 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 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.
[0060] Exemplified herein are various homing molecules which
selectively home to tumor lymphatic vasculature but which do not
detectably home to the lymphatic vessels of several normal tissues
such as CGNKRTRGC (SEQ ID NO: 1), GNKRTRG (SEQ ID NO: 2), and
conservative variants or peptidomimetics thereof. Additional homing
molecules that selectively home to tumor lymphatics can be
identified using in vivo panning coupled, if desired, with ex vivo
selection, as disclosed in Examples 1 and 2 (see, also, U.S. Pat.
No. 5,622,699).
[0061] The term "homing molecule" as used herein, means any
molecule that selectively homes in vivo to the lymphatic
vasculature of one or more tumors in preference to normal lymphatic
vasculature. Similarly, the term "homing peptide" or "homing
peptidomimetic" means a peptide or peptidomimetic that selectively
homes in vivo to the lymphatic vasculature of one or more tumors in
preference to normal lymphatic vasculature. It is understood that a
homing molecule that selectively homes in vivo to tumor lymphatic
vasculature can home to the lymphatic vasculature of all tumors or
can exhibit preferential homing to the lymphatic vasculature of a
subset of tumor types.
[0062] By "selectively homes" is meant that, in vivo, the homing
molecule, peptide or peptidomimetic binds preferentially to tumor
lymphatic vasculature, such as breast tumor or osteosarcoma
lymphatic vasculature, as compared to non-tumoral lymphatic
vasculature. Selective homing generally is characterized by at
least a two-fold two-fold greater localization within tumor
lymphatic vasculature, such as breast or osteosarcoma lymphatic
vasculature as compared to several tissue types of non-tumoral
lymphatic vasculature. A homing molecule can be characterized by
5-fold, 10-fold, 20-fold or more preferential localization to tumor
lymphatic vasculature as compared to several tissue types of
non-tumoral lymphatic vasculature, or as compared to most or all
non-tumoral lymphatic vasculature. Thus, it is understood that a
homing molecule can home, in part, to the lymphatic vasculature of
one or more normal organs, in addition to tumor lymphatic
vasculature.
[0063] In one embodiment, a conjugate of the invention includes a
homing molecule that is not an antibody or antigen-binding fragment
thereof, which is an art-recognized term that refers to a peptide
or polypeptide containing one or more complementarity determining
regions (CDRs). See, for example, Borrabaeck, Antibody Engineering
2nd Edition, Oxford University Press, New York (1995).
[0064] In another embodiment, the peptide or peptidomimetic portion
of the conjugate has a defined length. The peptide or
peptidomimetic portion of the conjugate can have, for example, a
length of at most 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400,
500, 600, 700, 800, 900, 1000 or 2000 residues. It is understood
that the term "peptide or peptidomimetic portion of the conjugate"
means total number of residues in the homing peptide or
peptidomimetic and any contiguous protein, peptide or
peptidomimetic, such as a therapeutic protein or pro-apoptotic
peptide.
[0065] If desired, a conjugate of the invention can contain
multiple homing molecules which each selectively homes to tumor
lymphatic vasculature. In one embodiment, a conjugate of the
invention contains at least two homing molecules which each
selectively homes to tumor lymphatic vasculature. In further
embodiments, a conjugate of the invention contains at least 10
homing molecules, or at least 100 homing molecules, which each
selectively homes to tumor lymphatic vasculature. In yet a further
embodiment, the invention provides a conjugate containing a phage
linked to at least 100 homing molecules which each selectively
homes to tumor lymphatic vasculature.
[0066] A conjugate of the invention can contain, for example, a
moiety linked to at least two homing molecules which each
selectively homes to tumor lymphatic vasculature and which each
independently includes the amino acid sequence GNKRTRG (SEQ ID NO:
2), or a conservative variant or peptidomimetic thereof. In a
further embodiment, the invention provides a conjugate containing a
moiety linked to at least ten homing molecules which each
selectively homes to tumor lymphatic vasculature and which each
independently includes the amino acid sequence GNKRTRG (SEQ ID NO:
2) or a conservative variant or peptidomimetic thereof. In yet
another embodiment, the invention provides a conjugate containing a
moiety linked to at least 100 homing molecules which each
selectively homes to tumor lymphatic vasculature and which each
independently includes the amino acid sequence GNKRTRG (SEQ ID NO:
2) or a conservative variant or peptidomimetic thereof. Moieties
useful in a conjugate of the invention containing multiple homing
peptides include, but are not limited to, phage moieties.
[0067] Thus, a conjugate of the invention containing multiple
homing molecules 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 100 or more homing molecules. In
one embodiment, the homing molecules have an identical amino acid
sequence. In another embodiment, the conjugate includes homing
molecules having non-identical amino acid sequences. Moieties
useful in a conjugate of the invention that incorporates multiple
homing molecules 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.
[0068] A conjugate of the invention can contain, for example, a
liposome or other polymeric matrix linked to at least two homing
molecules which each selectively homes to tumor lymphatic
vasculature. If desired, the liposome or other polymeric matrix can
be linked to at least ten or at least 100 homing molecules which
each selectively homes to tumor lymphatic vasculature. Homing
molecules useful in such a conjugate can independently include, for
example, the amino acid sequence GNKRTRG (SEQ ID NO: 2), the amino
acid sequence CGNKRTRGC (SEQ ID NO: 1), or a conservative variant
or peptidomimetic of one of these sequences. Liposomes, for
example, which consist of phospholipids or other lipids, are
nontoxic, physiologically acceptable and metabolizable carriers
that are relatively simple to make and administer (Gregoriadis,
Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla. (1984)).
The liposome or other polymeric matrix additionally can include
another component if desired, for example, a therapeutic agent,
cancer chemotherapeutic agent, cytotoxic agent or
anti-lymphangiogenic agent.
[0069] A conjugate of the invention includes a moiety linked to a
homing molecule that selectively homes to tumor lymphatic
vasculature. As used herein, the term "moiety" is used broadly to
mean a physical, chemical, or biological material that can be
linked to a homing molecule of the invention and generally imparts
a biologically useful function to the homing molecule. A moiety can
be any natural or normatural 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.
Moieties useful in the invention include, without limitation,
therapeutic agents; cancer chemotherapeutic agents, cytotoxic
agents, pro-apoptotic agents, anti-lymphangiogenic agents,
detectable labels and imaging agents; and tags or other insoluble
supports. Moieties useful in the invention further include, for
example, phage and other viruses, cells, liposomes, polymeric
matrices, non-polymeric matrices or particles such as gold
particles, microdevices and nanodevices, and nano-scale
semiconductor materials. These and other moieties known in the art
can be components of a conjugate of the invention, as disclosed
herein.
[0070] In one embodiment, a moiety useful in a conjugate of the
invention is an anti-lymphangiogenic agent. As used herein, the
term "anti-lymphangiogenic agent" is a molecule that reduces or
inhibits the growth of lymphatic vessels. Stimulation of the
vascular endothelial growth factor receptor-3 (VEGFR-3) signal
transduction pathway is sufficient to specifically induce
lymphangiogenesis in vivo, and VEGFR-3 expression mainly is
restricted to lymphatic vessels in later development. Furthermore,
the VEGFR-3 ligand, VEGF-C, is mitogenic towards lymphatic
endothelial cells and can induce a lymphangiogenic response in
differentiated avian chorioallantoic membrane and mouse skin
(Karkkainen and Petrova, Oncogene 19:5598-5605 (2000); and Veikkola
et al., EMBO J. 20:1223-1231 (2001)). Thus, an anti-lymphangiogenic
agent can be, for example, a VEGFR-3 inhibitor, which is a molecule
that inhibits VEGFR-3 expression, activity, or signaling.
[0071] A VEGFR-3 inhibitor can be selective for VEGFR-3 and can
exhibit, for example, at least 10-fold greater inhibition of
VEGFR-3 expression or activity as compared to the expression or
activity of other vascular endothelial growth factor receptors
(VEGFRs). Such a selective VEFGR-3 inhibitor can exhibit, for
example, at least 20-fold, 50-fold, or 100-fold greater inhibition
of VEGFR-3 expression or activity as compared to the expression or
activity of other VEGFRs. It is understood that a non-selective
VEGFR-3 inhibitor also can be useful in the invention. Such a
VEGFR-3 inhibitor inhibits the expression or activity of one or
more other VEGFRs such as VEGFR-1 or VEGFR-2 or other tyrosine
kinases in addition to inhibiting VEGFR-3. It further is understood
that a VEGFR-3 inhibition or other anti-lymphangiogenic agent also
can have additional activity, for example, as an anti-angiogenic
agent.
[0072] A variety of anti-lymphangiogenic agents are known in the
art including for example, VEGFR-3 antagonists, which bind to but
do not activate VEGFR-3; soluble receptors or other dominant
negative VEGFR-3 receptors such as kinase-inactive receptors;
inhibitory anti-VEGFR-3 antibodies; competitors of VEGFR-3 ligand
binding, for example, VEGF-C or VEGF-D binding; small molecules;
antisense nucleic acid molecules; ribozymes; transcription factors
or their encoding nucleic acid molecules; or other molecules which
reduce VEGFR-3 expression; selective VEGFR-3 kinase inhibitors such
as ATP analogs; and selective inhibitors of the VEGFR-3 signaling
pathway. Thus, it is understood that various types of molecules can
function as an anti-lymphangiogenic agent, including a small
molecule; a protein, for example, a dominant negative receptor,
transcription factor or antibody; a peptide or peptidomimetic; a
ribozyme; or a nucleic acid molecule such as an antisense
oligonucleotide or nucleic acid molecule encoding a dominant
negative receptor, transcription factor or antibody.
[0073] In one embodiment, a moiety contained in a conjugate of the
invention is a therapeutic agent. As used herein, the term
"therapeutic agent" means a molecule which alters biological
activity in a normal or pathologic tissue. A therapeutic agent,
therefore, is potentially useful for the treatment of disease
conditions. A variety of therapeutic agents can be contained in a
conjugate of the invention. In another embodiment, a conjugate of
the invention contains a cancer chemotherapeutic agent. As used
herein, a "cancer chemotherapeutic agent" is a chemical agent that
inhibits the proliferation, growth, life-span or metastatic
activity of cancer cells. Such a cancer chemotherapeutic agent can
be, without limitation, a taxane such as docetaxel; an anthracyclin
such as doxorubicin; an alkylating agent; a vinca alkaloid; an
anti-metabolite; a platinum agent such as cisplatin or carboplatin;
a selective estrogen receptor modulator; an antibody such as
trastuzumab; a steroid such as methotrexate; an antibiotic such as
adriamycin; and a chemotherapeutic such as isofamide.
[0074] A taxane compound useful as a cancer chemotherapeutic agent
in a conjugate of the invention can be, for example, docetaxel
(Taxotere; Aventis Pharmaceuticals, Inc.; Parsippany, N.J.) or
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).
[0075] A cancer chemotherapeutic 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 and can be useful, for example, for treating
breast cancer (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 (Folkman, supra, 1997; Steiner, In
"Angiogenesis: Key principles-Science, technology and medicine,"
pp. 449-454 (eds. Steiner et al.; Birkhauser Verlag, 1992)), which
can contribute to its effectiveness in treating cancer.
[0076] An alkylating agent such as melphalan or chlorambucil also
can be a cancer chemotherapeutic 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
cancer chemotherapeutic agent useful in a conjugate of the
invention.
[0077] Another cancer chemotherapeutic agent useful in conjugates
of the invention is a platinum agent. 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 cancer
chemotherapeutic agents useful in a conjugate of the invention
include, without limitation, methotrexate, mitomycin-C, adriamycin,
ifosfamide and ansamycins.
[0078] A cancer chemotherapeutic agent for treatment of breast
cancer and other hormonally-dependent cancers also can be an agent
that antagonizes the effect of estrogen, such as a selective
estrogen receptor modulator or an anti-estrogen. The selective
estrogen receptor modulator, tamoxifen, is a cancer
chemotherapeutic agent that can be used in a conjugate of the
invention for treatment of breast cancer (Fisher et al., J. Natl.
Cancer Instit. 90:1371-1388 (1998)).
[0079] A therapeutic agent useful in a conjugate of the invention
can be an antibody such as a humanized monoclonal antibody. For
example, the anti-epidermal growth factor receptor 2 (HER2)
antibody, trastuzumab (Herceptin; Genentech, South San Francisco,
Calif.) is a therapeutic agent useful in a conjugate of the
invention for treating HER2/neu overexpressing breast cancers
(Burris et al., supra, 2001; White et al., Annu Rev. Med.
52:125-141 (2001)).
[0080] In another embodiment, a moiety useful in a conjugate of the
invention is a cytotoxic agent. As used herein, the term "cytotoxic
agent" refers to any molecule that results in cell death by any
mechanism. Exemplary cytotoxic agents useful in a conjugate of the
invention are doxorubicin, docetaxel and trastuzumab and
antimicrobial peptides, described herein below.
[0081] A moiety useful in a conjugate of the invention also can be
an anti-angiogenic agent. As used herein, an "anti-angiogenic
agent" is a molecule that reduces or prevents angiogenesis, the
growth and development of blood vessels. Vascular endothelial
growth factor (VEGF) has been shown to be important for
angiogenesis in many types of cancer, including breast cancer
angiogenesis in vivo (Borgstrom et al., Anticancer Res.
19:4213-4214 (1999)). An anti-angiogenic agent can be, for example,
an inhibitor or neutralizing antibody that inhibits a growth factor
or other factor important for angiogenesis. In one embodiment, the
anti-angiogenic agent is an anti-VEGF neutralizing monoclonal
antibody (Borgstrom et al., supra, 1999). In another embodiment,
the anti-angiogenic agent is a steroid or teratogen. In a further
embodiment, the anti-angiogenic agent is a protein, peptide, or
peptide fragment, such as endostatin, anastellin, thrombospondin,
angiostatin, or a kringle peptide of angiostatin.
[0082] A moiety useful in a conjugate of the invention also can be
a detectable label. As used herein, the term "detectable label"
refers to any molecule which can be administered in vivo and
subsequently detected. Exemplary detectable labels useful in the
conjugates and methods of the invention include 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.
[0083] The invention further provides a conjugate in which a homing
molecule that selectively homes to tumor lymphatic vasculature is
linked to an antimicrobial peptide, where the conjugate is
selectively internalized by tumor lymphatic vasculature and
exhibits a high toxicity to the tumor lymphatic vasculature, and
where the antimicrobial peptide has low mammalian cell toxicity
when not linked to the homing molecule. 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 which has low
mammalian cell toxicity when not linked to a homing molecule. An
antimicrobial peptide, for example, can kill or slow the growth of
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.
[0084] 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., supra,
1996; Blondelle and Houghten, supra, 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).
[0085] 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.
[0086] An antimicrobial peptide incorporated within a conjugate of
the invention has low mammalian cell toxicity when not linked to a
tumor homing molecule. 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 (DM for lytic activity,
preferably concentrations greater than 200, 300, 500 or 1000
.PHI.M.
[0087] In one embodiment, the invention provides a conjugate in
which the antimicrobial peptide portion promotes disruption of
mitochondrial membranes when internalized by eukaryotic cells. In
particular, such an antimicrobial peptide preferentially disrupts
mitochondrial membranes as compared to eukaryotic membranes.
Mitochondrial membranes, like bacterial membranes but in contrast
to eukaryotic plasma membranes, have a high content of negatively
charged phospholipids. An antimicrobial peptide can be assayed for
activity in disrupting mitochondrial membranes using, for example,
an assay for mitochondrial swelling or another assay well known in
the art. .sub.D(KLAKLAK).sub.2, for example, is an antimicrobial
peptide which induces marked mitochondrial swelling at a
concentration of 10 .PHI.M, significantly less than the
concentration required to kill eukaryotic cells. An antimicrobial
peptide that induces significant mitochondrial swelling at, for
example, 50 .PHI.M, 40 .PHI.M, 30 .PHI.M, 20 .PHI.M, 10 .PHI.M, or
less, is considered a peptide that promotes disruption of
mitochondrial membranes.
[0088] An antimicrobial peptide portion can include, for example,
the sequence (KLAKLAK).sub.2 (SEQ ID NO: 5), (KLAKKLA).sub.2 (SEQ
ID NO: 6), (KAAKKAA).sub.2 (SEQ ID NO: 7), or (KLGKKLG).sub.3 (SEQ
ID NO: 8), and, in one embodiment, includes the sequence
.sub.D(KLAKLAK).sub.2. A conjugate of the invention, which contains
a homing molecule that selectively homes to tumor lymphatic
vasculature linked to an antimicrobial peptide, can have, for
example, the sequence CGNKRTRGC-GG-.sub.D(KLAKLAK).sub.2 or
GNKRTRG-GG-.sub.D(KLAKLAK).sub.2.
[0089] Antimicrobial peptides generally have random coil
conformations in dilute aqueous solutions, yet high levels of
helicity can be induced by helix-promoting solvents and amphipathic
media such as micelles, synthetic bilayers or cell membranes.
.alpha.-Helical structures are well known in the art, with an ideal
.alpha.-helix characterized by having 3.6 residues per turn and a
translation of 1.5 .ANG. per residue (5.4 .ANG. per turn; see
Creighton, Proteins: Structures and Molecular Properties W.H
Freeman, New York (1984)). In an amphipathic .alpha.-helical
structure, polar and non-polar amino acid residues are aligned into
an amphipathic helix, which is an .alpha.-helix in which the
hydrophobic amino acid residues are predominantly on one face, with
hydrophilic residues predominantly on the opposite face when the
peptide is viewed along the helical axis.
[0090] Antimicrobial peptides of widely varying sequence have been
isolated, sharing an amphipathic .alpha.-helical structure as a
common feature (Saberwal et al., Biochim. Biophys. Acta
1197:109-131 (1994)). Analogs of native peptides with amino acid
substitutions predicted to enhance amphipathicity and helicity
typically have increased antimicrobial activity. In general,
analogs with increased antimicrobial activity also have increased
cytotoxicity against mammalian cells (Maloy et al., Biopolymers
37:105-122 (1995)).
[0091] As used herein in reference to an antimicrobial peptide, the
term "amphipathic .alpha.-helical structure" means an .alpha.-helix
with a hydrophilic face containing several polar residues at
physiological pH and a hydrophobic face containing nonpolar
residues. A polar residue can be, for example, a lysine or arginine
residue, while a nonpolar residue can be, for example, a leucine or
alanine residue. An antimicrobial peptide having an amphipathic
.alpha.-helical structure generally has an equivalent number of
polar and nonpolar residues within the amphipathic domain and a
sufficient number of basic residues to give the peptide an overall
positive charge at neutral pH (Saberwal et al., Biochim. Biophys.
Acta 1197:109-131 (1994)). One skilled in the art understands that
helix-promoting amino acids such as leucine and alanine can be
advantageously included in an antimicrobial peptide of the
invention (see, for example, Creighton, supra, 1984). Synthetic,
antimicrobial peptides having an amphipathic .alpha.-helical
structure are known in the art, for example, as described in U.S.
Pat. No. 5,789,542 to McLaughlin and Becker.
[0092] It is understood by one skilled in the art of medicinal
oncology that these and other agents are useful therapeutic agents,
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 agents
and that additional components can be included as part of the
conjugate, if desired. For 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)).
[0093] The invention also provides a method of imaging tumor
lymphatic vasculature in a subject by administering to the subject
a conjugate which contains a detectable label linked to a homing
molecule that selectively homes to tumor lymphatic vasculature, and
detecting the conjugate, thereby imaging the tumor lymphatic
vasculature. In a method of the invention for imaging tumor
lymphatic vasculature, the homing peptide can be, for example,
cyclic or otherwise conformationally constrained and further can
be, for example, a peptide or peptidomimetic. In one embodiment,
the homing molecule is a peptide containing the amino acid sequence
GNKRTRG (SEQ ID NO: 2), or a conservative variant or peptidomimetic
thereof. In another embodiment, the homing molecule is a peptide
containing the amino acid sequence CGNKRTRGC (SEQ ID NO: 1), or a
conservative variant or peptidomimetic thereof. A detectable label
useful in an imaging method of the invention can be, for example, a
radionuclide or a fluorescent molecule. Examples of radionuclides
useful as detectable labels include, but are not limited to,
indium-111, technetium-99, carbon-11, and carbon-13.
[0094] The methods of the invention for imaging tumor lymphatic
vasculature can be useful for detecting the presence of tumor
lymphatic vasculature associated with a variety of tumors.
Following administration of a conjugate of the invention containing
a detectable label, tumor lymphatic vasculature is visualized. If
the image is positive for the presence of such tumor lymphatics,
the tumor can be evaluated for size and quantity of lymphatic
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.
[0095] In a method of imaging tumor lymphatic vasculature, the
conjugate administered contains a detectable label that allows
detection or visualization of lymphatic vasculature in tumors, for
example in breast tumors or in osteosarcomas. For in vivo
diagnostic imaging of such tumor lymphatic vasculature, a homing
molecule selective for the desired tumor is linked to a detectable
label that, upon administration to the subject, is detectable
external to the subject. Such a detectable label 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.
[0096] The present invention also provides a method for reducing or
inhibiting tumor metastasis in a subject. Metastasis occurs
primarily through the lymphatic system, and the extent of lymph
node involvement is a key prognostic factor for severity of
disease. Lymphangiogenesis and the quantity of intratumoral
lymphatic vessels in primary tumors have been correlated with tumor
metastasis in animal experiments, for example, in breast cancer.
(Skobe et al., Nature Medicine 7(2):192-198 (2001)). Intratumoral
lymphatic vasculature can play an important role in the metastasis
of many tumor types such as breast, colon, lung, thyroid, gastric,
squamous cell cancers, mesotheliomas, osteosarcomas, and
neuroblastomas.
[0097] According to the present invention, tumor metastasis is
reduced or inhibited by administering to the subject a conjugate
which contains a moiety linked to a homing molecule that
selectively homes to tumor lymphatic vasculature, thereby reducing
or inhibiting tumor metastasis. In such a method of the invention,
the homing molecule can be, for example, cyclic or otherwise
conformationally constrained and further can be, for example, a
peptide or peptidomimetic. In one embodiment, the homing molecule
is a peptide containing the amino acid sequence GNKRTRG (SEQ ID NO:
2), or a conservative variant or peptidomimetic thereof. In another
embodiment, the homing molecule is a peptide containing the amino
acid sequence CGNKRTRGC (SEQ ID NO: 1), or a conservative variant
or peptidomimetic thereof. A variety of moieties are useful in a
method of the invention for reducing or inhibiting tumor
metastasis. Such a moieties include, without limitation, cancer
chemotherapeutic agents, cytotoxic agents and anti-lymphangiogenic
agents.
[0098] The present invention further provides a method of reducing
the number of tumor lymphatic vessels in a subject by administering
to the subject a conjugate which contains a moiety linked to a
homing molecule that selectively homes to tumor lymphatic
vasculature, thereby reducing the number of tumor lymphatic vessels
in the subject. In a method of the invention, the homing molecule
can be, for example, cyclic or otherwise conformationally
constrained and further can be, for example, a peptide or
peptidomimetic. In one embodiment, the homing molecule is a peptide
containing the amino acid sequence GNKRTRG (SEQ ID NO: 2), or a
conservative variant or peptidomimetic thereof. In another
embodiment, the homing molecule is a peptide containing the amino
acid sequence CGNKRTRGC (SEQ ID NO: 1), or a conservative variant
or peptidomimetic thereof. A variety of moieties can be useful in a
method of the invention for reducing the number of tumor lymphatic
vessels including, without limitation, cancer chemotherapeutic
agents, cytotoxic agents and anti-lymphangiogenic agents.
[0099] In addition, the present invention provides a method of
treating cancer in a subject by administering to the subject a
conjugate which contains a moiety linked to a homing molecule that
selectively homes to tumor lymphatic vasculature. In a method of
the invention, the homing molecule can be, for example, cyclic or
otherwise conformationally constrained and further can be, for
example, a peptide or peptidomimetic. In one embodiment, the homing
molecule is a peptide containing the amino acid sequence GNKRTRG
(SEQ ID NO: 2), or a conservative variant or peptidomimetic
thereof. In another embodiment, the homing molecule is a peptide
containing the amino acid sequence CGNKRTRGC (SEQ ID NO: 1), or a
conservative variant or peptidomimetic thereof. A variety of
moieties can be useful in a method of the invention for treating
cancer in a subject including, but not limited to, cancer
chemotherapeutic agents, cytotoxic agents and anti-lymphangiogenic
agents.
[0100] As disclosed above, peptide SEQ ID NO: 1 selectively homes
to tumor lymphatic vasculature and further has cytotoxic activity.
Thus, the present invention further provides a method of treating
cancer in a subject by administering to the subject a cytotoxic
homing molecule that selectively homes to tumor lymphatic
vasculature. In a method of the invention, the cytotoxic homing
molecule can be, for example, cyclic or otherwise conformationally
constrained and further can be, for example, a peptide or
peptidomimetic. Furthermore, a cytotoxic homing molecule useful in
a method of the invention can be, for example, a cytotoxic peptide
that contains the amino acid sequence GNKRTRG (SEQ ID NO: 2), or a
conservative variant or peptidomimetic thereof, or a cytotoxic
peptide that contains the amino acid sequence CGNKRTRGC (SEQ ID NO:
1), or a conservative variant or peptidomimetic thereof.
[0101] The following examples are intended to illustrate but not
limit the present invention.
Example 1
Identification of Peptides that Bind to MDA-MB-435 Breast Carcinoma
Cells In Vitro
[0102] This example describes identification of a peptide that
selectively homes to MDA-MB-435 breast carcinoma xenograft tumors
using ex vivo and in vivo selections.
[0103] Several ex vivo selections were performed with 435 tumor
cell suspensions prepared from MDA-MB-435 breast carcinoma
xenografts grown in nude mice, as described below. Anti-mouse CD31
was used to deplete the tumor cell mixture of blood vessel
endothelial cells prior to rescuing phage bound to the
CD31-negative cell population. By the third ex vivo round, the
phage pool bound a tumor cell suspension approximately 350-fold
over control, nonrecombinant phage. The ex vivo preselected phage
pool then was subjected to an in vivo selection round by injection
into the tail vein of a nude mouse bearing an MDA-MB-435 tumor.
Phage were then rescued from harvested xenograft tumor tissue.
[0104] Selected phage were enriched 30-fold relative to
nonrecombinant T7 phage in the tumor. Forty-eight individual clones
were randomly chosen from the in vivo selected pool, and the
inserts sequenced as described below. Individual clones were
assayed for the ability to bind cultured 435 cells as well as cell
suspensions prepared from 435 tumors.
[0105] As shown in FIG. 1A, phage displaying the peptide CGNKRTRGC
(SEQ ID NO: 1) bound to primary 435 tumor cell suspensions about
5000 times better than nonrecombinant phage. In contrast, phage
displaying the spontaneous permutation CGEKRTRGC (SEQ ID NO: 3) or
CGNKRTRGV (SEQ ID NO: 4) did not bind 435 tumor cell suspensions
(see FIG. 1A). Addition of synthetic CGNKRTRGC (SEQ ID NO: 1)
peptide inhibited binding of phage displaying the same peptide
sequence.
[0106] Binding of the CGNKRTRGC (SEQ ID NO: 1)-displaying phage to
the 435 tumor cell suspension correlated with copy number of the
displayed peptide. As shown in FIG. 1B, there was a progressive
decrease in phage binding when the peptide was displayed at 415
copies, 10 copies, or 1 copy. Thus, binding of SEQ ID NO:
1-displaying phage to a 435 tumor cell suspension was specific. The
CGNKRTRGC (SEQ ID NO: 1)-displaying phage also bound to the 435
cells cultured for one to three days, although binding was weaker
than to cells isolated from tumors. On average, binding to cultured
435 cells was about 50-fold greater than the binding observed with
control, nonrecombinant phage.
[0107] These results demonstrate specific binding of phage
displaying peptide SEQ ID NO: 1 to tumor cell suspensions prepared
from breast carcinoma MDA-MB-435 xenografts.
[0108] A peptide display phage library with the general structure
CX.sub.7C, where C is cysteine and X is any amino acid, was
constructed in T7 phage essentially as follows. Briefly,
complementary oligonucleotides that encoded the random peptide
insert as NNK codons, and had 5' Eco RI and 3' Hind III overhangs,
were annealed. The resulting double stranded DNA was phosphorylated
with T4 polynucleotide kinase (Novagen; Madison, Wis.) and ligated
into 1 .mu.g of T7Select415-1b vector arms. The ligated product was
directly added to 50 .mu.l of packaging extract and incubated for
two hours, yielding 10.sup.8 pfu total recombinants. Following
amplification of recombinants in 500 ml of liquid culture,
purification of phage particles and sequencing of single stranded
phage DNA was performed by standard methods.
[0109] Xenografts, tumor cell suspensions and ex vivo selections
were performed essentially as follows. Nude Balb/c mice were
subcutaneously injected with 1.times.10.sup.6 MDA-MB-435 tumor
cells to generate breast carcinoma xenografts. Human MDA-MB-435
breast carcinoma xenograft tumors were harvested 9 to 12 weeks
after implantation, and tumor cell suspensions prepared using
collagenase (0.5 mg/ml, Sigma).
[0110] 435 tumor cell suspensions were incubated with the T7
phage-displayed CX.sub.7C library (3.7.times.10.sup.10 pfu)
overnight at 4.degree. C. The suspension was subjected to serial
washes with 1% BSA in DME to remove unbound phage. Blood vessel
endothelial cells were depleted from the tumor cell mixture using
magnetic beads (Dynal; Lake Success, N.Y.) coated with anti-mouse
CD31 antibody (MEC 13.3; Phaminogen; San Diego, Calif.) according
to the manufacturer=s instructions. Phage bound to the
CD31-negative cell population were rescued by adding bacteria, and
the rescued phage titered and amplified in liquid culture.
Example 2
Selective In Vivo Homing of Peptide CGNKRTRGC (SEQ ID NO: 1)
[0111] This example demonstrates that CGNKRTRGC (SEQ ID NO:
1)-displaying phage selectively home to MDA-MB-435 and KRIB human
osteosarcoma tumors in vivo.
[0112] In vivo homing selectivity was analyzed essentially as
follows. CGNKRTRGC (SEQ ID NO: 1)-displaying phage were injected
into the tail vein of nude mice bearing either an MDA-MB-435 tumor,
a KRIB osteosarcoma, a C8161 melanoma or an HL-60 leukemia cell
xenograft. Phage subsequently were rescued from the respective
tumors, as well as from several normal tissues (brain, kidney,
liver, spleen, skin and breast).
[0113] As shown in FIG. 1C, CGNRTRKGC (SEQ ID NO: 1)-displaying
phage homed to 435 and KRIB tumors in vivo. Although the strength
of homing varied, the mean phage titer in tumor tissue was about
60-fold greater than for nonrecombinant phage in 435 tumors and
15-fold greater than for nonrecombinant phage in KRIB
osteosarcomas. In contrast, the CGNKRTRGC (SEQ ID NO: 1)-displaying
phage did not home in vivo to C8161 melanoma xenografts or HL-60
human leukemia xenografts. Furthermore, the CGNKRTRGC (SEQ ID NO:
1)-displaying phage did not home to normal brain, spleen, skin,
kidney, or lung tissue. The CGNKRTRGC (SEQ ID NO: 1)-displaying
phage appeared to home weakly to normal breast tissue (see FIG.
1D).
[0114] Homing of a CGEKRTRGC (SEQ ID NO: 3)-displaying phage, in
which asparagine-3 is substituted with glutamate, to 435 tumors in
vivo was only about 8% of that of the CGNKRTRGC (SEQ ID NO: 1)
phage (see FIG. 1C). These in vivo homing results, which reproduced
the binding results exhibited in tumor-derived cell suspensions,
indicate that an asparagine or related residue at position three
can contribute to homing activity.
[0115] In sum, these results demonstrate that peptide SEQ ID NO: 1
has selective homing activity and homes to breast cancer and
osteosarcoma tumors in preference to normal tissues.
Example 3
Internalization of the CGNKRTRGC (SEQ ID NO: 1)-Displaying Phage by
Cells
[0116] This example demonstrates that CGNKRTRGC (SEQ ID NO:
1)-displaying phage are internalized by cells.
[0117] Increasing the time from the intravenous injection of the
CGNKRTRGC (SEQ ID NO: 1)-displaying phage to rescue from the 435
tumors decreased phage recovery. To determine whether or not this
decrease was due to internalization of phage by the cells, phage
were rescued by lysing 435 tumor cells with a 0.5% solution of the
detergent NP-40. As shown in FIG. 1E, 10 to 20 times more SEQ ID
NO: 1-displaying phage were recovered with detergent than without
detergent, indicating that phage were internalized by the tumor
cells.
[0118] A fluorescein-conjugated CGNKRTRGC (SEQ ID NO: 1) peptide
was utilized to further analyze cellular internalization and
subcellular localization. Fluorescein-conjugated CGNKRTRGC (SEQ ID
NO: 1) peptide and a fluorescein-conjugated control peptide
containing three basic residues were synthesized according to
Wender et al., Proc. Natl. Acad. Sci. 97:13003 (2000). The
fluorescein-conjugated peptides were incubated with cultured
MDA-MB-435 tumor cells for one to five hours at 37.degree. C. As
shown in FIG. 2A, fluorescein-labeled peptide CGNKRTRGC (SEQ ID NO:
1) was taken up by the 435 cells and translocated into the cell
nucleus. In contrast, there was no detectable uptake by the cells
of fluorescein-labeled control peptide (see FIG. 2B). Furthermore,
fluorescein-conjugated CGNKRTRGC (SEQ ID NO: 1) peptide was
internalized and transported into nuclei in vivo. Cultured 435
cells also internalized the CGNKRTRGC (SEQ ID NO: 1) phage,
although the phage accumulated in the cytoplasm of the cells.
[0119] These results demonstrate that CGNKRTRGC (SEQ ID NO:
1)-displaying phage and a fluorescein-SEQ ID NO: 1 conjugate were
internalized by cells.
Example 4
Localization of CGNKRTRGC (SEQ ID NO: 1) Peptide with Lymphatic
Markers VEGFR-3 and LYVE-1
[0120] This example demonstrates that peptide SEQ ID NO: 1
localizes to lymphatic vasculature.
A. Peptide SEQ ID NO: 1 Localizes to Vessels that are Negative for
Blood Vessel Markers
[0121] Localization of phage displaying SEQ ID NO: 1 was analyzed
with anti-T7 phage antibody following intravenous injection of
CGNKRTRGC (SEQ ID NO: 1)-displaying phage into the tail vein of
nude mice carrying an MDA-MB-435 tumor. CGNKRTRGC (SEQ ID NO:
1)-displaying phage localized in vessel-like structures and in some
single cells within the 435 tumors. The vessels in which the phage
localized were negative for Meca-32, a marker specific for blood
vessels, and also negative for CD31, which is expressed more
prominently in blood vessels than in lymphatic vessels. Similar
results were obtained by a phage overlay assay in which phage were
added onto frozen tissue sections, rather than being injected into
the mice.
[0122] When fluorescein-conjugated CGNKRTRGC (SEQ ID NO: 1) peptide
was injected into the tail vein of 435 tumor-bearing mice, the
fluorescein-conjugated peptide localized in vessel-like structures
and in individual cells within the tumor. These vessels were
negative for the CD31 and Meca-32 blood vessel markers. As seen in
FIGS. 3A-C, there was a notable lack of co-localization of
fluorescein-conjugated peptide with blood vessels labeled with
biotin-conjugated tomato lectin. Even where portions of the tumor
contained both peptide and blood vessels, their locations were
distinct (FIGS. 3G-I). These data indicate that the vessel-like
structures targeted by the CGNKRTRGC (SEQ ID NO: 1) peptide were
not blood vessels.
[0123] Several normal tissues also were studied for localization of
the CGNKRTRGC (SEQ ID NO: 1) peptide. Fluorescence was seen only in
the kidney tubuli, which may be a result of uptake of peptide from
the glomerular filtrate. The fluorescein-labeled control peptide
was also detected to the same extent in the kidney tubuli after
intravenous injection, but was not detected in tumor tissue,
indicating that peptide localization to kidney tubuli was
non-specific.
[0124] An anti-T7 antiserum was prepared by immunizing New Zealand
white rabbits with 10.sup.10 pfu of T7 nonrecombinant phage
(Novagen). The initial immunization performed in complete Freund's
Adjuvant, while boosters were administered in incomplete Freund's
Adjuvant. The antibody titer was estimated by ELISA, and the
antiserum was absorbed against BLT5615 bacterial and mouse liver
lysates. Phage were detected using anti-T7 phage antiserum (1:1000
dilution) and goat anti-rabbit secondary antibody conjugated to
fluorescein.
[0125] Detection of fluorescent peptides and biotin-conjugated
tomato lectin was performed as follows. Fluorescein-conjugated
peptide CGNKRTRGC (SEQ ID NO: 1) was prepared as described above.
The peptide (100 .mu.g in 200 .mu.l of PBS) was injected into the
tail vein of mice bearing MDA-MB-435 breast carcinoma tumors. After
10 minutes, biotin-conjugated lycopersicon esculentum (tomato)
lectin (100 .mu.g in 200 .mu.l of PBS; Vector; Burlingame, Calif.)
was also injected into the tail vein. After 5 minutes, the mouse
was perfused through the heart with 4% paraformaldehyde. Tissues
were removed and frozen in O.C.T. embedding medium (Tissue-Tek;
Torrence, Calif.). Blood vessels were visualized by detecting the
tomato lectin with streptavidin-conjugated Alexa 594 (Molecular
Probes; Eugene, Oreg.). Green staining indicated the presence of
peptide SEQ ID NO: 1, while red staining indicated the presence of
tomato lectin.
B. Peptide CGNKRTRGC (SEQ ID NO: 1) Homes to Lymphatic Vessels and
Cells within Tumors
[0126] MDA-MB-435 breast carcinoma tumors contain lymphatic vessels
that are positive for markers of lymphatic endothelial cells. To
determine whether CGNKRTRGC (SEQ ID NO: 1) homed to the 435 tumor
lymphatics, tumor sections were stained with the lymphatic markers
VEGFR-3 and LYVE-1. Lymphatic vessels were visualized using a rat
anti-mouse VEGFR-3 antibody or with rabbit anti-LYVE-1 antibody
produced as described below.
[0127] As shown in FIG. 4, the vessel-like structures in the tumor
stained with antibodies against both lymphatic markers. Only a
small number of these vessels were blood vessels, as shown by the
rare occurrence of overlap of VEGFR-3 and labeling of injected
tomato lectin. In contrast, intravenously injected
fluorescein-conjugated CGNKRTRGC peptide SEQ ID NO: 1 co-localized
with VEGFR-3 and LYVE-1 staining in 435 tumor tissue (see FIG. 5).
Furthermore, the fluorescein-conjugated peptide (SEQ ID NO: 1)
accumulated in the nuclei lining the vessel-like structures (FIG.
5C). These results indicate that peptide SEQ ID NO: 1 selectively
homes to tumor lymphatic vasculature.
[0128] The CGNKRTRGC (SEQ ID NO: 1) peptide also accumulated in
VEGFR-3 positive single cells within the tumor and in structures
that resembled the staining pattern observed in collapsed lymphatic
vessels in human tissues (Fukuda et al., The Prostate 44:332 (2000)
and Ebata et al., Microvasc. Res. 61: 40 (2001)); these structures
were only occasionally positive for VEGFR-3 or LYVE-1. The VEGFR-3
positive single cells were not macrophages, which can infiltrate
tumors, as evidenced by a lack of co-localization of SEQ ID NO: 1
and the macrophage marker F4/80. The VEGFR-3 positive single cells
can be involved in the development of lymphatic endothelial cells,
for example, lymphangioblasts (Schneider et al., Dev. Dyn. 216: 311
(1999) or migrating endothelial cells of a mixed type such as those
described by Wigle et al., Cell 98: 769 (1999).
[0129] LYVE-1 antibody was produced by immunizing New Zealand White
rabbits with a peptide encoding the 19 most C-terminal residues of
mouse LYVE-1 (Prevo et al., J. Biol. Chem. 276:19420 (2001))
conjugated to keyhole limpet hemocyanin (KLH; Pierce; Iselin,
N.J.). The initial immunization was done in complete Freund=s
Adjuvant, with boosters performed in incomplete Freund=s Adjuvant.
Specific antibody was obtained following affinity purification with
the peptide coupled to Sulfolink Gel (Pierce). Tissues were
processed for fifteen minutes following injection with
fluorescein-conjugated peptide using rat anti-mouse anti-VEGFR-3,
and rabbit anti-LYVE-1. The anti-LYVE-1 antibody was used at a
1:500 dilution on cryo sections, followed by detection with goat
anti-rabbit secondary antibody conjugated to Alexa 594 (Molecular
Probes).
Example 5
In Vivo Homing of Phage Displaying Peptide CGNKRTRGC (SEQ ID NO: 1)
Following Subcutaneous Injection
[0130] This example demonstrates in vivo homing of phage displaying
SEQ ID NO: 1 to MDA-MB-435 tumor lymphatics following subcutaneous
injection.
[0131] Subcutaneous and intravenous injection of phage was
performed as follows. CGNKRTRGC (SEQ ID NO: 1) phage
(5.times.10.sup.9 PFU) were subcutaneously (s.c.) injected about 3
cm from a 435 tumor or into the tail vein (i.v.) of a 435
tumor-bearing mouse. After 12 minutes, the mouse was perfused
through the heart with 20 ml of PBS, and the tumor and control
organ were removed. Unbound phage were removed by several washes,
and the bound phage were recovered by adding bacteria and
titrated.
[0132] As shown in FIG. 6, a striking enrichment of SEQ ID NO:
1-bearing phage was observed in the tumor relative to
nonrecombinant phage after subcutaneous injection. As further shown
in FIG. 6, a significantly lower background of CGNKRTRGC (SEQ ID
NO: 1) phage was present in the control organ following
subcutaneous injection as compared to the background resulting from
intravenous injection. These results further demonstrate the
lymphatic vessel specificity of the CGNKRTRGC (SEQ ID NO: 1)
peptide and indicate that SEQ ID NO:1-displaying phage and peptide
can accumulate in the lymphatic vessels of a tumor within minutes
after having been injected intravenously.
Example 6
Cytotoxic Activity of Peptide CGNKRTRGC (SEQ ID NO: 1)
[0133] This example demonstrates that peptide SEQ ID NO: 1 has
cytotoxic activity in cell culture and in vivo.
[0134] Cultured MDA-MB-435 cells were incubated with a control
peptide (CGEKRTRGC; SEQ ID NO: 3) or with CGNKRTRGC (SEQ ID NO: 1)
at 37.degree. C. for 1, 2 or 4 hours. After staining with trypan
blue, which is taken up by cells that are dead or dying, the total
number of cells were counted, and the percentage of trypan blue
stained cells determined. As shown in FIG. 7, a significantly
enhanced cytotoxic effect was observed with peptide SEQ ID NO: 1 as
compared to control peptide SEQ ID NO: 3 at the later time points.
In particular, after four hours incubation, at least 7-fold greater
trypan blue uptake was evident in cell cultures incubated with
peptide SEQ ID NO: 1 as compared to cultures incubated with control
peptide. These results indicate that peptide SEQ ID NO: 1 has
cytotoxic activity.
[0135] A tumor treatment study was performed using MDA-MB-435 human
breast carcinoma cell xenografts prepared by subcutaneously
injecting 1.times.10.sup.6 cells in 200 .mu.l PBS into nude mice.
Mice (five per group) were treated intravenously twice a week with
67 nmol CGNKRTRGC peptide (SEQ ID NO: 1) or with PBS starting four
weeks after tumor implantation. Tumor volumes were measured once a
week for four weeks. As shown in FIG. 8, the mean tumor volume was
at least several fold less in mice administered peptide SEQ ID NO:
1 than in mice administered PBS alone beginning after about two
weeks of treatment. These results demonstrate that peptide
CGNKRTRGC (SEQ ID NO: 1) has cytotoxic activity in vivo and
indicate that this peptide and peptides that bind the same receptor
can be useful for slowing or preventing tumor growth in vivo.
[0136] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0137] 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
claims.
Sequence CWU 1
1
819PRTArtificial SequenceSynthetic construct 1Cys Gly Asn Lys Arg
Thr Arg Gly Cys1 527PRTArtificial SequenceSynthetic construct 2Gly
Asn Lys Arg Thr Arg Gly1 539PRTArtificial SequenceSynthetic
construct 3Cys Gly Glu Lys Arg Thr Arg Gly Cys1 549PRTArtificial
SequenceSynthetic construct 4Cys Gly Asn Lys Arg Thr Arg Gly Val1
5514PRTArtificial SequenceSynthetic construct 5Lys Leu Ala Lys Leu
Ala Lys Lys Leu Ala Lys Leu Ala Lys1 5 10614PRTArtificial
SequenceSynthetic construct 6Lys Leu Ala Lys Lys Leu Ala Lys Leu
Ala Lys Lys Leu Ala1 5 10714PRTArtificial SequenceSynthetic
construct 7Lys Ala Ala Lys Lys Ala Ala Lys Ala Ala Lys Lys Ala Ala1
5 10821PRTArtificial SequenceSynthetic construct 8Lys Leu Gly Lys
Lys Leu Gly Lys Leu Gly Lys Lys Leu Gly Lys Leu1 5 10 15Gly Lys Lys
Leu Gly 20
* * * * *