U.S. patent application number 10/375992 was filed with the patent office on 2004-07-08 for tumor homing molecules, conjugates derived therefrom, and methods of using same.
This patent application is currently assigned to THE BURNHAM INSTITUTE. Invention is credited to Pasqualini, Renata, Ruoslahti, Erkki.
Application Number | 20040131623 10/375992 |
Document ID | / |
Family ID | 26740545 |
Filed Date | 2004-07-08 |
United States Patent
Application |
20040131623 |
Kind Code |
A9 |
Ruoslahti, Erkki ; et
al. |
July 8, 2004 |
Tumor homing molecules, conjugates derived therefrom, and methods
of using same
Abstract
The present invention provides tumor homing molecules, which
selectively home to a tumor. The invention also provides methods of
using a tumor homing molecule to target an agent such as a drug to
a selected tumor or to identify the target molecule expressed by
the tumor. The invention also provides methods of targeting a tumor
containing angiogenic vasculature by contacting the tumor with a
molecule that specifically binds an .alpha..sub.v-containing
integrin. The invention further provides molecules that can
selectively home to angiogenic vasculature. In addition, the
invention provides a target molecule, which is specifically bound
by a tumor homing molecule and is expressed by angiogenic
vasculature. The invention also provides antibodies that bind to
the target molecule and peptidomimetics that competitively inhibit
binding of a ligand to the target molecule.
Inventors: |
Ruoslahti, Erkki; (Rancho
Santa Fe, CA) ; Pasqualini, Renata; (Solana Beach,
CA) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Assignee: |
THE BURNHAM INSTITUTE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0152578 A1 |
August 14, 2003 |
|
|
Family ID: |
26740545 |
Appl. No.: |
10/375992 |
Filed: |
February 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10375992 |
Feb 27, 2003 |
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08926914 |
Sep 10, 1997 |
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6576239 |
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60060947 |
Sep 10, 1996 |
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Current U.S.
Class: |
424/178.1 ;
435/7.23 |
Current CPC
Class: |
A61K 47/64 20170801;
G01N 33/5011 20130101; G01N 2333/948 20130101; G01N 2333/70546
20130101; G01N 33/57492 20130101 |
Class at
Publication: |
424/178.1 ;
435/007.23 |
International
Class: |
G01N 033/574; A61K
039/395 |
Goverment Interests
[0002] This invention was made with government support under CA
42507, CA 62042, CA74238-01 and Cancer Center Support Grant CA
30199 awarded by the National Institutes of Health. The government
has certain rights in this invention.
Claims
We claim:
1. A conjugate, comprising a tumor homing peptide linked to a
moiety, said tumor homing peptide obtained by in vivo panning,
comprising the steps of: a) administering to a first subject having
a tumor a library of diverse peptides; b) collecting a sample of
the tumor; c) identifying a peptide that homes to said tumor; d)
collecting a sample of normal tissue corresponding to said tumor;
and e) determining that said peptide that homes to said tumor is
not present in said normal tissue, thereby obtaining said tumor
homing peptide, provided said tumor homing peptide is not an
antibody.
2. The conjugate of claim 1, wherein said peptide contains the
amino acid sequence RGD.
3. The conjugate of claim 1, wherein said peptide contains the
amino acid sequence NGR.
4. The conjugate of claim 1, wherein said peptide contains the
amino acid sequence GSL.
5. The conjugate of claim 1, wherein said tumor homing peptide is
NGRAHA (SEQ ID NO: 6) or CNGRC (SEQ ID NO: 8).
6. The conjugate of claim 1, wherein said tumor homing peptide is
CDCRGDCFC (SEQ ID NO: 1).
7. The conjugate of claim 1, wherein said tumor homing peptide is
CNGRCVSGCAGRC (SEQ ID NO: 3) or CGSLVRC (SEQ ID NO: 5).
8. The conjugate of claim 1, wherein said moiety is a cytotoxic
agent.
9. The conjugate of claim 1, wherein said moiety is a drug.
10. The conjugate of claim 9, wherein said drug is a cancer
chemotherapeutic agent.
11. The conjugate of claim 10, wherein said cancer chemotherapeutic
agent is doxorubicin.
12. The conjugate of claim 1, wherein said moiety is a detectable
moiety.
13. The conjugate of claim 1, wherein said moiety is selected from
the group consisting of a chambered microdevice, a liposome, a cell
and a virus.
14. The conjugate of claim 1, wherein said moiety is a grafted
polypeptide.
15. A conjugate, comprising a tumor homing molecule linked to a
moiety, said tumor homing molecule obtained by in vivo panning,
comprising the steps of: a) administering to a first subject having
a tumor a library of diverse molecules; b) collecting a sample of
the tumor; c) identifying a molecule that homes to said tumor; d)
collecting a sample of normal tissue corresponding to said tumor;
and e) determining that said molecule that homes to said tumor is
not present in said normal tissue, thereby obtaining said tumor
homing molecule, provided said tumor homing molecule is not an
antibody.
16. The conjugate of claim 15, wherein said molecule is a nucleic
acid molecule.
17. The conjugate of claim 15, wherein said molecule is a
peptidomimetic.
18. A conjugate, comprising a tumor homing peptide containing the
amino acid sequence RGD, said tumor homing peptide linked to a
moiety.
19. The conjugate of claim 18, wherein said tumor homing peptide is
CDCRGDCFC (SEQ ID NO: 1).
20. A conjugate, comprising a tumor homing peptide containing the
amino acid sequence NGR, said tumor homing peptide linked to a
moiety.
21. The conjugate of claim 20, wherein said tumor homing peptide is
NGRAHA (SEQ ID NO: 6).
22. The conjugate of claim 20, wherein said tumor homing peptide is
CNGRC (SEQ ID NO: 8).
23. The conjugate of claim 20, wherein said tumor homing peptide is
CNGRCVSGCAGRC (SEQ ID NO: 3).
24. A conjugate, comprising a tumor homing peptide containing the
amino acid sequence GSL, said tumor homing peptide linked to a
moiety.
25. The conjugate of claim 24, wherein said tumor homing peptide is
CGSLVRC (SEQ ID NO: 5).
26. A conjugate, comprising a tumor homing peptide selected from
the group consisting of NGRAHA (SEQ ID NO: 6) and CNGRC (SEQ ID NO:
8), said tumor homing peptide linked to a moiety.
27. A conjugate, comprising CDCRGDCFC (SEQ ID NO: 1) linked to a
moiety.
28. A conjugate, comprising a tumor homing peptide selected from
the group consisting of CNGRCVSGCAGRC (SEQ ID NO: 3) and CGSLVRC
(SEQ ID NO: 5), said tumor homing peptide linked to a moiety.
29. A tumor homing peptide identified by in vivo panning,
comprising the steps of: a) administering to a first subject having
a tumor a library of diverse peptides; b) collecting a sample of
the tumor; c) identifying a peptide that homes to said tumor; d)
collecting a sample of normal tissue corresponding to said tumor;
and e) determining that said peptide that homes to said tumor is
not present in said normal tissue, thereby identifying said peptide
as a tumor homing peptide, provided said peptide is not an
antibody.
30. The tumor homing peptide of claim 29, wherein said sample of
normal tissue corresponding to said tumor is collected from said
first subject.
31. The tumor homing peptide of claim 29, wherein said sample of
normal tissue corresponding to said tumor is collected from a
second subject.
32. The tumor homing peptide of claim 29, wherein said tumor is a
breast tumor.
33. The tumor homing peptide of claim 29, wherein said tumor is a
melanoma.
34. The tumor homing peptide of claim 29, wherein said tumor is a
Kaposi's sarcoma.
35. A tumor homing peptide selected from the group consisting of
CNGRCVSGCAGRC (SEQ ID NO: 3) and CGSLVRC (SEQ ID NO: 5).
36. A tumor homing molecule identified by in vivo panning,
comprising the steps of: a) administering to a first subject having
a tumor a library of diverse molecules; b) collecting a sample of
the tumor; c) identifying a molecule that homes to said tumor; d)
collecting a sample of normal tissue corresponding to said tumor;
and e) determining that said molecule that homes to said tumor is
not present in said normal tissue, thereby identifying said
molecule as a tumor homing molecule, provided said molecule is not
an antibody.
37. A method of directing a moiety to a tumor, comprising
contacting the tumor with the conjugate of claim 1.
38. The method of claim 37, wherein said contacting step is
performed in vitro.
39. The method of claim 37, wherein said contacting step is
performed in vivo.
40. A method of identifying the presence of a target molecule,
which specifically binds a tumor homing molecule, wherein said
tumor homing molecule is not an antibody, comprising contacting a
sample of a tumor with the tumor homing molecule and detecting
specific binding of said tumor homing molecule to a component of
said sample, said binding identifying the presence of a target
molecule.
41. A method of identifying a target molecule, which is expressed
in a tumor tissue, comprising the steps of: a) contacting a tumor
tissue sample with a tumor homing molecule that specifically binds
to said tumor tissue, wherein said tumor homing molecule is not an
antibody; b) identifying in said tumor tissue sample target
molecules bound by said tumor homing molecule; c) contacting a
corresponding nontumor tissue sample with said tumor homing
molecule; d) identifying in said corresponding nontumor tissue
sample target molecules bound by said tumor homing molecule; and e)
comparing said target molecules of said tumor tissue with said
target molecules of said corresponding nontumor tissue, thereby
identifying a target molecule expressed by said tumor tissue,
wherein said target molecule specifically binds said tumor homing
molecule.
42. A method of obtaining a substantially isolated target molecule,
which specifically binds a tumor homing molecule, comprising the
step of substantially isolating the target molecule identified by
the method of claim 41.
43. A substantially isolated target molecule, which specifically
binds a tumor homing molecule, obtained by the method of claim 42,
provided said target molecule is not an integrin.
44. A peptidomimetic, which competitively inhibits the binding of
the target molecule of claim 43 to a naturally occurring ligand of
the target molecule.
45. A molecule, which specifically binds to the target molecule of
claim 43.
46. A molecule, which competitively inhibits the binding of the
target molecule of claim 43 to the tumor homing molecule.
47. The molecule of claim 46, which is a peptide.
48. The peptide of claim 47, which contains the amino acid sequence
NGR.
49. The peptide of claim 48, which has the amino acid sequence
CNGRCVSGCAGRC (SEQ ID NO: 3).
50. The peptide of claim 47, which contains the amino acid sequence
GSL.
51. The peptide of claim 50, which has the amino acid sequence
CGSLVRC (SEQ ID NO: 5).
52. A target molecule, which is expressed in tumor vasculature,
wherein said target molecule binds CNGRC (SEQ ID NO: 8) with a
higher affinity than said target molecule binds CDCRGDCFC (SEQ ID
NO: 1).
53. An antibody that specifically binds the target molecule of
claim 43.
54. The antibody of claim 53, which is a monoclonal antibody.
55. A method of directing a moiety in vivo to a tumor containing
angiogenic vasculature, comprising contacting the tumor with the
conjugate of claim 1.
56. The method of claim 55, wherein said conjugate comprises a
peptide containing the amino acid sequence RGD.
57. The method of claim 55, wherein said conjugate comprises a
peptide containing the amino acid sequence NGR.
58. The method of claim 55, wherein said conjugate comprises a
peptide containing the amino acid sequence GSL.
59. The method of claim 55, wherein said conjugate comprises
CDCRGDCFC (SEQ ID NO 1).
60. The method of claim 55, wherein said conjugate comprises NGRAHA
(SEQ ID NO: 6) or CNGRC (SEQ ID NO: 8).
61. The method of claim 55, wherein said conjugate comprises
CNGRCVSGCAGRC (SEQ ID NO: 3) or CGSLVRC (SEQ ID NO: 5).
62. The method of claim 55, wherein said conjugate comprises a
moiety, which is a cytotoxic agent.
63. The method of claim 55, wherein said conjugate comprises a
moiety, which is a drug.
64. The method of claim 63, wherein said drug is a cancer
chemotherapeutic agent.
65. The method of claim 64, wherein said cancer chemotherapeutic
agent is doxorubicin.
66. The method of claim 55, wherein said conjugate comprises a
moiety, which is a detectable moiety.
67. The method of claim 55, wherein said conjugate comprises a
moiety selected from the group consisting of a chambered
microdevice, a liposome, a cell and a virus.
68. The method of claim 55, wherein said conjugate comprises a
moiety, which is a grafted polypeptide.
69. A method of targeting in vivo a tumor containing angiogenic
vasculature, comprising contacting the tumor with a molecule that
selectively binds an .alpha..sub.v-containing integrin.
70. The method of claim 69, said molecule selected from the group
of an RGD-containing peptide and an antibody that selectively binds
an .alpha..sub.v-containing integrin.
71. The method of claim 69, wherein said .alpha..sub.v-containing
integrin is .alpha..sub.v.beta..sub.3 integrin.
72. The method of claim 69, wherein said molecule is CDCRGDCFC (SEQ
ID NO: 14).
Description
[0001] This application claims the benefit of priority of U.S.
Provisional Application No. 60/______, which was converted from
U.S. Ser. No. 08/710,067, filed Sep. 10, 1996, the entire contents
of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[0003] The present invention relates generally to the fields of
cancer biology and drug delivery and, more specifically, to
peptides that selectively home to a tumor, particularly a malignant
tumor, to compositions comprising an agent such as a therapeutic
agent conjugated to such tumor homing molecules, and to methods of
using such molecules to target an agent to a tumor.
BACKGROUND INFORMATION
[0004] Continuous developments over the past quarter century have
resulted in substantial improvements in the ability of a physician
to diagnose a cancer in a patient. For example, antibody based
assays such as that for prostate specific antigen now allow early
diagnosis of cancers such as prostate cancer. More recently,
methods of genetic screening are becoming available to identify
persons that may be particularly susceptible to developing a
cancer. Genetic screening methods are based on the identification
of one or more mutations in a gene that correlates with the
development of a cancer. For example, the identification of genes
such as BRCA1 and BRCA2 allowed the further identification of
mutations in these genes that, in some cases, can correlate with
susceptibility to developing breast cancer.
[0005] Unfortunately, methods for treating cancer have not kept
pace with those for diagnosing the disease. Thus, while the death
rate from various cancers has decreased due to the ability of a
physician to detect the disease at an earlier stage, the ability to
treat patients presenting with more advanced disease has advanced
only minimally.
[0006] A major hurdle to advances in treating cancer is the
relative lack of agents that can selectively target the cancer,
while sparing normal tissue. For example, radiation therapy and
surgery, which generally are localized treatments, can cause
substantial damage to normal tissue in the treatment field,
resulting in scarring and, in severe cases, loss of function of the
normal tissue. Chemotherapy, in comparison, which generally is
administered systemically, can cause substantial damage to organs
such as bone marrow, mucosae, skin and the small intestine, which
undergo rapid cell turnover and continuous cell division. As a
result, undesirable side effects such as nausea, loss of hair and
drop in blood cell count occur as a result of systemically treating
a cancer patient with chemotherapeutic agents. Such undesirable
side effects often limit the amount of a treatment that can be
administered. Thus, cancer remains a leading cause of patient
morbidity and death.
[0007] Efforts have been made to increase the target specificity of
various drugs. For example, where a unique cell surface marker is
expressed by a population of cells making up a tumor, an antibody
can be raised against the unique marker and a drug can be linked to
the antibody. Upon administration of the drug/antibody complex to
the patient, the binding of the antibody to the marker results in
the delivery of a relatively high concentration of the drug to the
tumor. Similar methods can be used where a particular cancer cell
or the supporting cell or matrix expresses a unique cell surface
receptor or a ligand for a particular receptor. In these cases, the
drug can be linked to the specific ligand or to the receptor,
respectively, thus providing a means to deliver a relatively high
concentration of the drug to the tumor.
[0008] Tumors are characterized, in part, by a relatively high
level of active angiogenesis, resulting in the continual formation
of new blood vessels to support the growing tumor. Such angiogenic
blood vessels are distinguishable from mature vasculature. One of
the distinguishing features of angiogenic vasculature is that
unique endothelial cell surface markers are expressed. Thus, the
blood vessels in a tumor provide a potential target for directing a
chemotherapeutic agent to the tumor, thereby reducing the
likelihood that the agent will kill sensitive normal tissues.
Furthermore, if agents that target the angiogenic blood vessels in
a tumor can be identified, there is a likelihood that the agents
can be useful against a variety of different types of tumors, since
it is the target molecules in the angiogenic vessels that are
recognized by such agents and not receptors specific for the tumor
cells. However, the use of molecules that can bind specifically to
tumor vasculature and target a chemotherapeutic agent to the tumor
has not been demonstrated.
[0009] While linking a drug to a molecule that homes to a tumor can
provide significant advantages for treatment over the use of a
drug, alone, use of this method is severely limited by the scarcity
of useful cell surface markers expressed in a tumor. Thus, a need
exists to identify molecules that can selectively home to a tumor,
particularly to the vasculature supporting the tumor. The present
invention satisfies this need and provides related advantages as
well.
SUMMARY OF THE INVENTION
[0010] The present invention relates to molecules that selectively
home to tumors, generally to the vasculature supporting the tumor.
For example, the invention provides tumor homing peptides that
contain, for example, the motif asparagine-glycine-arginine (NGR)
or glycine-serine-leucine (GSL), or the .alpha..sub.v-containing
integrin binding motif, arginine-glycine-aspartic acid (RGD).
[0011] The invention also relates to compositions comprising a
tumor homing molecule, such as a tumor homing peptide, linked to a
moiety to produce a tumor homing molecule/moiety conjugate. Such a
moiety can be a drug, for example, a cancer therapeutic agent such
as doxorubicin, taxol, cis-platinum, or the like, in which case the
tumor homing molecule/moiety conjugate provides a therapeutic
reagent. A moiety conjugated to a tumor homing molecule also can be
a detectable label, for example, a radionuclide or paramagnetic
spin label, such that the molecule/moiety conjugate provides a
diagnostic reagent.
[0012] The invention also relates to methods of targeting a moiety
such as a drug to a tumor by contacting the tumor homing
molecule/moiety conjugate with the tumor. Thus, the invention
provides methods of diagnosing or treating a cancer in a subject by
administering a composition comprising a tumor homing molecule
conjugated to a cancer therapeutic agent to the subject. For
example, administration of a composition comprising a
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate to a mouse bearing a
transplanted breast carcinoma substantially reduced the growth of
the breast cancer and the number of metastases and resulted in
substantially greater survival as compared to tumor bearing mice
treated with doxorubicin, alone, or with doxorubicin conjugated to
an unrelated peptide.
[0013] The invention further relates to methods of identifying a
target molecule in a tumor by detecting selective binding of the
target molecule to a tumor homing molecule. For example, a peptide
that selectively homes to a tumor can be attached to a solid matrix
for use in affinity chromatography. A sample of the tumor can be
obtained and passed over the affinity matrix under conditions that
allow specific binding of the target molecule, which then can be
collected and identified using well known biochemical methods.
Thus, the invention also provides a target molecule, which acts as
a receptor for a tumor homing molecule. Such a target molecule can
be useful, for example, for raising an antibody specific for the
target molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A to 1V show the immunohistochemical staining of
phage expressing the NGR tumor homing peptide, CNGRCVSGCAGRC (SEQ
ID NO: 3; "NGR phage"), in tumors and normal tissues following
intravenous injection into nude mice bearing a human breast
carcinoma or a human Kaposi's sarcoma. Samples were taken 4 min
(FIGS. 1E, 1G, 1H and 1J) or 24 hr (FIGS. 1A to 1D, 1F, 1I, and 1K
to 1V) after administration of the phage. FIGS. 1A, 1C, 1G and 1J
are from mice receiving insertless phage (control phage) and FIGS.
1B, 1D, 1E, 1F, 1H, 1I and 1K to 1V are from mice receiving NGR
phage. FIGS. 1A, 1B, 1E, 1F and 1G are breast tumor samples; FIGS.
1C, 1D, 1H, 1I and 1J are Kaposi's sarcoma samples; FIG. 1K is
brain; FIG. 1L is lymph node; FIG. 1M is kidney; FIG. 1N is
pancreas; FIG. 1O is uterus; FIG. 1P is mammary fat pad; FIG. 1Q is
lung; FIG. 1R is intestine; FIG. 1S is skin; FIG. 1T is skeletal
muscle; FIG. 1U is heart and FIG. 1V is urinary tract epithelium.
Magnification: FIGS. 1A to 1D, 40.times.; FIGS. 1E to 1V,
200.times..
[0015] FIGS. 2A to 2F show the hematoxylin and eosin stained tumor
samples from representative pairs of mice treated two times with 5
.mu.g equivalent of doxotubicin (FIGS. 2A to 2C) or
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate (FIGS. 2D to 2F).
Paired mice had size matched tumors at the time treatment was
initiated.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention relates to molecules that selectively
home to tumors. For example, the invention provides tumor homing
peptides such as the peptides CGRECPRLCQSSC (SEQ ID NO: 2) and
CNGRCVSGCAGRC (SEQ ID NO: 3), which were identified based on their
ability to home to a breast carcinoma, and the peptide CLSGSLSC
(SEQ ID NO: 4, which was identified based on its ability to home to
a melanoma. Such tumor homing peptides were identified using in
vivo panning (see U.S. Pat. No. 5,622,699, issued Apr. 22, 1997;
Pasqualini and Ruoslahti, Nature 380:364-366 (1996), each of which
is incorporated herein by reference).
[0017] The disclosed tumor homing peptides were identified based on
their homing to various particular tumors. For example, in vivo
panning was performed using a mouse bearing a human breast
carcinoma xenograft and peptides that homed to the breast tumor
were identified. However, as disclosed herein, such tumor homing
peptides generally homed to other types of tumors, including a
mouse melanoma and a human Kaposi's sarcoma. Thus, while the tumor
homing peptide CNGRCVSGCAGRC (SEQ ID NO: 3) was identified by its
ability to home in vivo to a breast tumor, this peptide also homed
in vivo to a melanoma and to a Kaposi's sarcoma, but not to
nontumor tissues.
[0018] Similarly, the tumor homing peptide CLSGSLSC (SEQ ID NO: 4)
was identified based on its homing to melanoma. However, further
examination of this peptide revealed that it also homed to a breast
tumor and to Kaposi's sarcoma. Immunohistological analysis revealed
that such tumor homing peptides initially are associated with the
vasculature of the various tumors, although at later time the
molecules are associated with tumor parenchymal cells. Thus, the
general tumor homing ability of a tumor homing molecule of the
invention is due, at least in part, to the ability of the tumor
homing molecules to target angiogenic vasculature associated with a
tumor. These results indicate that specific target molecules are
expressed by the cells comprising the vasculature in a tumor as
compared to the cell surface molecule expressed by vasculature in
nontumor tissues. Using methods as disclosed herein, the artisan
readily can determine whether a tumor homing molecule homes
generally to the angiogenic vasculature associated with a tumor or
homes specifically to a particular type of tumor cell.
[0019] As used herein, the term "tumor" means a mass of cells that
are characterized, at least in part, by containing angiogenic
vasculature. The term "tumor" is used broadly to include the tumor
parenchymal cells as well as the supporting stroma, including the
angiogenic blood vessels that infiltrate the tumor parenchymal cell
mass. Although a tumor generally is a malignant tumor, i.e., a
"cancer," a tumor also can be nonmalignant, provided that
neovascularization is associated with the tumor. The term "normal"
or "nontumor" tissue is used to refer to tissue that is not a
"tumor." As disclosed herein, a tumor homing molecule can be
identified based on its ability to home a tumor, but not to a
corresponding nontumor tissue.
[0020] As used herein, the term "corresponding," when used in
reference to tumors or tissues or both, means that two or more
tumors, or two or more tissues, or a tumor and a tissue are of the
same histologic type. The skilled artisan will recognize that the
histologic type of a tissue is a function of the cells comprising
the tissue. Thus, the artisan will recognize, for example, that a
nontumor tissue corresponding to a breast tumor is normal breast
tissue, whereas a nontumor tissue corresponding to a melanoma is
skin, which contains melanocytes. Furthermore, for purposes of the
invention, it is recognized that a tumor homing molecule can bind
specifically to a target molecule expressed by the vasculature in a
tumor, which generally contains blood vessels undergoing
neovascularization, in which case a tissue corresponding to the
tumor would comprise nontumor tissue containing blood vessels that
are not undergoing active angiogenesis.
[0021] The term "corresponding" also is used herein in reference to
the evolutionarily conserved nature of target molecules, which are
expressed in a tumor, for example, in a mouse as compared to a
human. Thus, reference to the corresponding target molecules in
mouse tumor vasculature as compared, for example, to human
vasculature, means target molecules having a similar function,
particularly the ability to specifically bind a tumor homing
molecule.
[0022] Identified tumor homing molecules are useful, for example,
for targeting a desired moiety such as a drug, a toxin or a
detectable label, which can be linked to the molecule, to a tumor.
Thus, the invention provides tumor homing molecule/moiety
conjugates, which are useful for targeting the moiety to a tumor.
Accordingly, the invention also provides methods of targeting a
moiety to a tumor and, therefore, methods of reducing the severity
of a tumor and of treating a subject having a cancer (see Example
VII). In addition, a tumor homing molecule is useful for
identifying the target molecule, to which the homing molecule binds
in the tumor.
[0023] A tumor homing molecule can be identified by screening a
library of molecules by in vivo panning as disclosed herein. As
used herein, the term "library" means a collection of molecules. A
library can contain a few or a large number of different molecules,
varying from about ten molecules to several billion molecules or
more. If desired, a molecule can be linked to a tag, which can
facilitate recovery or identification of the molecule.
[0024] As used herein, the term "molecule" is used broadly to mean
an organic chemical such as a drug; a nucleic acid molecule such as
an RNA, a cDNA or an oligonucleotide; a peptide, including a
variant or modified peptide or peptide-like molecules, referred to
herein as peptidomimetics, which mimic the activity of a peptide;
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,
which contains a binding domain. For convenience, the term
"peptide" is used broadly herein to mean peptides, proteins,
fragments of proteins and the like. A molecule also can be a
non-naturally occurring molecule, which does not occur in nature,
but is produced as a result of in vitro methods, or can be a
naturally occurring molecule such as a protein or fragment thereof
expressed from a cDNA library or a peptidomimetic.
[0025] As used herein, the term "peptidomimetic" is used broadly to
mean a peptide-like molecule that has the binding activity of the
tumor homing peptide. With respect to the tumor homing peptides of
the invention, peptidomimetics, which include chemically modified
peptides, peptide-like molecules containing non-naturally occurring
amino acids, peptoids and the like, have the binding activity of a
tumor homing peptide upon which the peptidomimetic is derived (see,
for example, "Burger's Medicinal Chemistry and Drug Discovery" 5th
ed., vols. 1 to 3 (ed. M. E. Wolff; Wiley Interscience 1995), which
is incorporated herein by reference). Peptidomimetics provide
various advantages over a peptide, including that a peptidomimetic
can be stable during passage through the digestive tract and,
therefore, useful for oral administration.
[0026] 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 tumor homing molecule, as
well as potential geometrical and chemical complementarity to a
target molecule bound by a tumor homing peptide. Where no crystal
structure of a tumor homing peptide or a target molecule, which
binds the tumor homing molecule, 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 tumor homing
molecule.
[0027] Methods for preparing libraries containing diverse
populations of various types of molecules such as peptides,
peptoids and peptidomimetics are well known in the art and various
libraries are commercially available (see, for example, Ecker and
Crooke, Biotechnology 13:351-360 (1995), and Blondelle et al.,
Trends Anal. Chem. 14:83-92 (1995), and the references cited
therein, each of which is incorporated herein by reference; see,
also, 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, and Gordon et al., J.
Med. Chem. 37:1385-1401 (1994), each of which is incorporated
herein by reference). Where a molecule is a peptide, protein or
fragment thereof, the molecule can be produced in vitro directly or
can be expressed from a nucleic acid, which can be produced in
vitro. Methods of synthetic peptide and nucleic acid chemistry are
well known in the art.
[0028] A library of molecules also can be produced, for example, by
constructing a cDNA expression library from mRNA collected from a
cell, tissue, organ or organism of interest. Methods for producing
such libraries are well known in the art (see, for example,
Sambrook et al., Molecular Cloning: A laboratory manual (Cold
Spring Harbor Laboratory Press 1989), which is incorporated herein
by reference). Preferably, a peptide encoded by the cDNA is
expressed on the surface of a cell or a virus containing the cDNA.
For example, cDNA can be cloned into a phage vector such as fuse 5
(Example I), wherein, upon expression, the encoded peptide is
expressed as a fusion protein on the surface of the phage.
[0029] In addition, a library of molecules can comprise a library
of nucleic acid molecules, which can be DNA or RNA or an analog
thereof. Nucleic acid molecules that bind, for example, to a cell
surface receptor are well known (see, for example, O'Connell et
al., Proc. Natl. Acad. Sci., USA 93:5883-5887 (1996); Tuerk and
Gold, Science 249:505-510 (1990); Gold et al., Ann. Rev. Biochem.
64:763-797 (1995), each of which is incorporated herein by
reference). Thus, a library of nucleic acid molecules can be
administered to a subject having a tumor and tumor homing molecules
can be identified by in vivo panning. If desired, the nucleic acid
molecules can be nucleic acid analogs that, for example, are less
susceptible to attack by nucleases (see, for example, Jelinek et
al., Biochemistry 34:11363-11372 (1995); Latham et al., Nucl. Acids
Res. 22:2817-2822 (1994); Tam et al., Nucl. Acids Res. 22:977-986
(1994); Reed et al., Cancer Res. 59:6565-6570 (1990), each of which
is incorporated herein by reference).
[0030] As disclosed herein, in vivo panning for the purpose of
identifying a tumor homing molecule comprises administering a
library to a subject, collecting a sample of a tumor and
identifying a tumor homing molecule. The presence of a tumor homing
molecule can be identified using various methods well known in the
art. Generally, the presence of a tumor homing molecule in a tumor
is identified based on one or more characteristics common to the
molecules present in the library, then the structure of a
particular tumor homing molecule is identified. For example, a
highly sensitive detection method such as mass spectrometry, either
alone or in combination with a method such as gas chromatography,
can be used to identify tumor homing molecules in a tumor. Thus,
where a library comprises diverse molecules based generally on the
structure of an organic molecule such as a drug, a tumor homing
molecule can be identified by determining the presence of a parent
peak for the particular molecule.
[0031] If desired, the tumor can be collected, then processed using
a method such as HPLC, which can provide a fraction enriched in
molecules having a defined range of molecular weights or polar or
nonpolar characteristics or the like, depending, for example, on
the general characteristics of the molecules comprising the
library. Conditions for HPLC will depend on the chemistry of the
particular molecule and are well known to those skilled in the art.
Similarly, methods for bulk removal of potentially interfering
cellular materials such as DNA, RNA, proteins, lipids or
carbohydrates are well known in the art, as are methods for
enriching a fraction containing an organic molecule using, for
example, methods of selective extraction. Where a library comprises
a population of diverse organic chemical molecules, each linked to
a specific oligonucleotide tag, such that the specific molecule can
be identified by determining the oligonucleotide sequence using
polymerase chain reaction (PCR), genomic DNA can be removed from
the sample of the collected tumor in order to reduce the potential
for background PCR reactions. In addition, a library can comprise a
population of diverse molecules such as organic chemical molecules,
each linked to a common, shared tag. Based on the presence and
properties of the shared tag, molecules of the library that
selectively home to a tumor can be substantially isolated from a
sample of the tumor. These and other methods can be useful for
enriching a sample of a collected tumor for the particular tumor
homing molecule, thereby removing potentially contaminating
materials from the collected tumor sample and increasing the
sensitivity of detecting a molecule.
[0032] Evidence provided herein indicates that a sufficient number
of tumor homing molecules selectively homes to a. tumor during in
vivo panning such that the molecules readily can be identified. For
example, various independent phage expressing the same peptide were
identified in tumors formed from implanted human breast cancer
cells (Table 1), from mouse melanoma cells (Table 2) or from human
Kaposi's sarcoma cells (Table 3).
[0033] Although a substantial fraction of the identified tumor
homing molecules have the same structure, the peptide inserts of
only a small number of isolated phage were determined. It should be
recognized, however, that hundreds of thousands to millions of
phage expressing organ homing peptides have been recovered
following in vivo pannings for organ homing molecules (see, for
example, U.S. Pat. No. 5,622,699; Pasqualini and Ruoslahti, supra,
1996). These results indicate that specific tumor homing molecules
will be present in substantial numbers in a tumor following in vivo
homing, thereby increasing the ease with which the molecules can be
identified.
[0034] Ease of identification of a tumor homing molecule,
particularly an untagged molecule, depends on various factors,
including the presence of potentially contaminating background
cellular material. Thus, where the tumor homing molecule is an
untagged peptide, a larger number must home to the tumor in order
to identify the specific peptides against the background of
cellular protein. In contrast, a much smaller number of an untagged
organic chemical homing molecule such as a drug is identifiable
because such molecules normally are absent from or present in only
small numbers in the body. In such a case, a highly sensitive
method such as mass spectrometry can be used to identify a tumor
homing molecule. The skilled artisan will recognize that the method
of identifying a molecule will depend, in part, on the chemistry of
the particular molecule.
[0035] Where a tumor homing molecule is a nucleic acid or is tagged
with a nucleic acid, an assay such as PCR can be particularly
useful for identifying the presence of the molecule because, in
principle, PCR can detect the presence of a single nucleic acid
molecule (see, for example, Erlich, PCR Technology: Principles and
Applications for DNA Amplification (Stockton Press 1989), which is
incorporated herein by reference). Preliminary studies have
demonstrated that, following intravenous injection of 10 ng of an
approximately 6000 base pair plasmid into a mouse and 2 minutes in
the circulation, the plasmid was detectable by PCR in a sample of
lung. These results indicate that nucleic acids are sufficiently
stable when administered into the circulation such that in vivo
panning can be used to identify nucleic acid molecules that
selectively home to a tumor.
[0036] The molecules of a library can be tagged, which can
facilitate recovery or identification of the molecule. As used
herein, the term "tag" means a physical, chemical or biological
moiety such as a plastic microbead, an oligonucleotide or a
bacteriophage, respectively, that is linked to a molecule of the
library. Methods for tagging a molecule are well known in the art
(Hermanson, Bioconjugate Techniques (Academic Press 1996), which is
incorporated herein by reference).
[0037] A tag, which can be a shared tag or a specific tag, can be
useful for identifying the presence or structure of a tumor homing
molecule of a library. As used herein, the term "shared tag" means
a physical, chemical or biological moiety that is common to each
molecule in a library. Biotin, for example, can be a shared tag
that is linked to each molecule in a library. A shared tag can be
useful to identify the presence of a molecule of the library in a
sample and also can be useful to substantially isolate the
molecules from a sample. For example, where the shared tag is
biotin, the biotin-tagged molecules in a library can be
substantially isolated by binding to streptavidin or their presence
can be identified by binding with a labeled streptavidin. Where a
library is a phage display library, the phage that express the
peptides are another example of a shared tag, since each peptide of
the library is linked to a phage. In addition, a peptide such as
the hemaglutinin antigen can be a shared tag that is linked to each
molecule in a library, thereby allowing the use of an antibody
specific for the hemaglutinin antigen to substantially isolate
molecules of the library from a sample of a selected tumor.
[0038] A shared tag also can be a nucleic acid sequence that can be
useful to identify the presence of molecules of the library in a
sample or to substantially isolate molecules of a library from a
sample. For example, each of the molecules of a library can be
linked to the same selected nucleotide sequence, which constitutes
the shared tag. An affinity column containing a nucleotide sequence
that is complementary to the shared tag then can be used to
hybridize molecules of the library containing the shared tag, thus
substantially isolating the molecules from a tumor sample. A
nucleotide sequence complementary to a portion of the shared
nucleotide sequence tag also can be used as a PCR primer such that
the presence of molecules containing the shared tag can be
identified in a sample by PCR.
[0039] A tag also can be a specific tag. As used herein, the term
"specific tag" means a physical, chemical or biological tag that is
linked to a particular molecule in a library and is unique for that
particular molecule. A specific tag is particularly useful if it is
readily identifiable. A nucleotide sequence that is unique for a
particular molecule of a library is an example of a specific tag.
For example, the method of synthesizing peptides tagged with a
unique nucleotide sequence provides a library of molecules, each
containing a specific tag, such that upon determining the
nucleotide sequence, the identity of the peptide is known (see
Brenner and Lerner, Proc. Natl. Acad. Sci., USA 89:5381-5383
(1992), which is incorporated herein by reference). The use of a
nucleotide sequence as a specific tag for a peptide or other type
of molecule provides a simple means to identify the presence of the
molecule in a sample because an extremely sensitive method such as
PCR can be used to determine the nucleotide sequence of the
specific tag, thereby identifying the sequence of the molecule
linked thereto. Similarly, the nucleic acid sequence encoding a
peptide expressed on a phage is another example of a specific tag,
since sequencing of the specific tag identifies the amino acid
sequence of the expressed peptide.
[0040] The presence of a shared tag or a specific tag can provide a
means to identify or recover a tumor homing molecule of the
invention following in vivo panning. In addition, the combination
of a shared tag and specific tag can be particularly useful for
identifying a tumor homing molecule. For example, a library of
peptides can be prepared such that each is linked to a specific
nucleotide sequence tag (see, for example, Brenner and Lerner,
supra, 1992), wherein each specific nucleotide sequence tag has
incorporated therein a shared tag such as biotin. Upon homing to a
tumor, the particular tumor homing peptides can be substantially
isolated from a sample of the tumor based on the shared tag and the
specific peptides can be identified, for example, by PCR of the
specific tag (see Erlich, supra, 1989).
[0041] A tag also can serve as a support. As used herein, the term
"supports" means a tag having a defined surface to which a molecule
can be attached. In general, a tag useful as a support is a shared
tag. For example, a support can be a biological tag such as a virus
or virus-like particle such as a bacteriophage ("phage"); a
bacterium such as E. coli; or a eukaryotic cell such as a yeast,
insect or mammalian cell; or can be a physical tag such as a
liposome or a microbead, which can be composed of a plastic,
agarose, gelatin or other biological or inert material. If desired,
a shared tag useful as a support can have linked thereto a specific
tag. Thus, the phage display libraries used in the exemplified
methods can be considered to consist of the phage, which is a
shared tag that also is a support, and the nucleic acid sequence
encoding the expressed peptide, the nucleic acid sequence being a
specific tag.
[0042] In general, a support should have a diameter less than about
10 .mu.m to about 50 .mu.m in its shortest dimension, such that the
support can pass relatively unhindered through the capillary beds
present in the subject and not occlude circulation. In addition, a
support can be nontoxic, so that it does not perturb the normal
expression of cell surface molecules or normal physiology of the
subject, and biodegradable, particularly where the subject used for
in vivo panning is not sacrificed to collect a selected tumor.
[0043] Where a molecule is linked to a support, the tagged molecule
comprises the molecule attached to the surface of the support, such
that the part of the molecule suspected of being able to interact
with a target molecule in a cell in the subject is positioned so as
to be able to participate in the interaction. For example, where
the tumor homing molecule is suspected of being a ligand for a
growth factor receptor, the binding portion of the molecule
attached to a support is positioned so it can interact with the
growth factor receptor on a cell in the tumor. If desired, an
appropriate spacer molecule can be positioned between the molecule
and the support such that the ability of the potential tumor homing
molecule to interact with the target molecule is not hindered. A
spacer molecule also can contain a reactive group, which provides a
convenient and efficient means of linking a molecule to a support
and, if desired, can contain a tag, which can facilitate recovery
or identification of the molecule (see Hermanson, supra, 1996).
[0044] As exemplified herein, a peptide suspected of being able to
home to a selected tumor such as a breast carcinoma or a melanoma
was expressed as the N-terminus of a fusion protein, wherein the
C-terminus consisted of a phage coat protein. Upon expression of
the fusion protein, the C-terminal coat protein linked the fusion
protein to the surface of a phage such that the N-terminal peptide
was in a position to interact with a target molecule in the tumor.
Thus, a molecule having a shared tag was formed by the linking of a
peptide to a phage, wherein the phage provided a biological
support, the peptide molecule was linked as a fusion protein, the
phage-encoded portion of the fusion protein acted as a spacer
molecule, and the nucleic acid encoding the peptide provided a
specific tag allowing identification of a tumor homing peptide.
[0045] As used herein, the term "in vivo panning," when used in
reference to the identification of a tumor homing molecule, means a
method of screening a library by administering the library to a
subject and identifying a molecule that selectively homes to a
tumor in the subject (see U.S. Pat. No. 5,622,699). The term
"administering to a subject", when used in referring to a library
of molecules or a portion of such a library, is used in its
broadest sense to mean that the library is delivered to a tumor in
the subject, which, generally, is a vertebrate, particularly a
mammal such as a human.
[0046] A library can be administered to a subject, for example, by
injecting the library into the circulation of the subject such that
the molecules pass through the tumor; after an appropriate period
of time, circulation is terminated by sacrificing the subject or by
removing a sample of the tumor (Example I; see, also, U.S. Pat. No.
5,622,699; Pasqualini and Ruoslahti, supra, 1996). Alternatively, a
cannula can be inserted into a blood vessel in the subject, such
that the library is administered by perfusion for an appropriate
period of time, after which the library can be removed from the
circulation through the cannula or the subject can be sacrificed to
collect the tumor, or the tumor can be sampled, to terminate
circulation. Similarly, a library can be shunted through one or a
few organs, including the tumor, by cannulation of the appropriate
blood vessels in the subject. It is recognized that a library also
can be administered to an isolated perfused tumor. Such panning in
an isolated perfused tumor can be useful to identify molecules that
bind to the tumor and, if desired, can be used as an initial
screening of a library.
[0047] The use of in vivo panning to identify tumor homing
molecules is exemplified herein by screening a phage peptide
display library in tumor-bearing mice and identifying specific
peptides that selectively homed to a breast tumor or to a melanoma
tumor (Example I). However, phage libraries that display protein
receptor molecules, including, for example, an antibody or an
antigen binding fragment of an antibody such an Fv, Fd or Fab
fragment; a hormone receptor such as a growth factor receptor; or a
cell adhesion receptor such as an integrin or a selectin also can
be used to practice the invention. Variants of such molecules can
be constructed using well known methods such as random,
site-directed or codon based mutagenesis (see Huse, U.S. Pat. No.
5,264,563, issued Nov. 23, 1993, which is incorporated herein by
reference) and, if desired, peptides can be chemically modified
following expression of the phage but prior to administration to
the subject. Thus, various types of phage display libraries can be
screened by in vivo panning.
[0048] Phage display technology provides a means for expressing a
diverse population of random or selectively randomized peptides.
Various methods of phage display and methods for producing diverse
populations of peptides are well known in the art. For example,
Ladner et al. (U.S. Pat. No. 5,223,409, issued Jun. 29, 1993, which
is incorporated herein by reference) describe methods for preparing
diverse populations of binding domains on the surface of a phage.
In particular, Ladner et al. describe phage vectors useful for
producing a phage display library, as well as methods for selecting
potential binding domains and producing randomly or selectively
mutated binding domains.
[0049] Similarly, Smith and Scott (Meth. Enzymol. 35 217:228-257
(1993); see, also, Scott and Smith, Science 49: 386-390 (1990),
each of which is incorporated herein by reference) describe methods
of producing phage peptide display libraries, including vectors and
methods of diversifying the population of peptides that are
expressed (see, also, Huse, WO 91/07141 and WO 91/07149, each of
which is incorporated herein by reference; see, also, Example I).
Phage display technology can be particularly powerful when used,
for example, with a codon based mutagenesis method, which can be
used to produce random peptides or randomly or desirably biased
peptides (Huse, U.S. Pat. No. 5,264,563, supra, 1993). These or
other well known methods can be used to produce a phage display
library, which can be subjected to the in vivo panning method of
the invention in order to identify a peptide that homes to a
tumor.
[0050] In addition to screening a phage display library, in vivo
panning can be used to screen various other types of libraries,
including, for example, an RNA or DNA library or a chemical
library. If desired, the tumor homing molecule can be tagged, which
can facilitate recovery of the molecule from the tumor or
identification of the molecule in the tumor. For example, where a
library of organic molecules, each containing a shared tag, is
screened, the tag can be a moiety such as biotin, which can be
linked directly to the molecule or can be linked to a support
containing the molecules. Biotin provides a shared tag useful for
recovering the molecule from a selected tumor sample using an
avidin or streptavidin affinity matrix. In addition, a molecule or
a support containing a molecule can be linked to a hapten such as
4-ethoxy-methylene-2-phenyl-2-oxazoline-5-one (phOx), which can be
bound by an anti-phOx antibody linked to a magnetic bead as a means
to recover the molecule. Methods for purifying biotin or phOx
labeled conjugates are known in the art and the materials for
performing these procedures are commercially available (e.g.,
Invitrogen, La Jolla Calif.; and Promega Corp., Madison Wis.). In
the case where a phage library is screened, the phage can be
recovered using methods as disclosed in Example I.
[0051] In vivo panning provides a method for directly identifying
molecules that can selectively home to a tumor. As used herein, the
term "home" or "selectively home" means that a particular molecule
binds relatively specifically to a target molecule present in the
tumor following administration to a subject. In general, selective
homing is characterized, in part, by detecting at least a two-fold
(2.times.) greater specific binding of the molecule to the tumor as
compared to a control organ or tissue.
[0052] It should be recognized that, in some cases, a molecule can
localize nonspecifically to an organ or tissue containing a tumor.
For example, in vivo panning of a phage display library can result
in high background in organs such as liver and spleen, which
contain a marked component of the reticuloendothelial system (RES).
Thus, where a tumor is present, for example, in the liver,
nonspecific binding of molecules due to uptake by the RES can make
identifying a tumor homing molecule more difficult.
[0053] Selective homing can be distinguished from nonspecific
binding, however, by detecting differences in the abilities of
different individual phage to home to a tumor. For example,
selective homing can be identified by combining a putative tumor
homing molecule such as a peptide expressed on a phage with a large
excess of non-infective phage or with about a five-fold excess of
phage expressing unselected peptides, injecting the mixture into a
subject and collecting a sample of the tumor. In the latter case,
for example, provided the umber of injected phage expressing tumor
homing peptide is sufficiently low so as to be nonsaturating for
the target molecule, a determination that greater than about 20% of
the phage in the tumor express the putative tumor homing molecule
is demonstrative evidence that the peptide expressed by the phage
is a specific tumor homing molecule. In addition, nonspecific
localization can be distinguished from selective homing by
performing competition experiments using, for example, phage
expressing a putative tumor homing peptide in combination with an
excess amount of the "free" peptide (Example IV).
[0054] In addition, various methods can be used to prevent
nonspecific binding of a molecule to an organ containing a
component of the RES. For example, a molecule that homes
selectively to a tumor present in an organ containing a component
of the RES can be obtained by first blocking the RES using, for
example, polystyrene latex particles or dextran sulfate (see Kalin
et al., Nucl. Med. Biol. 20:171-174 (1993); Illum et al., J. Pharm.
Sci. 75:16-22 (1986); Takeya et al., J. Gen. Microbiol. 100:373-379
(1977), each of which is incorporated herein by reference), then
administering the library to the subject. For example,
pre-administration of dextran sulfate 500 or polystyrene
microspheres prior to administration of a test substance has been
used to block nonspecific uptake of the test substance by Kupffer
cells, which are the RES component of the liver (Illum et al.,
supra, 1986). Similarly, nonspecific uptake of agents by the RES
has been blocked using carbon particles or silica (Takeya et al.,
supra, 1977) or a gelatine colloid (Kalin et al., supra, 1993).
Thus, various agents useful for blocking nonspecific uptake by the
RES are known and routinely used.
[0055] Nonspecific binding of phage to RES or to other sites also
can be prevented by coinjecting, for example, mice with a specific
phage display library together with the same phage made
noninfective (Smith et al., supra, 1990, 1993). In addition, a
peptide that homes to tumor in an organ containing an RES component
can be identified by preparing a phage display library using phage
that exhibit low background binding to the particular organ. For
example, Merrill et al. (Proc. Natl. Acad. Sci., USA 93:3188-3192
(1996), which is incorporated herein by reference) selected
lambda-type phage that are not taken up by the RES and, as a
result, remain in the circulation for a prolonged period of time. A
filamentous phage variant, for example, can be selected using
similar methods.
[0056] Selective homing can be demonstrated by determining the
specificity of a tumor homing molecule for the tumor as compared to
a control organ or tissue. Selective homing also can be
demonstrated by showing that molecules that home to a tumor, as
identified by one round of in vivo panning, are enriched for tumor
homing molecules in a subsequent round of in vivo panning. For
example, phage expressing peptides that selectively home to a
melanoma tumor were isolated by in vivo panning, then were
subjected to additional rounds of in vivo panning. Following a
second round of screening, phage recovered from the tumor showed a
3-fold enrichment in homing to the tumor as compared to brain.
Phage recovered from the tumor after a third round of screening
showed an average of 10-fold enrichment in homing to the tumor as
compared to brain. Selective homing also can be demonstrated by
showing that molecules that home to a selected tumor, as identified
by one round of in vivo panning, are enriched for tumor homing
molecules in a subsequent round of in vivo panning.
[0057] Tumor homing molecules can be identified by in vivo panning
using, for example, a mouse containing a transplanted tumor. Such a
transplanted tumor can be, for example, a human tumor that is
transplanted into immunodeficient mice such as nude mice or a
murine tumor that is maintained by passage in tissue culture or in
mice. Due to the conserved nature of cellular receptors and of
ligands that bind a particular receptor, it is expected that
angiogenic vasculature and histologically similar tumor cells in
various species can share common cell surface markers useful as
target molecules for a tumor homing molecule. Thus, the skilled
artisan would recognize that a tumor homing molecule identified
using, for example, in vivo panning in a mouse having a murine
tumor of a defined histological type such as a melanoma also would
bind to the corresponding target molecule in a tumor in a human or
other species. Similarly, tumors growing in experimental animals
require associated neovascularization, just as that required for a
tumor growing in a human or other species. Thus, a tumor homing
molecule that binds a target molecule present in the vasculature in
a tumor grown in a mouse likely also can bind to the corresponding
target molecule in the vasculature of a tumor in a human or other
mammalian subject. The general ability of a tumor homing molecule
identified, for example, by homing to a human breast tumor, also to
home to a human Kaposi's sarcoma or to a mouse melanoma indicates
that the target molecules are shared by many tumors. Indeed, the
results disclosed herein demonstrate that the target molecules are
expressed in the neovasculature, which is host tissue (see Examples
IV and VII).
[0058] A tumor homing molecule identified using in vivo panning in
an experimental animal such as a mouse readily can be examined for
the ability to bind to a corresponding tumor in a human patient by
demonstrating, for example, that the molecule also can bind
specifically to a sample of the tumor obtained from the patient.
For example, the CDCRGDCFC (SEQ ID NO: 1) phage and NGR peptides
have been shown to bind to blood vessels in microscopic sections of
human tumors, whereas little or no binding occurs in the blood
vessels of nontumor tissues. Thus, routine methods can be used to
confirm that a tumor homing molecule identified using in vivo
panning in an experimental animal also can bind the target molecule
in a human tumor.
[0059] The steps of administering the library to the subject,
collecting a selected tumor and identifying the molecules that home
to the tumor, comprise a single round of in vivo panning. Although
not required, one or more additional rounds of in vivo panning
generally are performed. Where an additional round of in vivo
panning is performed, the molecules recovered from the tumor in the
previous round are administered to a subject, which can be the same
subject used in the previous round, where only a part of the tumor
was collected.
[0060] By performing a second round of in vivo panning, the
relative binding selectivity of the molecules recovered from the
first round can be determined by administering the identified
molecules to a subject, collecting the tumor, and determining
whether more phage is recovered from the tumor following the second
round of screening as compared to those recovered following the
first round. Although not required, a control organ or tissue also
can be collected and the molecules recovered from the tumor can be
compared with those recovered from the control organ. Ideally, no
molecules are recovered from a control organ or tissue following a
second or subsequent round of in vivo panning. Generally, however,
a proportion of the molecules also will be present in a control
organ or tissue. In this case, the ratio of molecules in the
selected tumor as compared to the control organ (selected:control)
can be determined. For example, phage that homed to melanoma
following a first round of in vivo panning demonstrated a 3.times.
enrichment in homing to the selected tumor as compared to the
control organ, brain, following two additional rounds of panning
(Example V).
[0061] Additional rounds of in vivo panning can be used to
determine whether a particular molecule homes only to the selected
tumor or can recognize a target on the tumor that also is expressed
in one or more normal organs or tissues in a subject or is
sufficiently similar to the target molecule on the tumor. It is
unlikely that a tumor homing molecule also will home to a
corresponding normal tissue because the method of in vivo panning
selects only those molecules that home to the tumor, which is
selected. Where a tumor homing molecule also directs homing to one
or more normal organs or tissues in addition to the tumor, the
organs or tissues are considered to constitute a family of selected
organs or tissues. Using the method of in vivo panning, molecules
that home to only the selected tumor can be distinguished from
molecules that also home to one or more selected organs or tissues.
Such identification is expedited by collecting various organs or
tissues during subsequent rounds of in vivo panning.
[0062] The term "control organ or tissue" is used to mean an organ
or tissue other than the tumor for which the identification of a
tumor homing molecule is desired. A control organ or tissue is
characterized in that a tumor homing molecule does not selectively
home to the control organ. A control organ or tissue can be
collected, for example, to identify nonspecific binding of the
molecule or to determine the selectivity of homing of the molecule.
In addition, nonspecific binding can be identified by
administering, for example, a control molecule, which is known not
to home to a tumor but is chemically similar to a potential tumor
homing molecule. Alternatively, were the administered molecules are
linked to a support, administration of the supports, alone, also
can be used to identify nonspecific binding. For example, a phage
that expresses the gene III protein, alone, but that does not
contain a peptide fusion protein, can be studied by in vivo panning
to determine the level of nonspecific binding of the phage
support.
[0063] As disclosed herein, specific homing of a tumor homing
molecule readily can be identified by examining the selected tumor
tissue as compared to a corresponding nontumor tissue, as well as
to control organs or tissues. For example, immunohistological
analysis can be performed on a tumor tissue and corresponding
nontumor tissue using an antibody specific for a phage used to
display tumor homing peptides (see Example IV). Alternatively, an
antibody can be used that is specific for a shared tag that
expressed with the peptide, for example, a FLAG epitope or the
like, such detection systems being commercially available.
[0064] In general, a library of molecules, which contains a diverse
population of random or selectively randomized molecules of
interest, is prepared, then administered to a subject. At a
selected time after administration, the subject is sacrificed and
the tumor is collected such that the molecules present in the tumor
can be identified (see Example I). If desired, one or more control
organs or tissues or a part of a control organ or tissue can be
sampled. For example, mice bearing a breast tumor or a melanoma
tumor were injected with a phage peptide display library, then,
after about 1 to 5 minutes, the mice were anesthetized, either
frozen in liquid nitrogen or, preferably, are perfused through the
heart to terminate circulation of the phage, the tumor and one or
more control organs were collected from each, phage present in the
tumor and the control organs were recovered and peptides that
selectively homed to the respective tumors were identified (see
Examples I, II and V).
[0065] In the examples provided, the animals were sacrificed to
collect the selected tumor and control organ or tissue. It should
be recognized, however, that only a part of a tumor need be
collected to recover a support containing a molecule that homes to
that tumor and, similarly, only part of an organ or tissue need be
collected as a control. Thus, a part of a tumor, for example, can
be collected by biopsy, such that a molecule such as a peptide
expressed by a phage can be administered to the same subject a
second time or more, as desired. Where the molecule that is to be
administered a second time to the same subject is tagged or linked,
for example, to a support, the tag or support should be nontoxic
and biodegradable, so as not to interfere with subsequent rounds of
screening.
[0066] In vitro screening of phage libraries previously has been
used to identify peptides that bind to antibodies or to cell
surface receptors (Smith and Scott, supra, 1993). For example, in
vitro screening of phage peptide display libraries has been used to
identify novel peptides that specifically bound to integrin
adhesion receptors (Koivunen et al., J. Cell Biol. 124:373-380
(1994a), which is incorporated herein by reference) and to the
human urokinase receptor (Goodson et al., Proc. Natl. Acad. Sci.,
USA 91:7129-7133 (1994)). However, such in vitro studies provide no
insight as to whether a peptide that can specifically bind to a
selected receptor in vitro also will bind the receptor in vivo or
whether the binding peptide or the receptor are unique to a
specific organ in the body. Furthermore, the in vitro methods are
performed using defined, well-characterized target molecules in an
artificial system. For example, Goodson et al. (supra, 1994)
utilized cells expressing a recombinant urokinase receptor.
However, such in vitro methods are limited in that they require
prior knowledge of the target molecule and yield little if any
information regarding in vivo utility.
[0067] In vitro panning against cells in culture also has been used
to identify molecules that can specifically bind to a receptor
expressed by the cells (Barry et al., Nature Med. 2:299-305 (1996),
which is incorporated herein by reference). However, the cell
surface molecules that are expressed by a cell in vivo often change
when the cell is grown in culture. Thus, in vitro panning methods
using cells in culture also are limited in that there is no
guarantee a molecule that is identified due to its binding to a
cell in culture will have the same binding ability in vivo.
Furthermore, it is not possible using in vitro panning to
distinguish molecules that home only to the tumor cells used in the
screening, but not to other cell types.
[0068] In contrast, in vivo panning requires no prior knowledge or
availability of a target molecule and identifies molecules that
bind to cell surface target molecules that are expressed in vivo.
Also, since the "nontargeted" tissues are present during the
screening, the probability of isolating tumor homing molecules that
lack specificity of homing is greatly reduced. Furthermore, in
obtaining tumor homing molecules by in vivo panning, any molecules
that may be particularly susceptible to degradation in the
circulation in vivo due, for example, to a metabolic activity, are
not recovered. Thus, in vivo panning provides significant
advantages over previous methods by identifying molecules hat
selectively home in vivo and the target molecule resent in a
tumor.
[0069] Although mechanisms by which the disclosed ethod of in vivo
panning works have not been fully defined, one possibility is that
a molecule such as a peptide expressed on a phage recognizes and
binds to a target molecule present on endothelial cells lining the
blood vessels in a tumor. Evidence indicates, for example, that the
vascular tissues in various organs differ from one another and that
such differences can be involved in regulating cellular trafficking
in the body. For example, lymphocytes home to lymph nodes or other
lymphoid tissues due, in part, to the expression of specific
"address" molecules by the endothelial cells in those tissues
(Salmi et al., Proc. Natl. Acad. Sci., USA 89:11436-11440 (1992);
Springer, Cell 76:301-314 (1994)). Similarly, various leukocytes
can recognize sites of inflammation due, in part, to the expression
of endothelial cell markers induced by inflammatory signals (see
Butcher and Picker, Science 272:60-66 (1996); Springer, supra,
1994). Thus, endothelial cell markers provide a potential target
for directing, for example, a drug, which can be linked to a tumor
homing molecule, to a tumor in a subject.
[0070] In some cases, the metastasis of cancer cells to specific
organs also can be due to recognition by the tumor cell of an organ
specific marker, including organ specific endothelial cell markers
(Fidler and Hart, Science 217:998-1003 (1982)). The pattern of
metastasis of many cancers can be explained by assuming that
circulating tumor cells are preferentially trapped in the first
vascular bed encountered. Thus, the lungs and the liver are the
most frequent sites of cancer metastasis. However, some cancers
show patterns of metastasis that are not explained by circulatory
routing. Metastasis of such cancers may be due to the presence of
selectively expressed address molecules such as endothelial cell
surface molecules expressed in the organ to which the cancer
metastasizes (see Goetz et al., Int. J. Cancer 65:192-199 (1996);
Zhu et al., Proc. Natl. Acad. Sci., USA 88:9568-9572 (1991); Pauli
et al., Cancer Metast. Rev. 9:175-189 (1990); Nicolson, Biochim.
Biophys. Acta 948:175-224 (1988)). The identification of molecules
that bind to such organ-specific endothelial cell markers can
provide a means to prevent tumor cell metastasis to the particular
organ.
[0071] The vasculature within a tumor generally undergoes active
angiogenesis, resulting in the continual formation of new blood
vessels to support the growing tumor. Such angiogenic blood vessels
are distinguishable from mature vasculature in that angiogenic
vasculature expresses unique endothelial cell surface markers,
including the .alpha..sub.v.beta..sub.3 integrin. (Brooks, Cell
79:1157-1164 (1994); WO 95/14714, Int. Filing Date Nov. 22, 1994)
and receptors for angiogenic growth factors (Mustonen and Alitalo,
J. Cell Biol. 129:895-898 (1995); Lappi, Semin. Cancer Biol.
6:279-288 (1995)). Moreover, tumor vasculature is histologically
distinguishable from blood vessel in general in that tumor
vasculature is fenestrated (Folkman, Nature Med. 1:27-31 (1995);
Rak et al., Anticancer Drugs 6:3-18 (1995)). Thus, the unique
characteristics of tumor vasculature make it a particularly
attractive target for determining whether a molecule that homes
specifically to a tumor can be identified by in vivo panning. Such
a tumor homing molecule can be useful for directing an agent such
as a chemotherapeutic drug to a tumor, while reducing the
likelihood the agent will have a toxic effect on normal, healthy
organs or tissues (Example VII). Moreover, a molecule that homes
selectively to tumor vasculature also may have use in targeting
other types of neovasculature such as that present in inflammatory,
regenerating or wounded tissues.
[0072] Using in vivo panning to a breast carcinoma, a melanoma and
a Kaposi's sarcoma, phage expressing various peptides that
selectively homed to tumors were identified (see Tables 1, 2 and 3,
respectively). Due to the large size of the phage (300 nm or
900-1000 nm ? PLEASE CONFIRM SIZE OF PHAGE) and the short time the
phage were allowed to circulate (3 to 5 min), it is unlikely that a
substantial number of phage would have exited the circulatory
system, particularly in the brain and kidney. Tissue staining
studies indicated that the tumor homing molecules that were
identified primarily homed to and bound endothelial cell surface
markers, which likely are expressed in an organ-specific manner.
These results indicate that in vivo panning can be used to identify
and analyze endothelial cell specificities. Such an analysis is not
possible using endothelial cells in culture because the cultured
cells tend to lose their tissue-specific differences (Pauli and
Lee, Lab. Invest. 58:379-387 (1988)).
[0073] Although the conditions under which the in vivo pannings
were performed identified tumor homing peptides that generally bind
to endothelial cell markers, the specific presence of phage
expressing tumor homing peptides also was observed in tumor
parenchyma, particularly at later times after administration of the
peptides (Example IV). These results demonstrate that phage
expressing peptides can pass through the blood vessels in the
tumor, possibly due to the fenestrated nature of the blood vessels,
and indicate that the in vivo panning method can be useful for
identifying target molecules expressed by tumor cells, as well as
target molecules expressed by endothelial cells.
[0074] Phage peptide display libraries were constructed essentially
as described Smith and Scott (supra, 1993; see, also, Koivunen et
al., Biotechnology 13:265-270 (1995); Koivunen et al., Meth.
Enzymol. 245:346-369 (1994b), each of which is incorporated herein
by reference). Oligonucleotides encoding peptides having
substantially random amino acid sequences were synthesized based on
an "NNK" codon, wherein "N" is A, T, C or G and "K" is G or T.
"NNK" encodes 32 triplets, which encode the twenty amino acids and
an amber STOP codon (Scott and Smith, supra, 1990). In some
libraries, at least one codon encoding cysteine also was included
in each oligonucleotide so that cyclic peptides could be formed
through disulfide linkages (Example I). The oligonucleotides were
inserted in frame with the sequence encoding the gene III protein
(gIII) in the vector fuse 5 such that a peptide-gIII fusion protein
can be expressed. Following expression, the fusion protein is
expressed on the surface of the phage containing the vector
(Koivunen et al., supra, 1994b; Smith and Scott, supra, 1993).
[0075] Following in vivo panning, the phage isolated based on their
ability to selectively home to human breast carcinoma, mouse
melanoma or human Kaposi's sarcoma tumors displayed only a few
different peptide sequences (see Tables 1, 2 and 3, respectively).
One of the screenings revealed peptide sequences that contained the
arginine-glycine-aspartic acid (RGD) integrin recognition sequence
(Ruoslahti, Ann. Rev. Cell Devel. Biol. 12:697 (1996)) in the
context of a peptide previously demonstrated to bind selectively to
.alpha..sub.v-containing integrins (Koivunen et al., supra, 1995;
WO 95/14714). The sequences of most of the remaining tumor homing
peptides did not reveal any significant similarities with known
ligands for endothelial cell receptors. However, one of the tumor
homing peptides contained the asparagine-glycine-arginin- e (NGR)
motif, which is a weak integrin binding motif similar to the motifs
present in integrin-binding peptides (Ruoslahti et al., U.S. Pat.
No. 5,536,814, issued Jul. 16, 1996, which is incorporated herein
by reference; see, also, Koivunen et al., supra, 1994a). Other
screenings have revealed numerous NGR-containing peptides (see
Table 1). Despite the weak integrin binding ability of NGR
peptides, an integrin receptor may not be the target molecule
recognized by the NGR tumor homing peptides exemplified herein
(Example VII). As used herein, the term "integrin" means a
heterodimeric cell surface adhesion receptor.
[0076] The peptides expressed by the phage that homed to the breast
tumor included the peptides CGRECPRLCQSSC (SEQ ID NO: 2) and
CNGRCVSGCAGRC (SEQ ID NO: 3; see Table 1; Example II). Similarly,
tumor homing peptides, including the peptides CDCRGDCFC (SEQ ID NO:
1) and CGSLVRC (SEQ ID NO: 5), were identified from two other phage
libraries administered to breast tumor bearing mice (Table 1). Some
of these motifs, as well as novel one, also were isolated in the
screen with mouse melanoma and human Kaposi's sarcoma (see Tables 2
and 3). These results demonstrated that tumor homing molecules can
be identified using in vivo panning.
[0077] Three main tumor homing motifs emerged. As discussed above,
one motif contained the sequence RGD (Ruoslahti, supra, 1996)
embedded in the peptide structure, CDCRGDCFC (SEQ ID NO: 1), which
is known to bind selectively to .alpha..sub.v integrins (Koivunen
et al., supra, 1995; WO 95/14714). Since the
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins
are markers of angiogenic vessels (Brooks et al., supra, 1994;
Friedlander et al., Science 270:1500 (1995)), a phage expressing
the peptide CDCRGDCFC (SEQ ID NO: 1) was examined for tumor
targeting and, as disclosed herein, homed to tumors in a highly
selective manner (see Example III). Furthermore, homing by the
CDCRGDCFC (SEQ ID NO: 1) phage was inhibited by coadministration of
the free CDCRGDCFC (SEQ ID NO: 1) peptide.
[0078] Another breast tumor homing peptide had the sequence
CNGRCVSGCAGRC (SEQ ID NO: 3), which contains the NGR motif
previously shown to have weak integrin binding activity (Koivunen
et al., J. Biol. Chem. 268:20205-20210 (1993); Koivunen et al.,
supra, 1994a; WO 95/14714). Since an NGR containing peptide was
identified, two additional peptides, the linear peptide, NGRAHA
(SEQ ID NO: 6), and the cyclic peptide, CVLNGRMEC (SEQ ID NO: 7),
each of which contains the NGR motif, were examined for tumor
homing. Like the phage expressing CNGRCVSGCAGRC (SEQ ID NO: 3),
phage expressing NGRAHA (SEQ ID NO: 6) or CVLNGRMEC (SEQ ID NO: 7)
homed to the tumors. Furthermore, tumor homing was not dependent on
the tumor type or on species, as the phage accumulated selectively
in human breast carcinoma, as well as in the tumors of mice bearing
a mouse melanoma and mice bearing a human Kaposi's sarcoma
xenograft.
[0079] The various peptides, including RGD- and NGR-containing
peptides, generally were bound to the tumor blood vessels. The
minimal cyclic NGR peptide, CNGRC (SEQ ID NO: 8), was synthesized
based on the CNGRCVSGCAGRC (SEQ ID NO: 3) sequence. When the CNGRC
(SEQ ID NO: 8) peptide was co-injected with phage expressing either
CNGRCVSGCAGRC (SEQ ID NO: 3), NGRAHA (SEQ ID NO: 6) or CVLNGRMEC
(SEQ ID NO: 7), accumulation of the phage in the breast carcinoma
xenografts was inhibited. However, the CNGRC (SEQ ID NO: 8) peptide
did not inhibit the homing of phage expressing the CDCRGDCFC (SEQ
ID NO: 1) peptide, even when administered in amounts up to ten
times higher than those that inhibited the homing of the NGR phage.
In comparison, the CDCRGDCFC (SEQ ID NO: 1) peptide partially
inhibited the homing of the NGR phage, although the amount needed
was 5 to 10 fold higher than that of the CEGRC peptide (SEQ ID NO:
8). These results indicate that NGR peptides and RGD peptides bind
to different receptor sites in tumor vasculature.
[0080] A third motif, GSL (glycine-serine-leucine), also was
identified following in vivo panning in mice bearing breast
carcinoma, malignant melanoma or Kaposi's sarcoma. Homing of phage
expressing the GSL peptide, CGSLVRC (SEQ ID NO: 5), was inhibited
by coadministration of the free CGSLVRC (SEQ ID NO: 5) peptide.
Like the RGD and NGR peptides, phage expressing GSL peptides also
bound to blood vessels of tumors. In view of the identification of
the conserved RGD, NGR and GSL motifs present in tumor homing
peptides, as disclosed herein, it will be recognized that peptides
containing such motifs can be useful as tumor homing peptides and,
in particular, for forming conjugates that can target a moiety such
as a cancer chemotherapeutic agent or a diagnostic agent to a
tumor.
[0081] Various peptide libraries containing up to 13 amino acids
were constructed and the NGR peptide, CNGRCVSGCAGRC (SEQ ID NO: 3),
was obtained as a result of in vivo panning against a breast tumor.
This NGR peptide, which was obtained by screening a random peptide
library, was a tumor homing peptide (see Example VII). In addition,
when a peptide library was constructed based on the formula
CXXXNGRXX (SEQ ID NO: 13) or CXXCNGRCX (SEQ ID NO: 14), each of
which is biased toward NGR sequences, and used for in vivo panning
against a breast tumor, numerous NGR peptides were obtained (see
Table 1).
[0082] These results indicate that a tumor homing peptide of the
invention can comprise the amino acid sequence RGD or NGR or GSL.
Such tumor peptides can be as small as five amino acids, such as
CNGRC (SEQ ID NO: 8). Such tumor homing peptides also can be not
only at least 13 amino acids in length, which is the largest
peptide exemplified herein, but can be up to 20 amino acids, or 30
amino acids, or 50 to 100 amino acids in length, as desired. A
tumor homing peptide of the invention conveniently is produced by
chemical synthesis.
[0083] Immunohistochemical analysis was performed by comparing
tissue staining for phage allowed to circulate for about four
minutes, followed by perfusion through the heart of the mice, or
with tissues analyzed 24 hours after phage injection (see FIG. 1).
At 24 hours following administration, essentially no phage remain
in the circulation and, therefore, perfusion is not required
(Pasqualini et al., supra, 1997). Strong phage staining was
observed in tumor vasculature, but not in normal endothelium, in
samples examined four minutes after administration of the
CNGRCVSGCAGRC (SEQ ID NO: 3) phage (Example IV; compare FIGS. 1E,
1G, 1H and 1J). In comparison, staining of the tumor was strong at
24 hours and appeared to have spread outside the blood vessels into
the tumor parenchyma (compare FIGS. 1A to 1D and 1F (tumor) with
FIGS. 1I and 1K to 1V (nontumor)). The NGRAHA (SEQ ID NO: 6) and
CVLNGRMEC (SEQ ID NO: 7) phage showed similar staining patterns
(Example IV). In contrast, the control organs and tissues showed
little or no immunostaining, confirming the specificity of the NGR
motifs for tumor vessels. Spleen and liver, however, captured
phage, as expected, since uptake by the reticuloendothelial system
is a general property of phage particles, independent of the
presence of peptide expression by the phage (Pasqualini et al.,
supra, 1997).
[0084] Immunostaining also was observed following administration of
phage expressing the GSL motif containing peptide, CLSGSLSC (SEQ ID
NO: 4), and, like that of the NGR peptides, was localized to the
blood vessels, in this case, within a melanoma tumor (see below;
see, also, Examples IV and V). Similarly, immunostaining following
administration of phage expressing the RGD motif containing
peptide, CDCRGDCFC (SEQ ID NO: 1), to breast tumor bearing mice was
localized to the blood vessels in the tumor, but was not observed
in brain, kidney or various other nontumor tissues (see Examples
III and IV; see, also, Pasqualini et al., supra, 1997). These
results demonstrate that the various tumor homing peptides
generally home to tumor vasculature.
[0085] The general applicability of the in vivo panning method for
identifying molecules that home to a tumor was examined by
injecting mice bearing a syngeneic melanoma with phage expressing a
diverse population of peptides (Example V). The B16 mouse melanoma
model was selected for these studies because the tumors that form
are highly vascularized and because the biology of this tumor line
has been thoroughly characterized (see Miner et al., Cancer Res.
42:4631-4638 (1982)). Furthermore, because the B16 melanoma cells
are of mouse origin, species differences between the host and the
tumor cell donor will not affect, for example, the distribution of
phage into the tumor as compared to into normal organs. As
disclosed herein, in vivo panning against B16 melanoma cells
revealed tumor homing peptides, including, for example, the GSL
moiety containing peptide CLSGSLSC (SEQ ID NO: 4; see, also, Table
2) and immunohistochemical staining of the tumor and other organs
using an anti-phage antibody demonstrated that the CLSGSLSC (SEQ ID
NO: 4) expressing phage resulted in immunostaining in the melanoma,
but essentially no staining in skin, kidney or other control organs
(Example V). The staining pattern generally followed the blood
vessels within the melanoma, but was not strictly confined to the
blood vessels.
[0086] Although in vivo panning was performed in mice, at least the
peptides comprising an NGR, RGD or GSL motif also likely can target
human vasculature. The NGR phage binds to blood vessels in the
transplanted human breast tumor, but not to blood vessels in normal
tissues, indicating that this motif can be particularly useful for
tumor targeting in patients. The CDCRGDCFC (SEQ ID NO: 1) peptide
binds to human .alpha..sub.v-integrins (Koivunen et al., supra,
1995), which are selectively expressed in tumor blood vessels of
human patients (Max et al., Int. J. Cancer 71:320 (1997); Max et
al., Int. J. Cancer 72:706 (1997)). Use of a moiety/CDCRGDCFC. (SEQ
ID NO: 1) conjugate to target the moiety to a tumor also provides
the additional advantage that the moiety will be targeted to tumor
cells, themselves, because breast carcinoma cells, for example, can
express the .alpha..sub.v.beta..sub.3 integrin (Pasqualini et al.,
supra, 1997). In fact, many human tumors express this integrin,
which may be involved in the progression of certain tumors such as
malignant melanomas (Albelda et al., Cancer Res. 50:6757-6764
(1990); Danen et al., Int. J. Cancer 61:491-496 (1995);
Felding-Habermann et al., J. Clin. Invest. 89:2018-2022 (1992);
Sanders et al., Cold Spring Harb. Symp. Ouant. Biol. 58:233-240
(1992); Mitjans et al., J. Cell. Sci. 108:3067-3078 (1995)). Unlike
the CDCRGDCFC (SEQ ID NO: 1) peptide, the NGR peptides do not
appear to bind to MDA-MD-435 breast carcinoma cells. However, NGR
peptides were able to deliver a therapeutically effective amount of
doxorubicin to breast tumors (Example VII), indicating that, even
where a tumor homing molecule homes only to tumor vasculature,
i.e., not directly to the tumor cells, such vasculature targeting
in sufficient to confer the effect of the moiety linked to the
molecule.
[0087] Since the .alpha..sub.v.beta..sub.3 integrin is expressed by
endothelial cells in angiogenic vasculature, experiments were
performed to determine whether tumor vasculature that is undergoing
angiogenesis can be targeted in vivo using methods as disclosed
herein. Phage expressing the peptide, CDCRGDCFC (SEQ ID NO: 1; see,
Koivunen et al., supra, 1995), which is known to bind to the
.alpha..sub.v.beta..sub.3 integrin, were injected into mice bearing
tumors formed from human breast carcinoma cells, mouse melanoma
cells or human Kaposi's sarcoma cells (see Example IV). The
CDCRGDCFC (SEQ ID NO: 1) phage selectively homed to each of the
tumors, whereas such homing did not occur with control phage. For
example, in mice bearing tumors formed by implantation of human
breast carcinoma cells, a twenty-to eighty-fold greater number of
the CDCRGDCFC (SEQ ID NO: 1) phage, as compared to unselected
control phage, accumulated in the tumor.
[0088] Tissue staining for the phage showed accumulation of the
CDCRGDCFC (SEQ ID NO: 1) phage in the blood vessels within the
tumor, whereas no staining was observed in brain, kidney or other
control organs. Specificity of tumor homing by the CDCRGDCFC (SEQ
ID NO: 1) phage was demonstrated by competition experiments, in
which coinjection of the free CDCRGDCFC (SEQ ID NO: 1) peptide
greatly reduced tumor homing of the RGD phage, whereas coinjection
of a non-RGD-containing control peptide had no effect on homing of
the RGD phage (see Example III). These results demonstrate that the
.alpha..sub.v.beta..sub.3 target molecule is expressed on the
luminal surface of endothelial cells in a tumor and that a peptide
that binds to an .alpha..sub.v-containing integrin can bind
selectively to this integrin and, therefore, to vasculature
undergoing angiogenesis.
[0089] The results of these studies indicate that tumor homing
molecules can be identified by in vivo panning and that, in some
cases, a tumor homing molecule can home to vascular tissue in the
tumor as well as to tumor parenchyma, probably due to the
fenestrated nature of the blood vessels permitting ready exit of
the phage from the circulatory system. Due to the ability of such
tumor homing molecules to home to tumors, the molecules are useful
for targeting a linked moiety to tumors. Thus, the invention
provides conjugates comprising a tumor homing molecule linked to a
moiety, such conjugates being useful for targeting the moiety to
tumor cells.
[0090] The ability of a molecule that homes to a particular tumor
to selectively home to another tumor of the same or a similar
histologic type can be determined using, for example, human tumors
grown in nude mice or mouse tumors grown in syngeneic mice for
these experiments. For example, various human breast cancer cell
lines, including MDA-MB-435 breast carcinoma (Price et al., Cancer
Res. 50:717-721 (1990)), SKBR-1-II and SK-BR-3 (Fogh et al., J.
Natl. Cancer Inst. 59:221-226 (1975)), and mouse mammary tumor
lines, including EMT6 (Rosen et al., Int. J. Cancer 57:706-714
(1994)) and C3-L5 (Lala and Parhar, Int. J. Cancer 54:677-684
(1993)), are readily available and commonly used as models for
human breast cancer. Using such breast tumor models, for example,
information relating to the specificity of an identified breast
tumor homing molecule for diverse breast tumors can be obtained and
molecules that home to a broad range of different breast tumors or
provide the most favorable specificity profiles can be identified.
In addition, such analyses can yield new information, for example,
about tumor stroma, since stromal cell gene expression, like that
of endothelial cells, can be modified by the tumor in ways that
cannot be reproduced in vitro.
[0091] Selective homing of a molecule such as a peptide or protein
to a tumor can be due to specific recognition by the peptide of a
particular cell target molecule such as a cell surface receptor
present on a cell in the tumor. Selectivity of homing is dependent
on the particular target molecule being expressed on only one or a
few different cell types, such that the molecule homes primarily to
the tumor. As discussed above, the identified tumor homing
peptides, at least in part, can recognize endothelial cell surface
markers in the blood vessels present in the tumors. However, most
cell types, particularly cell types that are unique to an organ or
tissue, can express unique target molecules. Thus, in vivo panning
can be used to identify molecules that selectively home to a
particular type of tumor cell such as a breast cancer cell and
specific homing can be demonstrated by performing the appropriate
competition experiments.
[0092] A tumor homing molecule of the invention can be used to
target a moiety to a tumor by linking the moiety to the molecule to
produce a tumor homing molecule/moiety conjugate and administering
the conjugate to a subject having a tumor. As used herein, the term
"moiety" is used broadly to mean a physical, chemical, or
biological material that is linked to a tumor homing molecule for
the purpose of being targeted in vivo to a tumor or to angiogenic
vasculature expressing a target molecule recognized by the tumor
homing molecule. In particular, a moiety is a biologically useful
moiety such as therapeutic moiety, a diagnostic moiety or a drug
delivery vehicle. Thus, a moiety can be a therapeutic agent, for
example, a cancer chemotherapeutic agent such as doxorubicin,
which, when linked to a tumor homing molecule, provides a conjugate
useful for treating a cancer in a subject. In addition, a moiety
can be a drug delivery vehicle such as a chambered microdevice, a
cell, a liposome or a virus, which can contain an agent such as a
drug or a nucleic acid.
[0093] A moiety also can be a molecule such as a polypeptide or
nucleic acid, to which a tumor homing molecule is grafted for the
purpose of directing the polypeptide or nucleic acid to a selected
tumor (Smith et al., J. Biol. Chem. 269:32788-32795 (1994); Goldman
et al., Cancer Res. 15:1447-1451 (1997), each of which is
incorporated herein by reference). For example, a peptide tumor
homing molecule can be expressed as a fusion protein with a desired
polypeptide such that the peptide targets the grafted polypeptide
to a selected tumor. Such a desired polypeptide, which is grafted
to the tumor homing peptide, can be a polypeptide involved in
initiating a cell death pathway, for example, caspase 8, thus
providing a means to direct caspase 8 to a tumor, where it can
induce apoptosis of the tumor cells or of the vasculature supplying
the tumor. A tumor homing peptide also can be grafted to a
polypeptide expressed by a virus, for example, the adenovirus
penton base coat protein, thus providing a means to target a virus
to a tumor (Wickham et al., Gene Ther. 2:750-756 (1995); Weitzman
et al., In: "Gene Therapy and Vector Systems" 2:17-25 (1997), each
of which is incorporated herein by reference; see, also, Example
III). Such a grafted virus can contain an exogenous gene useful in
a method of gene therapy. Accordingly, the invention provides
compositions of matter comprising a tumor homing molecule/moiety
conjugate.
[0094] A moiety can be a detectable label such a radiolabel or can
be a cytotoxic agent, including a toxin such as ricin or a drug
such as a chemotherapeutic agent or can be a physical, chemical or
biological material such as a liposome, microcapsule, micropump or
other chambered microdevice, which can be used, for example, as a
drug delivery system. Generally, such microdevices, should be
nontoxic and, if desired, biodegradable. Various moieties,
including microcapsules, which can contain an agent, and methods
for linking a moiety, including a chambered microdevice, to a
molecule of the invention are well known in the art and
commercially available (see, for example, "Remington's
Pharmaceutical Sciences" 18th ed. (Mack Publishing Co. 1990),
chapters 89-91; Harlow and Lane, Antibodies: A laboratory manual
(Cold Spring Harbor Laboratory Press 1988), each of which is
incorporated herein by reference; see, also, Hermanson,
supra,.1996).
[0095] As disclosed herein, a moiety can be, for example, a cancer
chemotherapeutic agent linked to a tumor homing molecule to produce
a tumor homing molecule/moiety conjugate. Cytotoxic chemotherapy is
the basis of the systemic treatment of disseminated malignant
tumors. However, a major limitation of the currently used
chemotherapeutic agents is that these drugs have the narrowest
therapeutic index in all of medicine. As such, the dose of cancer
chemotherapeutic agents generally is limited by undesirable
toxicity to the patient being treated. Thus, the ability of tumor
homing peptides of the invention to target drugs into tumors was
examined. As disclosed herein, the linking of a cancer
chemotherapeutic agent, doxorubicin, to a tumor homing molecule
reduced the systemic toxicity of the doxorubicin and enhanced
anti-tumor activity of the agent (see Example VII).
[0096] A conjugate of the invention is exemplified herein by
doxorubicin linked to various tumor homing peptides (see Examples
VI and VII). In view of the exemplified method of linking
doxorubicin to various tumor homing peptides and the disclosed
efficacy of such conjugates of the invention, the skilled artisan
will recognize that various other chemotherapeutic agents also can
be linked to a tumor homing molecule to make a conjugate of the
invention. Cancer chemotherapeutic agents have been linked to
antibodies, for example, for the purpose of targeting the agents to
cells such as tumor cells that express the antigen recognized by
the antibodies. In addition, in such antibody/drug conjugates, the
agent can maintain its therapeutic function and the antibody can
maintain its antigen binding specificity. For example, the
anthracyclin, doxorubicin, has been linked to antibodies and the
antibody/doxorubicin conjugates have been therapeutically effective
in treating tumors (Sivam et al., Cancer Res. 55:2352-2356 (1995);
Lau et al., Bioorg. Med. Chem. 3:1299-1304 (1995); Shih et al.,
Cancer Immunol. Immunother. 38:92-98 (1994)). Similarly, other
anthracyclins, including idarubicin and daunorubicin, have been
chemically conjugated to antibodies, which have delivered effective
doses of the agents to tumors (Rowland et al., Cancer Immunol.
Immunother. 37:195-202 (1993); Aboud-Pirak et al., Biochem.
Pharmacol. 38:641-648 (1989)).
[0097] In addition to the anthracyclins, alkylating agents such as
melphalan and chlorambucil have been linked to antibodies to
produce therapeutically effective conjugates (Rowland et al.,
supra, 1994; Smyth et al., Immunol. Cell Biol. 65:315-321 (1987)),
as have vinca alkaloids such as vindesine and vinblastine
(Aboud-Pirak et al., supra, 1989; Starling et al., Bioconj. Chem.
3:315-322 (1992)). Similarly, conjugates of antibodies and
antimetabolites such as 5-fluorouracil, 5-fluorouridine and
derivatives thereof have been effective in treating tumors (Krauer
et al., Cancer Res. 52:132-137 (1992); Henn et al., J. Med. Chem.
36:1570-1579 (1993)). Other chemotherapeutic agents, including
cis-platinum (Schechter et al., Int. J. Cancer 48:167-172 (1991)),
methotrexate (Shawler et al., J. Biol. Resp. Mod. 7:608-618 (1988);
Fitzpatrick and Garnett, Anticancer Drug Des. 10:11-24 (1995)) and
mitomycin-C (Dillman et al., Mol. Biother. 1:250-255 (1989)) also
are therapeutically effective when administered as conjugates with
various different antibodies.
[0098] The results obtained using antibody/drug conjugates
demonstrate that a chemotherapeutic agent can be linked to an
antibody to produce a conjugate that maintains the antigen binding
specificity of the antibody and the therapeutic function of the
agent. As disclosed herein, a conjugate comprising doxorubicin and
a tumor homing peptide maintains the tumor homing specificity of
the tumor homing peptide as well as the therapeutic efficacy of the
chemotherapeutic agent (see Example VII). Such results are
remarkable, since, in the doxorubicin/CNGRC (SEQ ID NO: 8)
conjugate, for example, the doxorubicin component has only a
slightly lower molecular weight than the peptide and comprises
about 46% of the molecular weight of the conjugate.
[0099] Since the moiety component of a tumor homing molecule/moiety
conjugate can comprise a substantial portion of the conjugate
without adversely affecting the ability of the tumor homing
molecule to home to a tumor, 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 a
tumor homing peptide and the moiety (Fitzpatrick and Garnett,
Anticancer Drug Des. 10:1-9 (1995)). In this way, panels of
moiety/spacer complexes can be constructed, in which a common
spacer is linked to various different moieties. Such panels of
moiety/spacer conjugates can facilitate linkage of the moiety to a
tumor homing molecule such as a tumor homing peptide of choice.
[0100] Doxorubicin is one of the most commonly used cancer
chemotherapeutic agents and, particularly, is used 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. Thus,
treatment of human breast cancer xenografts in mice using
doxorubicin was selected as a model for exemplifying the present
invention.
[0101] CDCRGDCFC (SEQ ID NO: 1) and CNGRC (SEQ ID NO: 8) were
coupled to doxorubicin (Example VI) and the peptide/doxorubicin
conjugates were used to treat mice bearing tumors derived from
human MDA-MB-435 breast carcinoma cells (Example VII). Mice were
treated with 5 .mu.g/week of doxorubicin equivalent (i.e., either
free doxorubicin or the doxorubicin component of the
peptide/doxorubicin conjugate), as compared to the more commonly
used 50-200 .mu.g/mouse used in tumor bearing mice (Berger et al.,
In "The Nude Mouse in Oncology Research" (CRC Press 1991)). The
lower dose was selected because it was expected that the conjugate
would be more effective than the free drug.
[0102] MDA-MB-435 tumor-bearing mice treated with the
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate had significantly
smaller tumors, less spread to regional lymph nodes, and fewer
pulmonary metastasis than mice treated with free doxorubicin (see
Example VII). All of the mice treated with the
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate survived beyond the
time when all of the mice treated with free doxorubicin had died
from widespread disease. In a dose-escalation experiment, the tumor
bearing mice were treated with the doxorubicin/CDCRGDCFC (SEQ ID
NO: 1) conjugate at 30 .mu.g/mouse every three weeks for three
cycles, then were observed, without further treatment, for an
extended period of time. The conjugate treated mice all remained
alive more than 6 months after the control, doxorubicin treated
mice had died (Example VII). These results indicate that primary
tumor growth and metastasis significantly were inhibited in mice
treated with the conjugate and that cures may have occurred.
[0103] Many of the mice that received doxorubicin/CDCRGDCFC (SEQ ID
NO: 1) conjugate presented marked skin ulceration and tumor
necrosis; no such signs were observed in mice treated with free
doxorubicin or with doxorubicin conjugated to an unrelated peptide
(Example VII). Histopathological analysis disclosed a pronounced
destruction of the vasculature in the tumors treated with conjugate
as compared to mice treated with free doxorubicin. Furthermore,
when tumors were removed from the mice and the tumor cells plated
in culture, viability of cells from the tumors of mice receiving
the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate was about 3 fold
less than cells from tumors of mice treated with the free
doxorubicin (see Example VII). These results demonstrate that
administration to a tumor bearing mouse of a conjugate comprising a
chemotherapeutic agent linked to a tumor homing molecule is more
efficacious than administration of the agent, alone, in treating a
tumor.
[0104] Toxicity was determined by administration of 200
.mu.g/doxorubicin equivalent in mice with very large, size matched
breast tumors. All of the mice treated with the
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate survived more than a
week, while all of the mice treated with free doxorubicin died
within 48 hours of the administration of the drug (Example VII).
These results indicate that accumulation of the tumor homing
peptide/doxorubicin conjugate in the large tumors can reduce
systemic toxicity of the agent.
[0105] Similar toxicity and treatment efficacy results were
obtained when breast tumor bearing mice were treated using a
doxorubicin/CNGRC (SEQ ID NO: 8) conjugate. Tumors in the mice
treated with the CNGRC (SEQ ID NO: 8) conjugate were significantly
smaller than in the control groups; the conjugate suppressed tumor
growth almost completely. A strong effect on survival also
occurred. Free doxorubicin or doxorubicin conjugated to an
unrelated peptide, at the dose used, had little if any effect on
tumor growth relative to vehicle alone.
[0106] Cytotoxic activity of free doxorubicin and the
doxorubicin/peptide conjugates was compared in vitro using
MDA-MB-435 cells. When cells were exposed to free doxorubicin,
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) or doxorubicin conjugated to
an unrelated peptide for 30 minutes, cell death occurred only in
the cultures treated with the doxorubicin/CDCRGDCFC (SEQ ID NO: 1)
conjugate. In comparison, cells were killed by all of the
treatments after 24 hours of exposure. These results indicate that
enhanced cellular uptake of the doxorubicin/CDCRGDCFC (SEQ ID NO:
1) conjugate occurs.
[0107] As disclosed herein, tumor homing molecules of the invention
can bind to the endothelial lining of small blood vessels of
tumors. The vasculature within tumors is distinct, presumably due
to the continual neovascularization, resulting in the formation of
new blood vessels required for tumor growth. The distinct
properties of the angiogenic neovasculature within tumors are
reflected in the presence of specific markers in endothelial cells
and pericytes (Folkman, Nature Biotechnol. 15:510 (1997); Risau,
FASEB J. 9:926-933 (1995); Brooks et al., supra, 1994); these
markers likely are being targeted by the tumor homing molecules of
the invention.
[0108] The ability of a tumor homing molecule to target the blood
vessels in a tumor provides substantial advantages over methods of
systemic treatment or methods that directly target the tumor cells.
For example, tumor cells depend on a vascular supply for survival
and the endothelial lining of blood vessels is readily accessible
to a circulating probe. Conversely, in order to reach solid tumor
cells, a chemotherapeutic agent must overcome potentially long
diffusion distances, closely packed tumor cells, and a dense
fibrous stroma with a high interstitial pressure that impedes
extravasation (Burrows and Thorpe, Pharmacol. Ther. 64:155-174
(1994)).
[0109] In addition, where the tumor vasculature is targeted, the
killing of all target cells may not be required, since partial
denudation of the endothelium can lead to the formation of an
occlusive thrombus halting the blood flow through the entirety of
the affected tumor vessel (Burrows and Thorpe, supra, 1994).
Furthermore, unlike direct tumor targeting, there is an intrinsic
amplification mechanism in tumor vasculature targeting. A single
capillary loop can supply nutrients to up to 100 tumor cells, each
of which is critically dependent on the blood supply (Denekamp,
Cancer Metast. Rev. 9:267-282 (1990); Folkman, supra, 1997).
[0110] Endothelial cells in a tumor also are unlikely to lose a
cell surface target receptor or develop a drug resistance
phenotype, as can develop through mutation and clonal evolution of
tumor cells, because endothelial cells are genetically stable
despite their high proliferation rates (Burrows and Thorpe, supra,
1994; Folkman, supra, 1995; Folkman, supra, 1997). In this regard,
it has been long recognized by medical oncologists that, while
tumors treated with chemotherapeutic agents commonly develop drug
resistance, normal tissues such as bone marrow do not develop such
resistance. Thus, toxicity to normal tissues such as chemotherapy
induced myelosuppression continues to occur during a treatment,
even after tumor cells have become drug resistant. Since the
endothelial cells in blood vessels supplying a tumor are nontumor
cells, it is expected that they will not develop resistance to
chemotherapeutic agents, in a manner analogous to bone marrow
cells. In fact, drug resistance has not been observed during long
term anti-angiogenic therapy in either experimental animals or in
clinical trials (Folkman, supra, 1997).
[0111] Linking of a moiety larger than an agent such as a drug or
other organic or biologic molecule to a tumor homing molecule for
the purpose of directing homing of the moiety to the selected tumor
is exemplified by expressing an RGD-containing peptide on a phage,
wherein the peptide directed homing of the phage to breast tumor
vasculature (Example IV). These results indicate that a tumor
homing molecule of the invention can be linked to other moieties
including, for example, a chambered microdevice or a liposome or a
cell such as a white blood cell (WBC), which can be a cytotoxic T
cell or a killer cell, wherein upon administration of the tumor
homing molecule/WBC conjugate, the molecule directs homing of the
WBC to the tumor, where the WBC can exert its effector
function.
[0112] The linking of a moiety to a tumor homing molecule can
result in the molecule directing homing of the linked moiety to a
tumor. For example, the linking of a brain homing peptide to a RBC
directed homing of the RBC to brain (see U.S. Pat. No. 5,622,699;
Pasqualini and Ruoslahti, supra, 1996). This result indicates that
a tumor homing molecule of the invention also can be linked to cell
type or to a physical, chemical or biological delivery system such
as a liposome or other encapsulating device, which can contain an
agent such as drug, in order to direct the cell type or the
delivery system to a selected tumor. For example, a tumor homing
molecule identified by in vivo panning can be linked to a white
blood cell (WBC) such as a cytotoxic T cell or a killer cell,
wherein upon administration of the tumor homing molecule/WBC
conjugate, the molecule directs homing of the WBC to the tumor,
where the WBC can exert its effector function. Similarly, a tumor
homing molecule can be linked to a liposome or to a chambered
microdevice comprising, for example, a permeable or semipermeable
membrane, wherein an agent such as a drug to be delivered to a
selected tumor is contained within the liposome or microdevice.
Such compositions also can be useful, for example, for delivering a
nucleic acid molecule to a tumor cells, thereby providing a means
for performing in vivo targeted gene therapy.
[0113] In one embodiment, a tumor homing molecule is linked to a
moiety that is detectable external to the subject, thereby
providing a composition useful to perform an in vivo diagnostic
imaging study. For example, in vivo imaging using a detectably
labeled tumor homing peptide can identify the presence of a tumor
in a subject. For such studies, a moiety such as a gamma ray
emitting radionuclide, for example, indium-111 or technitium-99,
can be linked to the tumor homing molecule and, following
administration to a subject, can be detected using a solid
scintillation detector. Similarly, a positron emitting radionuclide
such as carbon-11 or a paramagnetic spin label such as carbon-13
can be linked to the molecule and, following administration to a
subject, the localization of the moiety/molecule can be detected
using positron emission transaxial tomography or magnetic resonance
imaging, respectively. Such methods can identify a primary tumor as
well as a metastatic lesion, which may not be detectable using
other methods. Having identified the presence of a cancer in a
subject, in another embodiment of the invention, the tumor homing
molecule is linked to a cytotoxic agent such as ricin or a cancer
chemotherapeutic agent such as doxorubicin in order to direct the
moiety to the tumor or can be linked to a chambered microdevice,
which can contain a chemotherapeutic drug or other cytotoxic agent.
Use of such a composition provides a means to selectively killing
of the tumor, while substantially sparing normal tissues in a
cancer patient and, therefore, the conjugates of the invention
provide useful medicaments for diagnosing or treating a cancer
patient.
[0114] The skilled artisan would recognize that various tumor
homing molecules can selectively home only to a tumor or can
selectively home to a tumor and to a family of selected organs,
including, in some cases, the normal tissue counterpart to the
tumor. Thus, the artisan would select a tumor homing peptide for
administration to a subject based on the procedure being performed.
For example, a tumor homing molecule that homes only to a tumor can
be useful for directing a therapy to the tumor. In comparison, a
tumor homing molecule that selectively homes not only to the tumor,
but also to one or more normal organs or tissues, can be used in an
imaging method, whereby homing to an organ or tissue other than the
tumor provides an internal imaging control. Such an internal
control can be useful, for example, for detecting a change in the
size of a tumor in response to a treatment, since the normal organ
is not expected to change in size and, therefore, can be compared
with the tumor size.
[0115] Tumor homing peptides, which are identified by in vivo
panning, can be synthesized in required quantities using routine
methods of solid state peptide synthesis or can be purchased from
commercial sources (for example, Anaspec; San Jose Calif.) and a
desired moiety can be linked to the molecule. Several methods
useful for linking a moiety to a molecule are known in the art,
depending on the particular chemical characteristics of the
molecule. For example, methods of linking haptens to carrier
proteins as used routinely in the field of applied immunology (see,
for example, Harlow and Lane, supra, 1988; Hermanson, supra,
1996).
[0116] It is recognized that, in some cases, a drug can lose
cytotoxic efficacy upon conjugation or derivatization depending,
for example, on the conjugation procedure or the chemical group
utilized (Hurwitz et al., Cancer Res. 35:1175-1181 (1975); Trail et
al., Science 261;212-215 (1993); Nagy et al., Proc. Natl. Acad.
Sci., USA 93:7269-7273 (1996)). Moreover, it is recognized that a
phage that yields a tumor homing peptide of the invention displays
as many as five of the peptides. Thus, there is a possibility that
the affinity of an individual peptide is too low for effective
tumor homing and that multivalent, rather than univalent, peptide
conjugates must be used. However, as disclosed herein, doxorubicin
maintained cytotoxic activity when used as a conjugate with tumor
homing peptides (see Example VII), thus allaying the potential
concerns discussed above.
[0117] A moiety such as a therapeutic or diagnostic agent can be
conjugated to a tumor homing peptide using, for example,
carbodiimide conjugation (Bauminger and Wilchek, Meth. Enzymol.
70:151-159 (1980), which is incorporated herein by reference).
Carbodiimides comprise a group of compounds that have the general
formula R--N.dbd.C.dbd.N--R', where R and R'0 can be aliphatic or
aromatic, and are used for synthesis of peptide bonds. The
preparative procedure is simple, relatively fast, and is carried
out under mild conditions. Carbodiimide compounds attack carboxylic
groups to change them into reactive sites for free amino groups.
Carbodiimide conjugation has been used to conjugate a variety of
compounds to carriers for the production of antibodies.
[0118] The water soluble carbodiimide,
1-ethyl-3-(3-dimethylaminopropyl)ca- rbodiimide (EDC) is
particularly useful for conjugating a moiety to a tumor homing
peptide and was used to conjugate doxorubicin to tumor homing
peptides (Example VI). The conjugation of doxorubicin and a tumor
homing peptide requires the presence of an amino group, which is
provided by doxorubicin, and a carboxyl group, which is provided by
the peptide. EDC coupling of doxorubicin to the CNGRC (SEQ ID NO:
8) peptide was performed using a 1:1 molar ratio of the peptide
(carboxyl groups) to obtain a doxorubicin/CNGRC (SEQ ID NO: 8; see
Example VI).
[0119] In addition to using carbodlimides for the direct formation
of peptide bonds, EDC also can be used to prepare active esters
such as N-hydroxysuccinimide (NHS) ester. The NHS ester, which
binds only to amino groups, then can be used to induce the
formation of an amide bond with the single amino group of the
doxorubicin. The use of EDC and NHS in combination is commonly used
for conjugation in order to increase yield of conjugate formation
(Bauminger and Wilchek, supra, 1980).
[0120] Other methods for conjugating a moiety to a tumor homing
molecule also can be used. For example, sodium periodate oxidation
followed by reductive alkylation of appropriate reactants can be
used, as can glutaraldehyde crosslinking. However, it is recognized
that, regardless of which method of producing a conjugate of the
invention is selected, a determination must be made that the tumor
homing molecule maintains its targeting ability and that the moiety
maintains its relevant function. Methods as disclosed in Example
VII or otherwise known in the art can confirm the activity of the
moiety/tumor homing molecule conjugate.
[0121] The yield of moiety/tumor homing molecule conjugate formed
is determined using routine methods. For example, HPLC or capillary
electrophoresis or other qualitative or quantitative method can be
used (see, for example, Liu et al., J. Chromatogr. 735:357-366
(1996); Rose et al., J. Chromatogr. 425:419-412 (1988), each of
which is incorporated herein by reference; see, also, Example V).
In particular, the skilled artisan will recognize that the choice
of a method for determining yield of a conjugation reaction
depends, in part, on the physical and chemical characteristics of
the specific moiety and tumor homing molecule. Following
conjugation, the reaction products are desalted to remove any free
peptide and free drug.
[0122] When administered to a subject, the tumor homing
molecule/moiety conjugate is administered as a pharmaceutical
composition containing, for example, the conjugate and a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are well known in the art and include, for example,
aqueous solutions such as water or physiologically buffered saline
or other solvents or vehicles such as glycols, glycerol, oils such
as olive oil or injectable organic esters.
[0123] A pharmaceutically acceptable carrier can contain
physiologically acceptable compounds that act, for example, to
stabilize or to increase the absorption of the conjugate. Such
physiologically acceptable compounds include, for example,
carbohydrates, such as glucose, sucrose or dextrans, antioxidants,
such as ascorbic acid or glutathione, chelating agents, low
molecular weight proteins or other stabilizers or excipients. One
skilled in the art would know that the choice of a pharmaceutically
acceptable carrier, including a physiologically acceptable
compound, depends, for example, on the route of administration of
the composition. The pharmaceutical composition also can contain an
agent such as a cancer therapeutic agent.
[0124] One skilled in the art would know that a pharmaceutical
composition containing a tumor homing molecule can be administered
to a subject by various routes including, for example, orally or
parenterally, such as intravenously. The composition can be
administered by injection or by intubation. The pharmaceutical
composition also can be a tumor homing molecule linked to liposomes
or other polymer matrices, which can have incorporated therein, for
example, a drug such as a chemotherapeutic agent (Gregoriadis,
Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla. 1984),
which is incorporated herein by reference). 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.
[0125] For the diagnostic or therapeutic methods disclosed herein,
an effective amount of the tumor homing molecule/moiety conjugate
must be administered to the subject. As used herein, the term
"effective amount" means the amount of the conjugate that produces
the desired effect. An effective amount often will depend on the
moiety linked to the tumor homing molecule. Thus, a lesser amount
of a radiolabeled molecule can be required for imaging as compared
to the amount of a drug/molecule conjugate administered for
therapeutic purposes. An effective amount of a particular
molecule/moiety for a specific purpose can be determined using
methods well known to those in the art.
[0126] The route of administration of a tumor homing molecule will
depend, in part, on the chemical structure of the molecule.
Peptides, for example, are not particularly useful when
administered orally because they can be degraded in the digestive
tract. However, methods for chemically modifying peptides to render
them less susceptible to degradation by endogenous proteases or
more absorbable through the alimentary tract are well known (see,
for example, Blondelle et al., supra, 1995; Ecker and Crooke,
supra, 1995; Goodman and Ro, supra, 1995). Such modifications can
be performed on peptides identified by in vivo panning. In
addition, methods for preparing libraries of peptidomimetics, which
can contain D-amino acids, other non-naturally occurring amino
acids, or chemically modified amino acids; or can be organic
molecules that mimic the structure of peptide; or can be peptoids
such as vinylogous peptoids, are known in the art and can be used
to identify molecules that home to a tumor and are stable for oral
administration.
[0127] Tumor homing molecules obtained using the methods disclosed
herein also can be useful for identifying a target molecule such as
a cell surface receptor or a ligand for a receptor, which is
recognized by the tumor homing peptide, or for substantially
isolating the target molecule. For example, a tumor homing peptide
can be linked to a solid support such as a chromatography matrix.
The linked peptide then can be used for affinity chromatography by
passing an appropriately processed sample of a tumor over the
column in order to bind a particular target molecule. The target
molecule, which forms a complex with the tumor homing molecule,
then can be eluted from the column and collected in a substantially
isolated form. The substantially isolated target molecule then can
be characterized using well known methods. A tumor homing peptide
also can be linked to a detectable moiety such as a radionuclide, a
fluorescent molecule, an enzyme or biotin and can be used, for
example, to screen a sample in order to detect the presence of the
target molecule in a tumor or to follow the target molecule during
various isolation steps.
[0128] It follows that, upon identifying the presence of a target
molecule in a tumor sample, the skilled artisan readily can obtain
the target molecule in a substantially isolated form. For example,
the sample containing the target molecule can be passed over a
column containing attached thereto the relevant tumor homing
molecule, thereby providing a means to obtain the target molecule
in substantially isolated form. Thus, the invention further
provides a substantially isolated target molecule, which
specifically binds a tumor homing molecule and which can be
obtained using the methods disclosed herein.
[0129] The methods of the present invention were used to identify
tumor homing peptides, which can selectively home to various
tumors. It should be recognized that cysteine residues were
included in some peptides such that cyclization of the peptides
could be effected. In fact, the peptides containing at least two
cysteine residues cyclize spontaneously. However, such cyclic
peptides also can be active when present in a linear form (see, for
example, Koivunen et al., supra, 1993) and, as disclosed herein, a
linear peptide, NGRAHA (SEQ ID NO: 6), also was useful as tumor
homing molecule (Example VII; see, also, Table 1). Thus, in some
cases one or more cysteine residues in the peptides disclosed
herein or otherwise identified as tumor homing peptides can be
deleted without significantly affecting the tumor homing activity
of the peptide. Methods for determining the necessity of a cysteine
residue or of amino acid residues N-terminal or C-terminal to a
cysteine residue for tumor homing activity of a peptide of the
invention are routine and well known in the art.
[0130] A tumor homing peptide is useful, for example, for targeting
a desired moiety to the selected tumor as discussed above. In
addition, a tumor homing peptide can be used to identify the
presence of a target molecule in a sample. As used herein, the term
"sample" is used in its broadest sense to mean a cell, tissue,
organ or portion thereof, including a tumor, that is isolated from
the body. A sample can be, for example, a histologic section or a
specimen obtained by biopsy or cells that are placed in or adapted
to tissue culture. If desired, a sample can be processed, for
example, by homogenization, which can be an initial step for
isolating the target molecule to which a tumor homing molecule
binds.
[0131] A tumor homing peptide such as a breast tumor homing peptide
can be used to identify the target molecule expressed in a breast
tumor. For example, a breast tumor homing peptide can be attached
to a matrix such as a chromatography matrix to produce a peptide
affinity matrix. A homogenized sample of a breast tumor can be
applied to the peptide-affinity matrix under conditions that allow
specific binding of the target molecule to the tumor homing peptide
(see, for example, Deutshcer, Meth. Enzymol., Guide to Protein
Purification (Academic Press, Inc., ed. M. P. Deutscher, 1990),
Vol. 182, which is incorporated herein by reference; see, for
example, pages 357-379). Unbound and nonspecifically bound material
can be removed and the specifically bound breast tumor-derived
target molecule can be isolated in substantially purified form. The
presence or absence of the target molecule in normal breast tissue
also can be determined. Such an analysis can provide insight into
methods of treating the tumor.
[0132] As disclosed herein, a target molecule, which specifically
binds a tumor homing molecule, can be identified by contacting a
sample of a tumor with such a tumor homing molecule and identifying
a target molecule bound by the tumor homing molecule. In parallel,
the tumor homing molecule is contacted with a sample of a nontumor
tissue corresponding to the tumor. The presence of the target
molecule in the tumor sample can be identified by determining that
the tumor homing molecule does not bind to a component of the
corresponding nontumor tissue sample. Thus, the invention provides
methods for identifying the presence of a target molecule, which is
expressed in a tumor and specifically bound by a tumor homing
molecule.
[0133] Since numerous tumor homing peptides containing the NGR
motif have been identified, for example, a tumor homing peptide
comprising an NGR sequence can be used to isolate the NGR receptor.
Thus, an NGR tumor homing peptide can be linked to a solid matrix
and an appropriately processed sample of a tumor, which
specifically binds the NGR peptide, can be passed over the NGR
peptide-matrix. The NGR receptor, which is the target molecule for
the NGR tumor homing peptide, then can be obtained in a
substantially isolated form. When used in reference to a target
molecule, the term "substantially isolated" means that the target
molecule comprises at least 30% of the total protein present,
although the target molecule can comprise at least 50% of the total
protein, or 80% of the total protein, or 90% or 95% of the total
protein, or more. A method such as gel electrophoresis and silver
staining can be used to determine the relative amount of a target
molecule in a sample, following a purification protocol, and,
therefore, can be used to identify a substantially isolated target
molecule.
[0134] The skilled artisan will recognize that a substantially
isolated target molecule can be used as an immunogen to obtain
antibodies that specifically bind the target molecule. As used
herein, the term "antibody" is used in its broadest sense to
include polyclonal and monoclonal antibodies, as well as antigen
binding fragments of such antibodies. With regard to an antibody of
the invention, which specifically binds a target molecule targeted
by a tumor homing molecule, the term "antigen" means the target
molecule polypeptide or peptide portion thereof. An antibody or
antigen binding fragment of an antibody that binds a target
molecule is characterized by having specific binding activity for
the target molecule or a peptide portion thereof of at least about
1.times.10.sup.5 M.sup.-1, preferably at least about
1.times.10.sup.6 M.sup.-1, and more preferably at least about
1.times.10.sup.8 M.sup.-1. Thus, Fab, F(ab').sub.2, Fd and Fv
fragments of the antibody, which retain specific binding activity
for a target molecule, which is expressed by angiogenic
vasculature, are included within the definition of an antibody.
[0135] In addition, the term "antibody" as used herein includes
naturally occurring antibodies as well as non-naturally occurring
antibodies, including, for example, single chain antibodies,
chimeric, bifunctional and humanized antibodies, as well as
antigen-binding fragments thereof. Such non-naturally occurring
antibodies can be constructed using solid phase peptide synthesis,
can be produced recombinantly or can be obtained, for example, by
screening combinatorial libraries consisting of variable heavy
chains and variable light chains as described by Huse et al.,
Science 246:1275-1281 (1989), which is incorporated herein by
reference. These and other methods of making, for example,
chimeric, humanized, CDR-grafted, single chain, and bifunctional
antibodies are well known to those skilled in the art (Winter and
Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature
341:544-546 (1989); Hilyard et al., Protein Engineering: A
practical approach (IRL Press 1992); Borrabeck, Antibody
Engineering, 2d ed. (Oxford University Press 1995); each of which
is incorporated herein by reference; see, also, Harlow and Lane,
supra, 1988).
[0136] Antibodies that specifically bind a target molecule of the
invention can be raised using as an immunogen a substantially
isolated target molecule, which can be obtained as disclosed
herein, or a peptide portion of the target molecule, which can be
obtained, for example, by enzymatic degradation of the target
molecule and gel purification. A non-immunogenic peptide portion of
a target molecule can be made immunogenic by coupling the hapten to
a carrier molecule such as bovine serum albumin (BSA) or keyhole
limpet hemocyanin (KLH). Various other carrier molecules and
methods for coupling a hapten to a carrier molecule are well known
in the art and described, for example, by Harlow and Lane, supra,
1988).
[0137] Particularly useful antibodies of the invention are those
that bind to the tumor homing molecule binding site on the target
molecule, such antibodies being readily identifiable by detecting
competitive inhibition of binding of the antibody and the
particular tumor homing molecule that binds to the target molecule.
Conversely, antibodies that bind to an epitope of the target
molecule that is not involved in binding the tumor homing molecule
also are valuable, since such antibodies, which, themselves, can be
"tumor homing molecules," can be bind to target molecules having
another tumor homing molecule bound thereto.
[0138] An antibody that specifically binds a target molecule, for
example, the NGR receptor, is useful for determining the presence
or level of the target molecule in a tissue sample, which can be a
lysate or a histological section. The identification of the
presence or level of the target molecule in the sample can be made
using well known immunoassay and immunohistochemical methods
(Harlow and Lane, supra, 1988). An antibody specific for a target
molecule also can be used to substantially isolate the target
molecule from a sample. In addition, an antibody of the invention
can be used in a screening assay to identify, for example,
peptidomimetics of a tumor homing molecule that bind to the target
molecule or as a tool for tumor targeting.
[0139] Upon obtaining a target molecule, which, due to the nature
of a tumor homing molecule, is expressed in angiogenic vasculature,
for example, the angiogenic vasculature in a tumor, the naturally
occurring ligand for the target molecule, where it exists, can be
identified. Methods for identifying a ligand for such a target
molecule, which is akin to an "orphan receptor," are well known in
the art and include, for example, screening biological samples to
identify the ligand. A convenient screening assay to identify a
natural ligand for the target molecule can utilize the ability of a
putative natural ligand to competitively inhibit the binding to the
target molecule of a tumor homing molecule that specifically binds
the target molecule, for example, the tumor homing peptide used to
obtain the substantially isolated target molecule.
[0140] A screening assay comprising a competitive binding assay for
the target molecule and, for example, the natural ligand for the
target molecule or a tumor homing peptide that specifically binds
the target molecule, also provides a means to identify
peptidomimetics of a tumor homing molecule. As discussed above,
such peptidomimetics can provide advantages over tumor homing
peptides in that they can be small, relatively stable for storage,
conveniently produced in suitable quantities, and capable of being
administered orally. A peptidomimetic of a tumor homing peptide can
be identified by screening libraries of peptidomimetics in a
competitive binding assay as described above.
[0141] The disclosed in vivo panning method can be used to detect
four different kinds of target molecules in tumors. First, because
tumor vasculature undergoes active angiogenesis, target molecules
that are characteristic of angiogenic vasculature, in general, or
angiogenic tumor vasculature, in particular, can be identified.
Second, vascular target molecules that are characteristic of the
tissue of origin of the tumor can be identified. Third, target
molecules that are expressed in the vasculature of a particular
type of tumor can be identified. Fourth, tumor stroma or tumor cell
target molecules can be identified due to the fenestrated nature of
tumor vasculature, which allows the potential tumor homing
molecules to leave the circulation and contact the tumor
parenchyma.
[0142] As further disclosed herein, some, but not all, tumor homing
molecules also can home to angiogenic vasculature that is not
contained within a tumor. For example, tumor homing molecules
containing either the RGD motif or the GSL motif specifically homed
to retinal neovasculature (Smith et al., Invest. Ophthamol. Vis.
Sci. 35:101-111 (1994), which is incorporated herein by reference),
whereas tumor homing peptides containing the NGR motif did not
accumulate substantially to this angiogenic vasculature. Thus, the
present invention also provides peptides that home to nontumor
angiogenic vasculature. Furthermore, these results indicate that
tumor vasculature expressing target molecules that are not
substantially expressed by other kinds of angiogenic vasculature.
Thus, the present invention provides a means to identify target
molecules expressed specifically by angiogenic vasculature present
in a tumor, as well as for target molecules expressed by angiogenic
vasculature not associated with a tumor. Methods as disclosed
herein can be used to distinguish such homing peptides and to
isolate the various target molecules.
[0143] As an alternative to using a tumor sample to obtain the
target molecule, extracts of cultured tumor cells or endothelial
cells, depending on which cell type expresses the target molecule,
can be used as the starting material in order to enhance the
concentration of the target molecule in the sample. It is
recognized, however, that the characteristics of such cells can
change upon adaptation to tissue culture. Thus, care must be
exercised if such a preselection step is attempted. The presence of
the target molecule can be established, for example, by using phage
binding and cell attachment assays (see, for example, Barry et al.,
supra, 1996).
[0144] A cell line expressing a particular target molecule can be
identified and surface iodination of the cells can be used to label
the target molecule. The cells then can be extracted, for example,
with octylglucoside and the extract can be fractionated by affinity
chromatography using a tumor homing peptide (see Tables 1 and 2)
coupled to a matrix such as SEPHAROSE (see Hermanson, supra, 1996).
The purified target molecule can be microsequenced and antibodies
can be prepared. If desired, oligonucleotide probes can be prepared
and used to isolate cDNA clones encoding the target molecule.
Alternatively, an anti-target molecule antibody can be used to
isolate a cDNA clone from an expression library (see Argraves et
al., J. Cell Biol. 105:1183-1190 (1987), which is incorporated
herein by reference).
[0145] As an alternative to isolating the target molecule, a
nucleic acid encoding the target molecule can be isolated using a
mammalian cell expression cloning system such as the COS cell
system. An appropriate library can be prepared, for example, using
mRNA from primary tumor cells. The nucleic acids can be cloned into
the pcDNAIII vector (Invitrogen), for example. Cells expressing a
cDNA for the target molecule can be selected by binding to the
tumor homing peptide. Purified phage can be used as the carrier of
the peptide and can be attached to magnetic beads coated, for
example, with anti-M13 antibodies (Pharmacia Biotech; Piscataway
N.J.). Cells that bind to the peptide coating can be recovered
using a magnet and the plasmids can be isolated. The recovered
plasmid preparations then can be divided into pools and examined in
COS cell transfections. The procedure can be repeated until single
plasmids are obtained that can confer upon the COS cells the
ability to bind the tumor homing peptide.
[0146] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
In Vivo Panning
[0147] This example demonstrates methods for preparing a phage
library and screening the library using in vivo panning to identify
phage expressing peptides that home to a tumor.
[0148] A. Preparation of Phage Libraries
[0149] Phage display libraries were constructed using the fuse 5
vector as described by Koivunen et al. (supra, 1995; Koivunen et
al., supra, 1994b). Libraries encoding peptides designated
CX.sub.5C (SEQ ID NO: 9), CX.sub.6C (SEQ ID NO: 10), CX.sub.7C (SEQ
ID NO: 11) and CX.sub.3CX.sub.3CX.sub.3C (SEQ ID NO: 12) were
prepared, where "C" indicates cysteine and "X.sub.N" indicates the
given number of individually selected amino acids. These libraries
can display cyclic peptides when at least two cysteine residues are
present in the peptide. In addition, a library that did not contain
defined cysteine residues also was constructed. Such a library
results in the production primarily of linear peptides, although
cyclic peptides also can occur due to random probability.
[0150] A biased library based on the sequence CXXXNGRXX (SEQ ID NO:
13) also was constructed. Furthermore, in some cases the CXXXNGRXX
(SEQ ID NO: 13) library was further biased by in the incorporation
of cysteine residues flanking the NGR sequence, i.e., CXXCNGRCX
(SEQ ID NO: 14; see Table 1).
[0151] The libraries containing the defined cysteine residues were
generated using oligonucleotides constructed such that "C" was
encoded by the codon TGT and "X.sub.N" was encoded by NNK, where
"N" is equal molar mixtures of A, C, G and T, and where "K" is
equal molar mixtures of G and T. Thus, the peptide represented by
CX.sub.5C (SEQ ID NO: 9) can be represented by an oligonucleotide
having the sequence TGT(NNK).sub.5TGT (SEQ ID NO: 14).
Oligonucleotides were made double stranded by 3 cycles of PCR
amplification, purified and ligated to the nucleic acid encoding
the gene III protein in the fuse 5 vector such that, upon
expression, the peptide is present as a fusion protein at the
N-terminus of the gene III protein.
[0152] The vectors were transfected by electroporation into MC1061
cells. Bacteria were cultured for 24 hr in the presence of 20
.mu.g/ml tetracycline, then phage were collected from the
supernatant by precipitation twice using polyethylene glycol. Each
library contained about 5.times.10.sup.9 to 5.times.10.sup.14
transducing units (TU; individual recombinant phage).
[0153] B. In Vivo Panning of Phage
[0154] Tumors were transplanted into mice as described in Examples
II and III, below. A mixture of phage libraries containing
1.times.10.sup.9 to 1.times.10.sup.14 TU was diluted in 200 .mu.l
DMEM and injected into the tail vein of anesthetized mice (AVERTIN
(0.015 ml/g); see U.S. Pat. No. 5,622,699; Pasqualini and
Ruoslahti, supra, 1996). After 1-4 minutes, mice were snap frozen
in liquid nitrogen. To recover the phage, carcasses were partially
thawed at room temperature for 1 hr, tumors and control organs were
collected and weighed, then were ground in 1 ml DMEM-PI (DMEM
containing protease inhibitors (PI); phenylmethylsulfonyl fluoride
(PMSF; 1 mM), aprotinin (20 .mu.g/ml), leupeptin (1 .mu.g/ml)).
[0155] Alternatively, following introduction of a library into a
mouse, circulation of the library is terminated by perfusion
through the heart. Briefly, mice were anesthetized with AVERTIN,
then the heart was exposed and a 0.4 mm needle connected through a
0.5 mm cannula to a 10 cc syringe was inserted into the left
ventricle. An incision was made on the right atrium and 5 to 10 ml
of DMEM was slowly administered, perfusing the whole body over
about a 5 to 10 min period. Efficiency of the perfusion was
monitored directly by histologic analysis.
[0156] Tumor and organ samples were washed 3 times with ice cold
DMEM-PI containing 1% bovine serum albumin (BSA), then directly
incubated with 1 ml K91-kan bacteria for 1 hr. Ten ml NZY medium
containing 0.2 .mu.g/ml tetracycline (NZY/tet) was added to the
bacterial culture, the mixture was incubated in a 37.degree. C.
shaker for 1 hr, then 10 .mu.l or 100 .mu.l aliquots were plated in
agar plates containing 12.5 .mu.g/ml tetracycline (tet/agar).
[0157] Individual colonies containing phage recovered from a tumor
were grown for 16 hr in 5 ml NZY/tet. The bacterial cultures
obtained from the individual colonies were pooled and the phage
were purified and re-injected into mice as described above for a
second round of in vivo panning. In general, a third round of
panning also was performed. Phage DNA was purified from individual
bacterial colonies obtained from the final round of in vivo panning
and the DNA sequences encoding the peptides expressed by selected
phage were determined (see Koivunen et al., supra, 1994b).
EXAMPLE II
Identification of Tumor Homing Peptides By In Vivo Panning against
a Breast Tumor
[0158] This example demonstrates that in vivo panning can be
performed against a breast tumor to identify tumor homing peptides
that home to various tumors.
[0159] Human 435 breast carcinoma cells (Price et al., Cancer Res.
50:717-721 (1990)) were inoculated into the mammary fat pad of nude
mice. When the tumors attained a diameter of about 1 cm, either a
phage targeting experiment was performed, in which phage expressing
a specific peptide were administered to the tumor bearing mouse, or
in vivo panning was performed.
[0160] The breast tumor bearing mice were injected with
1.times.10.sup.9 phage expressing a library of
CX.sub.3CX.sub.3CX.sub.3C (SEQ ID NO: 12) peptides, where X.sub.3
indicates three groups of independently selected, random amino
acids. The phage were allowed to circulate for 4 min, then the mice
were anesthetized, snap frozen in liquid nitrogen while under
anesthesia, and the tumor was removed. Phage were isolated from the
tumor and subjected to two additional rounds of in vivo
panning.
[0161] Following the third round of panning, phage were quantitated
and the peptide sequences expressed by the cloned phage were
determined. The cloned phage expressed various different peptides,
including those shown in Table 1. Similarly, CX.sub.7C (SEQ ID NO:
11) and CX.sub.5C (SEQ ID NO: 9) libraries were screened and breast
tumor homing peptides were identified (Table 1). These results
demonstrate that in vivo panning against a breast tumor can
identify tumor homing molecules.
EXAMPLE III
In Vivo Targeting of a Phage Expressing an an RGD Peptode to a
Tumor
[0162] Human 435 breast carcinoma cells were inoculated into the
mammary fat pad of nude mice. When the tumors attained a diameter
of about 1 cm, phage expressing a specific RGD-containing peptide
were administered to the tumor bearing mouse. Similar results
1TABLE 1 PEPTIDES FROM PHAGE RECOVERED FROM HUMAN BREAST CANCER
CGRECPRLCQSSC (2*) CNGRCVSGCAGRC (3) CGEACGGQCALPC (20) IWSGYGVYW
(21) PSCAYMCIT (22) WESLYFPRE (23) SKVLYYNWE (24) CGLMCQGACFDVC
(25) CERACRNLCREGC (26) CPRGCLAVCVSQC (27) CKVCNGRCCG (28)
CEMCNGRCMG (29) CPLCNGRCAL (30) CPTCNGRCVR (31) CGVCNGRCGL (32)
CEQCNGRCGQ (33) CRNCNGRCEG (34) CVLCNGRCWS (35) CVTCNGRCRV (36)
CTECNGRCQL (37) CRTCNGRCLE (38) CETCNGRCVG (39) CAVCNGRCGF (40)
CRDLNGRKVM (41) CSCCNGRCGD (42) CWGCNGRCRM (43) CPLCNGRCAR (44)
CKSCNGRCLA (45) CVPCNGRCHE (46) CQSCNGRCVR (47) CRTCNGRCQV (48)
CVQCNGRCAL (49) CRCCNGRCSP (50) CASNNGRVVL (51) CGRCNGRCLL (52)
CWLCNGRCGR (53) CSKCNGRCGH (54) CVWCNGRCGL (55) CIRCNGRCSV (56)
CGECNGRCVE (57) CEGVNGRRLR (58) CLSCNGRCPS (59) CEVCNGRCAL (60)
CGSLVRC (5) GRSQMQI (61) HHTRFVS (62) SKGLRHR (63) VASVSVA (64)
WRVLAAF (65) KMGPKVW (66) IFSGSRE (67) SPGSWTW (68) NPRWFWD (69)
GRWYKWA (70) IKARASP (71) SGWCYRC (72) ALVGLMR (73) LWAEMTG (74)
CWSGVDC (75) DTLRLRI (76) SKSSGVS (77) IVADYQR (78) VWRTGHL (79)
VVDRFPD (80) LSMFTRP (81) GLPVKWS (82) IMYPGWL (83) CVMVRDGDC (84)
CVRIRPC (85) CQLAAVC (86) CGVGSSC (87) CVSGPRC (88) CGLSDSC (89)
CGEGHPC (90) CYTADPC (91) CELSLISKC (92) CPEHRSLVC (93) CLVVHEAAC
(94) CYVELHC (95) CWRKFYC (96) CFWPNRC (97) CYSYFLAC (98) CPRGSRC
(99) CRLGIAC (100) CDDSWKC (101) CAQLLQVSC (102) CYPADPC (103)
CKALSQAC (104) CTDYVRC (105) CGETMRC (106) *numbers in parentheses
indicate SEQ ID NO:.
[0163] to those discussed below also were obtained with nude mice
bearing tumors formed by implantation of human melanoma C8161 cells
or by implantation of mouse B16 melanoma cells.
[0164] 1.times.10.sup.9 phage expressing the RGD-containing
peptide, CDCRGDCFC (SEQ ID NO: 1; see, Koivunen et al., supra,
1995) or control (insertless) phage were injected intravenously
(iv) into the mice and allowed to circulate for 4 min. The mice
then were snap frozen or perfused through the heart while under
anesthesia, and various organs, including tumor, brain and kidney,
were removed and the phage present in the organs was quantitated
(see U.S. Pat. No. 5,622,699; Pasqualini and Ruoslahti, supra,
1996).
[0165] Approximately 2-3 times more phage expressing the CDCRGDCFC
(SEQ ID NO: 1) peptide were detected in the breast tumor as
compared to brain and kidney, indicating the CDCRGDCFC (SEQ ID NO:
1; RGD phage) peptide resulted in selective homing of the phage to
the breast tumor. In a parallel study, unselected phage, which
express various, diverse peptides, were injected into tumor-bearing
mice and various organs were examined for the presence of phage.
Far more phage were present in kidney and, to a lesser extent,
brain, as compared to the tumor. Thus, the 80-fold more
RGD-expressing phage than unselected phage concentrated in the
tumor. These results indicate that phage expressing the
RGD-containing peptide home to a tumor, possibly due to the
expression of the .alpha..sub.v.beta..sub.3 integrin on blood
vessels forming in the tumor.
[0166] Specificity of the breast tumor homing peptide was
demonstrated by competition experiments, in which coinjection of
500 .mu.g free peptide, ACDCRGDCFCG (SEQ ID NO: 16; see Pasqualini
et al., supra, 1997) with the phage expressing the tumor homing
peptide reduced the amount of phage in the tumor by about tenfold,
whereas coinjection with the inactive control peptide, GRGESP (SEQ
ID NO: 17) essentially had no effect. These results demonstrate
that phage displaying a peptide that can bind to an integrin
expressed on angiogenic vasculature can selectively home in vivo to
an organ or tissue such as a tumor containing such vasculature.
EXAMPLE IV
Immunohistologic Analysis of Tumor Homing Peptides
[0167] This example provides a method of identifying the
localization of tumor homing molecules by immunohistologic
examination.
[0168] Localization of phage expressing a tumor homing peptide was
identified by immunochemical methods in histologic sections
obtained either 5 min or 24 hr after administration of phage
expressing a tumor homing peptide ("peptide-phage") to a tumor
bearing mouse (FIG. 1). For samples obtained 5 min following
administration of the peptide-phage, mice were perfused with DMEM
and various organs, including the tumor, were removed and fixed in
Bouin's solution. For samples obtained at 24 hr, no peptide-phage
remains in the circulation and, therefore, perfusion was not
required. Histologic sections were prepared and reacted with
anti-M13 (phage) antibodies (Pharmacia Biotech; see U.S. Pat. No.
5,622,699; Pasqualini and Ruoslahti, supra, 1996). Visualization of
the bound anti-M13 antibody was performed using a
peroxidase-conjugated second antibody (Sigma; St. Louis Mo.)
according to the manufacturer's instructions.
[0169] As discussed in Example III, phage expressing the tumor
homing peptide, CDCRGDCFC (SEQ ID NO: 1; "RGD phage), were
administered intravenously to mice bearing the breast tumor. In
addition, the RGD phage were administered to mice bearing a mouse
melanoma or a human Kaposi's sarcoma. Circulation of the phage was
terminated and mice were sacrificed as described above and samples
of the tumor and of skin adjacent to the tumor, brain, kidney, lung
and liver were collected. Immunohistochemical staining for the
phage showed accumulation of the RGD phage in the blood vessels
present in the breast tumor as well as in the melanoma and the
Kaposi's sarcoma, whereas little or no staining was observed in the
control organs.
[0170] Similar experiments were performed using phage expressing
the tumor homing peptide, CNGRCVSGCAGRC (SEQ ID NO: 3; "NGR
phage"), which was identified by in vivo panning against a tumor
formed by the MDA-MB-435 breast carcinoma. In these experiments,
NGR phage or control phage, which do not express a peptide, were
administered to mice bearing tumors formed by the MDA-MB-435 breast
carcinoma or by a human SLK Kaposi's sarcoma xenograft, then the
mice were sacrificed as described above and tumors were collected
as well as control organs, including brain, lymph node, kidney,
pancreas, uterus, mammary fat pad, lung, intestine, skin, skeletal
muscle, heart and epithelium of the renal calices, bladder and
ureter (see FIG. 1). Histological samples were prepared and
examined by immunostaining as described above.
[0171] In samples obtained from mice sacrificed 4 min after
administration of the NGR phage, immunostaining of the vasculature
of both the breast tumor (FIG. 1E) and the Kaposi's sarcoma (FIG.
1H) was observed. Very little or no staining was observed in the
endothelium of the these tumors in mice administered an insertless
control phage (FIGS. 1G and 1J, respectively). In the samples
obtained from mice sacrificed 24 hr after administration of the NGR
phage, staining of the tumor samples appeared to have spread
outside of the vessels, into the breast tumor parenchyma (FIGS. 1B
and 1F) and the Kaposi's sarcoma parenchyma (FIGS. 1D and 1I).
Again, little or no staining was observed in samples prepared from
these tumors in mice administered the insertless control phage
(FIGS. A and C, respectively). In addition, little or no staining
was observed in various control organs in mice administered the NGR
phage (FIGS. 1K to 1V).
[0172] In other experiments, similar results were obtained
following administration of phage expressing the NGR tumor homing
peptides, NGRAHA (SEQ ID NO: 61) or CVLNGRMEC (SEQ ID NO: 7), to
tumor bearing mice. Also, as discussed below, similar results were
obtained using phage expressing the GSL tumor homing peptide,
CLSGSLSC (SEQ ID NO: 4), which was identified by in vivo panning of
a melanoma (see Example V, below).
[0173] These results demonstrate that tumor homing peptides
selectively home to tumors, particularly to the vasculature in the
tumors and that tumor homing peptides identified, for example, by
in vivo panning against a breast carcinoma also selectively home to
other tumors, including Kaposi's sarcoma and melanoma. In addition,
these results demonstrate that immunohistochemical analysis
provides a convenient assay for identifying the localization of
phage expressing tumor homing peptides.
EXAMPLE V
Identification of Tumor Homing Peptides by In Vivo Panning against
a Melanoma Tumor
[0174] The general applicability of the in vivo panning method to
identify tumor homing peptides was examined by performing in vivo
panning against an implanted mouse melanoma tumor.
[0175] Mice bearing a melanoma were produced by implantation of
B16B15b mouse melanoma cells, which produce highly vascularized
tumors. B16B15b mouse melanoma cells were injected subcutaneously
into the mammary fat pad of nude mice (2 months old) and tumors
were allowed to grow until the diameter was about 1 cm. In vivo
panning was performed as disclosed above. Approximately
1.times.10.sup.12 transducing units of phage expressing the
CX.sub.5C (SEQ ID NO: 9), CX.sub.6C (SEQ ID NO: 10) or CX.sub.7C
(SEQ ID NO: 11) library were injected, iv, and allowed to circulate
for 4 min. Mice then were snap frozen in liquid nitrogen or
perfused through the heart while under anesthesia, tumor tissue and
brain (control organ) were removed, and phage were isolated as
described above. Three rounds of in vivo panning were
performed.
[0176] The amino acid sequences were determined for the inserts in
89 cloned phage recovered from the B16B15b tumors. The peptides
expressed by these phage were represented by two predominant
sequences, CLSGSLSC (SEQ ID NO: 4; 52% of the clones sequenced) and
WGTGLC (SEQ ID NO: 18; 25% of the clones; see Table 2). Reinfection
of phage expressing one of the selected peptides resulted in
approximately three-fold enrichment of phage homing to the tumor
relative to brain.
2TABLE 2 PEPTIDES FROM PHAGE RECOVERED FROM MOUSE B16B15b MELANOMA
CLSGSLSC (4*) GICKDDWCQ (107) TSCDPSLCE (108) KGCGTRQCW (109)
YRCREVLCQ (110) CWGTGLC (111) WSCADRTCM (112) AGCRLKSCA (113)
SRCKTGLCQ (114) PICEVSRCW (115) WTCRASWCS (116) GRCLLMQCR (117)
TECDMSRCM (118) ARCRVDPCV (119) CIEGVLGGC (120) CSVANSC (121)
CSSTMRC (122) SIDSTTF (123) GPSRVGG (124) WWSGLEA (125) LGTDVRQ
(126) LVGVRLL (127) GRPGDIW (128) TVWNPVG (129) GLLLVVP (130)
FAATSAE (131) WCCRQFN (132) VGFGKAL (133) DSSLRLP (134) KLWCAMS
(135) SLVSFLG (136) GSFAFLV (137) IASVRWA (138) TWGHLRA (139)
QYREGLV (140) QSADRSV (141) YMFWTSR (142) LVRRWYL (143) TARGSSR
(144) TTREKNL (145) PKWLLFS (146) LRTNVVH (147) AVMGLAA (148)
VRMSLRN (149) *numbers in parentheses indicate SEQ ID NO:.
[0177] Localization of the phage expressing a tumor homing peptide
in the mouse organs also was examined by immunohistochemical
staining of the tumor and various other tissues (see Example IV).
In these experiments, 1.times.10.sup.9 pfu of a control
(insertless) phage or a phage expressing the tumor homing peptide,
CLSGSLSC (SEQ ID NO: 4), were injected, iv, into tumor bearing mice
and allowed to circulate for 4 min.
[0178] Immunostaining was evident in the melanoma obtained from a
mouse injected with phage expressing the CLSGSLSC (SEQ ID NO: 4)
tumor homing peptide. Staining of the melanoma generally was
localized to the blood vessels within the tumor, although some
staining also was present in the tumor parenchyma. Essentially no
staining was observed in a tumor obtained from a mouse injected
with the insertless control phage or in skin or in kidney samples
obtained from mice injected with either phage. However,
immunostaining was detected in the liver sinusoids and in spleen,
indicating that phage can be trapped nonspecifically in organs
containing RES.
[0179] Using similar methods, in vivo panning was performed in mice
bearing a SLK human Kaposi's sarcoma. Tumor homing peptides were
identified and are disclosed in Table 3. Together, these results
demonstrate that the in vivo panniang method is a generally
applicable method for screening a phage library to identify phage
expressing tumor homing peptides.
EXAM4PLE VI
Preparation and Characterization of Tumor HOming
Peptide/Doxorubicin Conjugates
[0180] This example provides methods for conjugating a moiety such
as the chemotherapeutic agent, doxorubicin, to a tumor homing
peptide and for characterizing the conjugation reaction.
[0181] The peptides CDCRGDCFC (SEQ ID NO: 1; Koivunen et al.,
supra, 1995; Pasqualini et al., supra, 1997), CNGRC (SEQ ID NO: 8),
CGSLVRC (SEQ ID NO: 5) and GACVFSIAHECGA (SEQ ID NO: 19) were
synthesized, cyclized under high dilution and purified to
homogeneity by HPLC. Conjugation of the peptides to doxorubicin
(Aldrich; Milwaukee Wis.) was performed using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC;
Sigma; St. Louis Mo.) and N-hydroxysuccinimide (NHS;
3TABLE 3 PEPTIDES FROM PHAGE RECOVERED FROM HUMAN KAPOSI'S SARCOMA
TDCTPSRCT (150*) SWCQFEKCL (151) VPCRFKQCW (152) CTAMRNTDC (153)
CRESLKNC (154) CMEMGVKC (155) VTCRSLMCQ (156) CNNVGSYC (157)
CGTRVDHC (158) CISLDRSC (159) CAMUSMED (160) CYLGVSNC (161)
CYLVNVDC (162) CIRSAVSC (163) LVCLPPSCE (164) RHCFSQWCS (165)
FYCPGVGCR (166) ISCAVDACL (167) EACEMAGCL (168) PRCESQLCP (169)
RSCIKHQCP (170) QWCSRRWCT (171) MFCRMRSCD (172) GICKDLWCQ (173)
NACESAICG (174) APCGLLACI (175) NRCRGVSCT (176) FPCEGKKCL (177)
ADCRQKPCL (178) FGCVMASCR (179) AGCINGLCG (180) RSCAEPWCY (181)
DTCRALRCN (182) KGCGTRQCW (109) GRCVDGGCT (183) YRCIARECE (184)
KRCSSSLCA (185) ICLLAHCA (186) QACPMLLCM (187) LDCLSELCS (188)
AGCRVESC (189) HTCLVALCA (190) IYCPGQECE (191) RLCSLYGCV (192)
RKCEVPGCQ (193) EDCTSRFCS (194) LECVVDSCR (195) EICVDGLCV (196)
RWCREKSCW (197) FRCLERVCT (198) RPCGDQACE (199) CNKTDGDEGVTC (200)
*numbers in parentheses indicate SEQ ID NO:.
[0182] Sigma) as described (Bauminger and Wilchek, supra, 1980;
Harlow and Lane, supra, 1988; Hurwitz et al., supra, 1975).
Unreacted doxorubicin and peptide were removed from the
doxorubicin/peptide conjugates by SEPHADEX G25 column
chromatography using phosphate buffered saline. The conjugates were
lyophilized for storage and were resuspended in sterile water prior
to use.
[0183] HPLC, capillary electrophoresis and NMR analyses were
performed to characterize the conjugates. HPLC-fluorescence was
performed using an INTERSIL ODS-2 column (4.6.times.150 mm) and a
mobile phase composed of 0.08% triethanolamine/0.02% phosphoric
acid (85%)/27% acetonitrile at 1 ml/min. Fluorescence detection was
performed with excitation at 490 nm and emission at 560 nm
wavelength and the retention time (RT) and the area under the
curves (AUC) for doxorubicin (dox) and for the major peaks was
determined. Each of the conjugates has a unique retention time,
depending on the peptide, as follows: dox/CDCRGDCFC (SEQ ID NO: 1),
RT 7.4 min, AUC 26%; dox/CNGRC (SEQ ID NO: 8), RT 4.7 min, AUC 56%;
and dox/GACVFSIAHECGA (SEQ ID NO: 19), RT 7.7 min, AUC 43%. In
comparison, the retention time of doxorubicin is 10.6 min and, in
the various reactions, the AUC was about 5%.
[0184] Capillary electrophoresis (CE; Liu et al., supra, 1996) was
performed in uncoated fused-silica capillaries with 75 .mu.m
internal diameter and an effective separation length of 50 cm. The
CE detection system was equipped with an UV absorbance detector and
an argon laser emitting at 488 nm. The laser beam is transmitted
via a fiber optic cable to the detector and illuminates the
capillary window and the fluorescence signal is collected through
an emission filter. Conjugation of doxorubicin to the peptides
changed the electrophoretic characteristics of each of the
conjugates, indicating that this method can be used as a fast
screening method to identify progress of the conjugation
reaction.
[0185] One dimensional NMR analysis of the doxorubicin/CNGRC
conjugate revealed no evidence of resonances arising from free
doxorubicin. Two dimension NMR analysis can allow a determination
of the precise molecular structure of the doxorubicin-peptide
species.
[0186] These results demonstrate that a moiety such as the cancer
chemotherapeutic agent, doxorubicin, can be efficiently linked to
tumor homing peptides of the invention to produce doxorubicin/tumor
homing peptide conjugates.
EXAMPLE VII
Tumor Therapy Using Doxorubicin/Tumor Homing Peptide Conjugates
[0187] This example demonstrates that doxorubicin/tumor homing
peptide conjugates-provide a therapeutic advantage over the use of
doxorubicin, alone, for treating tumors.
[0188] Doxorubicin concentration of the conjugates (see Example V)
was determined by measuring the optical absorbance of the solution
at 490 nm in a standard spectrophotometer; this wavelength detects
only the doxorubicin, not the peptides. A calibration curve for
doxorubicin was generated and used to calculate the concentration
prior to use. Conjugation of doxorubicin to the various peptides
did not affect this curve. This procedure ensures that each of the
administered conjugates contained the same amounts of doxorubicin
equivalent.
[0189] In addition, the viability of tumor cells obtained from
tumors of mice treated with a tumor homing peptide/doxorubicin
conjugate was compared to that of tumors from mice treated with
free doxorubicin. In these experiments, breast tumor bearing mice
were size matched with regard to the tumors, then treated
intravenously with 30 .mu.g equivalent of doxorubicin/CDCRGDCFC
(SEQ ID NO: 1) or of free doxorubicin. Five days after treatment,
the mice were euthanized and the tumors were removed. The tumor
pairs were weighed and ground and the cell suspensions were plated
(2 g tumor tissue per 150 mm plate).
[0190] Cell numbers were determined at 24 hours and 7 days after
plating. Viability of tumor cells from the tumors of mice receiving
the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate was about 3 fold
less than cells from tumors of mice treated with the free
doxorubicin. These results demonstrate that administration to a
tumor bearing mouse of a conjugate comprising a chemotherapeutic
agent linked to a tumor homing molecule is more efficacious than
administration of the agent, alone, in reducing the viability of
tumor cells.
[0191] A. In Vitro Characterization of Cytotoxicity
[0192] MDA-MB-435 human breast carcinoma cells were plated at
1.times.10.sup.5 cells/well in 96 well plates. Cells were incubated
with increasing amounts of doxorubicin, the doxorubicin/CDCRGDCFC
(SEQ ID NO: 1) conjugate, or the doxorubicin/GACVFSIAHECGA (SEQ ID
NO: 19; control) conjugate (0.1 to 10 .mu.g/well of
doxorubicin-equivalent) for either 30 min or overnight. Following
incubation, the agents were removed by extensive washing with PBS,
then fresh medium added and incubation was continued. The number of
surviving cells was determined at 24 hours with crystal violet
staining (see Koivunen et al., supra, 1994).
[0193] In cells exposed to free doxorubicin, the
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate, or the
doxorubicin/GACVFSIAHECGA (SEQ ID NO: 19) for 30 min, cell death
was present only in the cultures treated with the
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate. However, if the
agents were not removed after 30 min, the cells were killed by all
of the treatments after 24 hr. These results indicate that enhanced
cellular uptake occurs for the doxorubicin/CDCRGDCFC (SEQ ID NO: 1)
conjugate.
[0194] B. In Vivo Characterization of Doxorubicin/tumor Homing
Peptide Conjugates
[0195] Female 2-month old Balb c nu/nu mice (Harlan Sprague Dawley;
San Diego Calif.) were used for these studies and were cared for
according to the Burnham Institute animal facility guidelines.
MDA-MB-435 breast carcinoma cells (Price et al., Cancer Res.
50:717-721 (1993)) were injected in the mammary fat pad of the nude
mice and tumor growth was monitored (Pasqualini et al., supra,
1997). Tumors were allowed to grow to a size of about 1 cm.sup.3
(about 5% of the mouse's body weight) before starting the treatment
experiments, except for the toxicity experiments as discussed
below.
[0196] Weekly doxorubicin/peptide conjugate or control treatments
(5 .mu.g/mouse/week of doxorubicin-equivalent) were administered
intravenously. In some experiments, as indicated, a dose of 30
.mu.g/mouse was administered every 3 weeks. Treatment with
doxorubicin, alone, is referred to as "dox control" and treatment
with doxorubicin conjugated to the non-tumor homing control
peptide, GACVFSIAHECGA (SEQ ID NO: 19), is referred to as
"conjugate control." The results obtained in the dox control groups
as compared to the conjugate control groups were not significantly
different. As an additional control, in some experiments the tumor
homing peptide was mixed with doxorubicin, without linking, and the
mixture was administered to tumor bearing mice. Such treatment
produced results that were not statistically different from those
obtained with the above described dox controls.
[0197] Mice were anesthetized with a tribromoethanol-based
anesthetic mixture (AVERTIN; Papaioannou and Fox, Lab. Anim.
43:189-192 (1993)) before each treatment. Anesthetization
facilitated the tail vein injections (final volume, 200 l) and
allowed precise serial three dimensional tumor size measurements.
Tumor volume calculations were based on the equation for the volume
of an ovaloid: V=4/3(.PI.abc), where a, b, and c are 1/2 of the
measured diameters in each of the three dimensions.
[0198] At necropsy, MDA-MB-435 tumor-bearing mice treated with the
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate had significantly
smaller tumors (t test, p=0.02), less spread to regional lymph
nodes (t test, p<0.0001), a lower incidence of pulmonary
metastasis and fewer metastatic lesions (t test, p<0.0001) than
the dox control treated mice. All of the mice treated with the
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate survived beyond the
time when the dox control and conjugate control mice had died
(Log-Rank test, p<0.0001; Wilcoxon test, p=0.0007). Essentially
the same results were obtained in five separate experiments. These
results indicate that a doxorubicin/tumor homing peptide provides a
therapeutic advantage over doxorubicin, alone, in reducing the
growth of a primary tumor and preventing metastasis of the
tumor.
[0199] Gross and histopathologic examination was performed on the
mice. Many of the tumors in the mice treated with 5 .mu.g
doxorubicin equivalent of doxorubicin/CDCRGDCFC (SEQ ID NO: 1)
presented marked skin ulceration and tumor necrosis, whereas no
such signs were observed in dox control group or conjugate control
group. Histopathological analysis disclosed a pronounced
destruction of the vasculature in the tumors treated with
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate (see FIGS. 2D to 2F)
as compared to the dox control group (FIGS. 2A to 2C).
[0200] In a dose escalation experiment, tumor bearing mice were
treated with the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) at 30
.mu.g/mouse every three weeks for three cycles and were observed,
without further treatment, for an extended period of time. The
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) treated mice all remained
alive more than 6 months after the dox control and conjugate
control mice had died. The results indicate that treatment with a
doxorubicin/tumor homing peptide conjugate can have a curative
effect.
[0201] Acute toxicity studies also were performed. In these
experiments, mice bearing extremely large tumors (about 25% of body
weight) were treated with 200 .mu.g/mouse doxorubicin or
doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate. All of the mice
treated with the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate
survived for longer than one week, whereas all of the dox control
mice had died within 48 hr of treatment. These results suggest that
accumulation of the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate
in the large tumors reduced the circulating level of the conjugated
doxorubicin, thus reducing its toxicity.
[0202] Similar results were obtained using the doxorubicin/CNGRC
(SEQ ID NO: 8) conjugate. In each of three series of experiments,
tumors in the mice treated with doxorubicin/CNGRC (SEQ ID NO: 8)
were significantly smaller than tumor is the dox control and
conjugate control groups. Treatment with the doxorubicin/CNGRC (SEQ
ID NO: 8) conjugate almost completely suppressed tumor growth,
whereas free doxorubicin and doxorubicin conjugated to the control
peptide had essentially no effect on tumor growth relative to
treatment with the vehicle, alone. A marked effect on survival also
was observed and some of the doxorubicin/CNGRC (SEQ ID NO: 8)
treated animals survived for extended periods of time (Log-Rank
test, p=0.0064; Wilcoxon test, p=0.0343). In addition, the
doxorubicin/CNGRC (SEQ ID NO: 8) conjugate was less toxic than free
doxorubicin. These results confirm that conjugates comprising a
chemotherapeutic agent and a tumor homing molecule provide a
therapeutic advantage in treating cancer.
[0203] Although the invention has been described with reference to
the disclosed examples, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
* * * * *