U.S. patent application number 17/240714 was filed with the patent office on 2021-08-26 for methods and compositions related to annexin 1-binding compounds.
The applicant listed for this patent is Hamamatsu University School of Medicine, Sanford-Burnham Medical Research Institute. Invention is credited to Michiko Fukuda, Naohira Kanayama, Kazuhiro Sugihara.
Application Number | 20210260202 17/240714 |
Document ID | / |
Family ID | 1000005579501 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210260202 |
Kind Code |
A1 |
Fukuda; Michiko ; et
al. |
August 26, 2021 |
Methods and Compositions Related to Annexin 1-Binding Compounds
Abstract
Compositions and methods related to annexin 1-binding compounds.
Also compositions comprising a moiety and a peptide comprising an
amino acid sequence that can bind to a carbohydrate receptor on a
cell. Isolated nucleic acids comprising a nucleic acid sequence
encoding a peptide comprising an amino acid sequence that can bind
to a carbohydrate receptor on a cell. Methods comprising
administering to a subject a composition comprising a moiety and a
peptide comprising an amino acid sequence that can bind to a
carbohydrate receptor on a cell. Methods of targeting a tumor cell
in a subject comprising administering to the subject a peptide
comprising an amino acid sequence that can bind to a carbohydrate
receptor on a cell. Methods of targeting a tumor cell in a subject
comprising administering to the subject a composition comprising a
moiety and a peptide comprising an amino acid sequence that can
bind to a carbohydrate receptor on a cell.
Inventors: |
Fukuda; Michiko; (La Jolla,
CA) ; Sugihara; Kazuhiro; (Higashi-ku, JP) ;
Kanayama; Naohira; (Higashi-ku, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanford-Burnham Medical Research Institute
Hamamatsu University School of Medicine |
La Jolla
Higashi-ku |
CA |
US
JP |
|
|
Family ID: |
1000005579501 |
Appl. No.: |
17/240714 |
Filed: |
April 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15478699 |
Apr 4, 2017 |
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17240714 |
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13515930 |
Jun 14, 2012 |
9610359 |
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PCT/US10/62072 |
Dec 23, 2010 |
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15478699 |
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61289833 |
Dec 23, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/64 20170801 |
International
Class: |
A61K 47/64 20060101
A61K047/64 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under NIH
grant P01CA071932 (MF and MNF), DoD Breast Cancer Research IDEA
grant DAMD17-02-1-0311 (MNF), and a Susan Komen Breast Cancer
Research grant BCTR0504175 (MNF). The government has certain rights
in this invention.
Claims
1. An isolated nucleic acid comprising a nucleic acid sequence
encoding a peptide and moiety wherein the peptide comprises an
amino acid sequence that can bind to a carbohydrate receptor on a
cell.
2. A method comprising administering to a subject a composition
comprising a nucleic acid sequence encoding a peptide and moiety
wherein the peptide comprises an amino acid sequence that can bind
to a carbohydrate receptor on a cell.
3. The method of claim 2, wherein the subject comprises a cell,
wherein the cell is a tumor cell.
4. The method of claim 2, wherein the subject is in need of
treatment, wherein the administration treats the subject.
5. The method of claim 4, wherein the subject has cancer, wherein
the administration treats the cancer of the subject.
6. A method of targeting a tumor cell in a subject, the method
comprising administering to the subject a composition comprising a
nucleic acid sequence encoding a peptide and moiety wherein the
peptide comprises an amino acid sequence that can bind to a
carbohydrate receptor on a cell.
7. A method comprising administering to the subject a composition
comprising a nucleic acid sequence encoding a peptide and moiety
wherein the peptide comprises an amino acid sequence that can bind
to a carbohydrate receptor on a cell and detecting the composition
in the subject.
8. The method of claim 7, wherein detecting the composition thereby
detects a tumor in the subject.
9. The method of claim 7, wherein detecting the composition thereby
diagnoses cancer in the subject.
10. The method of claim 7, wherein detecting the composition
comprises detecting the level, amount, concentration, or a
combination of binding of the composition to cancer tissue in the
subject, wherein the level, amount, concentration, or a combination
of binding of the composition to cancer tissue in the subject
indicates the prognosis of the cancer in the subject.
11. The method of claim 7, further comprising repeating the
administration and detection at a later time, wherein a change in
the level, amount, concentration, or a combination of binding of
the composition to cancer tissue in the subject indicates the
progress of the endometriosis in the subject.
12. The method of claim 7, further comprising repeating the
administration and detection following treatment, wherein a change
in the level, amount, concentration, or a combination of binding of
the composition to cancer tissue in the subject indicates the
progress the treatment of the cancer in the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/478,699 filed Apr. 4, 2017, which claims
benefit of U.S. National Stage Patent application Ser. No.
13/515,930 filed Jun. 14, 2012, now U.S. Pat. No. 9,610,359, which
claims the benefit of priority under 371 of International Patent
Application No. PCT/US10/62072 filed Dec. 23, 2010, which claims
the benefit of U.S. Provisional Application No. 61/289,833, filed
Dec. 23, 2009, all of which are herein incorporated by
reference.
REFERENCE TO SEQUENCE LISTING
[0003] The Sequence Listing submitted Dec. 23, 2010 as a text file
named
"24520_47_9001_2010_12_23_AMD_AFD_Sequence_Listing_Text_File.txt,"
created on Dec. 23, 2010, and having a size of 9357 bytes is hereby
incorporated by reference pursuant to 37 C.F.R. .sctn.
1.52(e)(5).
FIELD OF THE INVENTION
[0004] The present invention relates generally to the fields of
molecular medicine and cancer biology, and, more specifically, to
annexin 1-binding compounds that selectively home to tumor
vasculature.
BACKGROUND OF THE INVENTION
[0005] 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 loss of normal tissue. Chemotherapy, in
comparison, which generally is administered systemically, can cause
substantial damage to organs such as the bone marrow, mucosae, skin
and 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 often
occur when a cancer patient is treated intravenously with a
chemotherapeutic drug. Such undesirable side effects can limit the
amount of a drug that can be safely administered, thereby hampering
survival rate and impacting the quality of patient life.
BRIEF SUMMARY OF THE INVENTION
[0006] Disclosed are isolated peptides comprising an amino acid
sequence that can bind to a carbohydrate receptor on a cell. Also
disclosed are compositions comprising a moiety and a peptide
comprising an amino acid sequence that can bind to a carbohydrate
receptor on a cell. Also disclosed are isolated nucleic acids
comprising a nucleic acid sequence encoding a peptide comprising an
amino acid sequence that can bind to a carbohydrate receptor on a
cell.
[0007] Also disclosed are methods comprising administering to a
subject a composition comprising a moiety and a peptide comprising
an amino acid sequence that can bind to a carbohydrate receptor on
a cell. Also disclosed are methods of targeting a tumor cell in a
subject comprising administering to the subject a peptide
comprising an amino acid sequence that can bind to a carbohydrate
receptor on a cell. Also disclosed are methods of targeting a tumor
cell in a subject comprising administering to the subject a
composition comprising a moiety and a peptide comprising an amino
acid sequence that can bind to a carbohydrate receptor on a cell.
Also disclosed are methods comprising administering to the subject
a composition comprising a peptide comprising an amino acid
sequence that can bind to a carbohydrate receptor on a cell and
detecting the composition in the subject.
[0008] The carbohydrate receptor can be annexin 1. The amino acid
sequence can selectively bind the carbohydrate receptor. The
subject can comprise a cell. The cell can be a tumor cell. The
peptide can be an annexin 1-binding compound. The amino acid
sequence can be an annexin 1-binding compound.
[0009] The amino acid sequence can comprise SEQ ID NO:2 having one
or more conservative amino acid substitutions. The amino acid
sequence can have at least 55% sequence identity to SEQ ID NO:2,
wherein differences between the amino acid sequence and SEQ ID NO:2
consist of conservative amino acid substitutions. The amino acid
sequence can have at least 70% sequence identity to SEQ ID NO:2.
The amino acid sequence can have at least 80% sequence identity to
SEQ ID NO:2. The amino acid sequence can comprise SEQ ID NO:2. The
amino acid sequence can consist of SEQ ID NO:2. The amino acid
sequence can comprise at least 5 consecutive amino acids of SEQ ID
NO:2. The amino acid sequence can comprise at least 6 consecutive
amino acids of SEQ ID NO:2.
[0010] The peptide can comprise SEQ ID NO:2. The peptide can
comprise at least 6 amino acids. The peptide can comprise at least
7 amino acids. The peptide can comprise at least 8 amino acids. The
peptide can comprise at least 9 amino acids. The peptide can
further comprise a moiety peptide.
[0011] The moiety can be a small molecule, pharmaceutical drug,
toxin, fatty acid, detectable marker, conjugating tag, nanoshell,
or enzyme. The moiety can be covalently linked to the peptide. The
moiety can be linked to the amino terminal end of the peptide. The
moiety can be linked to the carboxy terminal end of the peptide.
The moiety can be linked to an amino acid within the peptide. The
moiety can be SN-38. The moiety can comprise a detectable agent.
The moiety can comprise a therapeutic agent.
[0012] The composition can further comprise a linker connecting the
moiety and the peptide. The composition can further comprise a
pharmaceutically acceptable carrier. The composition can further
comprise a detectable agent. The composition can further comprise a
therapeutic agent. The composition can further comprise an
anti-cancer agent.
[0013] Detecting the composition in the method can thereby detect a
tumor in the subject. Detecting the composition in the method can
thereby diagnose cancer in the subject. Detecting the composition
in the method can comprise detecting the level, amount,
concentration, or a combination of binding of the composition to
cancer tissue in the subject, wherein the level, amount,
concentration, or a combination of binding of the composition to
cancer tissue in the subject indicates the prognosis of the cancer
in the subject. The method can further comprise repeating the
administration and detection at a later time, wherein a change in
the level, amount, concentration, or a combination of binding of
the composition to cancer tissue in the subject indicates the
progress of the endometriosis in the subject. The method can
further comprise repeating the administration and detection
following treatment, wherein a change in the level, amount,
concentration, or a combination of binding of the composition to
cancer tissue in the subject indicates the progress the treatment
of the cancer in the subject.
[0014] The peptides selectively bind to tumor vasculature. The
amino acid sequences selectively bind to tumor vasculature. The
peptides can comprise a plurality of amino acid sequences, wherein
the amino acid sequences selectively bind to tumor vasculature. The
peptides can bind to tumor vasculature. The amino acid sequences
can bind to tumor vasculature. The peptides can comprise a
plurality of amino acid sequences, wherein the amino acid sequences
can bind to tumor vasculature.
[0015] The peptides selectively bind to a carbohydrate receptor on
a cell. The amino acid sequences selectively bind to a carbohydrate
receptor on a cell. The peptides can comprise a plurality of amino
acid sequences, wherein the amino acid sequences selectively bind
to a carbohydrate receptor on a cell. The peptides can bind to a
carbohydrate receptor on a cell. The amino acid sequences can bind
to a carbohydrate receptor on a cell. The peptides can comprise a
plurality of amino acid sequences, wherein the amino acid sequences
can bind to a carbohydrate receptor on a cell.
[0016] The peptides selectively bind to a carbohydrate receptor on
a cell. The amino acid sequences selectively bind to annexin 1 on a
cell. The peptides can comprise a plurality of amino acid
sequences, wherein the amino acid sequences selectively bind to
annexin 1 on a cell. The peptides can bind to a carbohydrate
receptor on a cell. The amino acid sequences can bind to annexin 1
on a cell. The peptides can comprise a plurality of amino acid
sequences, wherein the amino acid sequences can bind to annexin 1
on a cell.
[0017] The peptides selectively bind to a carbohydrate receptor on
a cell. The amino acid sequences selectively bind to annexin 1 on
tumor vasculature. The peptides can comprise a plurality of amino
acid sequences, wherein the amino acid sequences selectively bind
to annexin 1 on tumor vasculature. The peptides can bind to a
carbohydrate receptor on tumor vasculature. The amino acid
sequences can bind to annexin 1 on tumor vasculature. The peptides
can comprise a plurality of amino acid sequences, wherein the amino
acid sequences can bind to annexin 1 on tumor vasculature.
[0018] The peptides selectively bind to a carbohydrate receptor on
a cell. The amino acid sequences selectively bind to annexin 1 on
tumor vasculature. The peptides can comprise a plurality of amino
acid sequences, wherein the amino acid sequences selectively bind
to annexin 1 on tumor vasculature. The peptides can bind to a
carbohydrate receptor on tumor vasculature. The amino acid
sequences can bind to annexin 1 on tumor vasculature. The peptides
can comprise a plurality of amino acid sequences, wherein the amino
acid sequences can bind to annexin 1 on tumor vasculature.
[0019] The composition can comprise a plurality of peptides,
wherein the peptides selectively bind to tumor vasculature. The
peptide can comprise a plurality of amino acid sequences, wherein
the amino acid sequences selectively bind to tumor vasculature. The
composition can comprise a plurality of amino acid sequences,
wherein the amino acid sequences selectively bind to tumor
vasculature. The composition can comprise a plurality of peptides,
wherein at least one of the peptides comprises an amino acid
sequence that selectively binds to tumor vasculature. The
composition can comprise a plurality of peptides, wherein a
plurality of the peptides each comprises an amino acid sequence
that selectively binds to tumor vasculature. The composition can
comprise a plurality of peptides, wherein the peptides each
comprise an amino acid sequence that selectively binds to tumor
vasculature.
[0020] The composition can comprise a plurality of peptides,
wherein the peptides selectively bind to a carbohydrate receptor on
a cell. The peptide can comprise a plurality of amino acid
sequences, wherein the amino acid sequences selectively bind to a
carbohydrate receptor on a cell. The composition can comprise a
plurality of amino acid sequences, wherein the amino acid sequences
selectively bind to a carbohydrate receptor on a cell. The
composition can comprise a plurality of peptides, wherein at least
one of the peptides comprises an amino acid sequence that bind to a
carbohydrate receptor on a cell. The composition can comprise a
plurality of peptides, wherein a plurality of the peptides each
comprise an amino acid sequence that selectively bind to a
carbohydrate receptor on a cell. The composition can comprise a
plurality of peptides, wherein the peptides each comprise an amino
acid sequence that selectively bind to a carbohydrate receptor on a
cell.
[0021] The composition can comprise a plurality of peptides,
wherein the peptides can bind to a carbohydrate receptor on a cell.
The peptide can comprise a plurality of amino acid sequences,
wherein the amino acid sequences can bind to a carbohydrate
receptor on a cell. The composition can comprise a plurality of
amino acid sequences, wherein the amino acid sequences can bind to
a carbohydrate receptor on a cell. The composition can comprise a
plurality of peptides, wherein at least one of the peptides
comprises an amino acid sequence that s can bind to a carbohydrate
receptor on a cell. The composition can comprise a plurality of
peptides, wherein a plurality of the peptides each comprise an
amino acid sequence that can bind to a carbohydrate receptor on a
cell. The composition can comprise a plurality of peptides, wherein
the peptides each comprise an amino acid sequence that can bind to
a carbohydrate receptor on a cell.
[0022] The composition can comprise a plurality of peptides,
wherein the peptides selectively bind to annexin 1 on a cell. The
peptide can comprise a plurality of amino acid sequences, wherein
the amino acid sequences selectively bind to annexin 1 on a cell.
The composition can comprise a plurality of amino acid sequences,
wherein the amino acid sequences selectively bind to annexin 1 on a
cell. The composition can comprise a plurality of peptides, wherein
at least one of the peptides comprises an amino acid sequence that
bind to annexin 1 on a cell. The composition can comprise a
plurality of peptides, wherein a plurality of the peptides each
comprise an amino acid sequence that selectively bind to annexin 1
on a cell. The composition can comprise a plurality of peptides,
wherein the peptides each comprise an amino acid sequence that
selectively bind to annexin 1 on a cell.
[0023] The composition can comprise a plurality of peptides,
wherein the peptides can bind to annexin 1 on a cell. The peptide
can comprise a plurality of amino acid sequences, wherein the amino
acid sequences can bind to annexin 1 on a cell. The composition can
comprise a plurality of amino acid sequences, wherein the amino
acid sequences can bind to annexin 1 on a cell. The composition can
comprise a plurality of peptides, wherein at least one of the
peptides comprises an amino acid sequence that can bind to annexin
1 on a cell. The composition can comprise a plurality of peptides,
wherein a plurality of the peptides each comprise an amino acid
sequence that can bind to annexin 1 on a cell. The composition can
comprise a plurality of peptides, wherein the peptides each
comprise an amino acid sequence that can bind to annexin 1 on a
cell.
[0024] The composition can comprise a plurality of peptides,
wherein the peptides selectively bind to annexin 1 on tumor
vasculature. The peptide can comprise a plurality of amino acid
sequences, wherein the amino acid sequences selectively bind to
annexin 1 on tumor vasculature. The composition can comprise a
plurality of amino acid sequences, wherein the amino acid sequences
selectively bind to annexin 1 on tumor vasculature. The composition
can comprise a plurality of peptides, wherein at least one of the
peptides comprises an amino acid sequence that bind to annexin 1 on
tumor vasculature. The composition can comprise a plurality of
peptides, wherein a plurality of the peptides each comprise an
amino acid sequence that selectively bind to annexin 1 on tumor
vasculature. The composition can comprise a plurality of peptides,
wherein the peptides each comprise an amino acid sequence that
selectively bind to annexin 1 on tumor vasculature.
[0025] The composition can comprise a plurality of peptides,
wherein the peptides can bind to annexin 1 on tumor vasculature.
The peptide can comprise a plurality of amino acid sequences,
wherein the amino acid sequences can bind to annexin 1 on tumor
vasculature. The composition can comprise a plurality of amino acid
sequences, wherein the amino acid sequences can bind to annexin 1
on tumor vasculature. The composition can comprise a plurality of
peptides, wherein at least one of the peptides comprises an amino
acid sequence that can bind to annexin 1 on tumor vasculature. The
composition can comprise a plurality of peptides, wherein a
plurality of the peptides each comprise an amino acid sequence that
can bind to annexin 1 on tumor vasculature. The composition can
comprise a plurality of peptides, wherein the peptides each
comprise an amino acid sequence that can bind to annexin 1 on tumor
vasculature.
[0026] The amino acid sequences, peptides, and compositions can
bind inside tumor blood vessels. The composition can reduce tumor
growth. The composition can comprise at least 100 annexin 1-binding
amino acid sequences. The composition can comprise at least 1000
annexin 1-binding amino acid sequences. The composition can
comprise at least 10,000 annexin 1-binding amino acid
sequences.
[0027] The composition can comprise one or more moieties. The
moieties can be independently selected from the group consisting
of, for example, an anti-angiogenic agent, a pro-angiogenic agent,
a cancer chemotherapeutic agent, a cytotoxic agent, an
antiinflammatory agent, an anti-arthritic agent, a polypeptide, a
nucleic acid molecule, and a small molecule. At least one of the
moieties can be a therapeutic agent. The therapeutic agent can
comprise a compound or composition for treating cancer. The
therapeutic agent can comprise a compound or composition to induce
programmed cell death or apoptosis. The therapeutic agent can be
Abraxane. The therapeutic agent can be paclitaxel. The therapeutic
agent can be docetaxel. At least one of the moieties can be a
detectable agent. The detectable agent can be FAM.
[0028] The amino acid sequences, peptides, and compositions can
selectively home to tumor vasculature. The composition can have a
therapeutic effect. The therapeutic effect can be a slowing in the
increase of or a reduction of tumor burden. The therapeutic effect
can be a slowing of the increase of or a reduction of tumor size.
The therapeutic effect can be a reduction or blocking of blood
circulation in a tumor.
[0029] The subject can have one or more sites to be targeted,
wherein the composition homes to one or more of the sites to be
targeted. The subject can have a tumor, wherein the composition has
a therapeutic effect on the tumor.
[0030] Sufficiency of the number and composition of annexin
1-binding amino acid sequences can be determined by assessing
accumulation of the composition in tumors in, for example, a
subject or a non-human animal.
[0031] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0033] FIGS. 1A-1H show the essential role of annexin 1 (Anxa1) in
tumor growth and identification of a peptide sequence with tumor
vasculature targeting activity in vivo. A and B. Growth of B16
tumors subcutaneously injected into Anxa1(+/-) and Anxa1(-/-)
mutant mice. C. Histology of B16 tumors produced in Anxa1(+/-) and
Anxa1(-/-) mice. Each scale bar represents 2.00 mm. D.
Immunohistochemistry of B16 tumors for CD31 showing endothelial
cells and vasculature. Each scale bar represents 200 .mu.m. E. In
vivo phage targeting in B16 tumor-bearing mice. Numbers of
transformed colonies recovered from the tumor or lung were
determined. Peptide sequences displayed by clones 1-10 are: IELLQAR
(1; SEQ ID NO:1), IFLLWQR (2; SEQ ID NO:2), IILLQAR (3; SEQ ID
NO:3), IDLMQAR (4; SEQ ID NO:4), ISLLQAR (5; SEQ ID NO:5), FSLLDAR
(6; SEQ ID NO:6), ISLLGAR (7; SEQ ID NO:7), PLWRPSR (8; SEQ ID
NO:8), LLLMQLR (9; SEQ ID NO:9), and LYLQRLR (10; SEQ ID NO: 10).
F. In vivo tumor and organ targeting activity of IFLLWQR (SEQ ID
NO:2) displaying phage. Phage was injected into B16 tumor bearing
mice pre-injected with anti-Anxa1 antibody or with control rabbit
IgG. G. In vitro plate assay for binding of IF7-A488 (upper line)
and control RQ7-A488C (lower line) to recombinant IF7-His.sub.6
protein produced by bacteria. H. Effect of IF7 (upper line) and
control RQ7 (lower line) on binding of FITC-labeled poly
acrylamide-LeA oligosaccharide to Anxa1-His.sub.6 protein.
[0034] FIG. 2 shows conjugation of IF7C peptide (SEQ ID NO:14) with
GA and with Alexa 488. A geldanamycin analogue, 17-GMB-APA-GA (GA),
was purchased from Invivogen (San Diego, Calif.). GA was also
synthesized from geldanamycin (LC labs, Wobum, Mass.) as described
by Mandler et al (J Natl Cancer Inst 92, 1573-81, 2000). Briefly,
GA was dissolved in chloroform and mixed with 1,3-diaminopropane
(Sigma-Aldrich) under argon gas at room temperature for 20 hours.
Diaminopropane cross-linked GA was precipitated with hexane. The
precipitate was dissolved in chloroform, and was reacted with
N-[g-maleimidobutyryloxy] succinimide ester (Pierce, Rockford,
Ill.) at room temperature for 2 hours. The product or 17-GMB-APA-GA
was purified by thin layer chromatography using preparative TLC
plate (1.5 mm silica gel, Analtech, Newark, Del.) in solvent
system, dichloromethane:methanol (92:8, v/v). The structure of
17-GMB-APA-GA was verified by ESI mass spectrometry (Micromass ZQ)
with MASSLYNX ver3.5 (Waters Corp., Milford, Mass.). To conjugate
IF7C peptide (SEQ ID NO:14) with 17-GMB-APA-GA, they were dissolved
in methanol at 1:1 molar ratio. Equal volume of purified water was
added for the mixture, and was left at room temperature for 2 hrs.
The product, IF7C-GA, was purified by C18 reverse-phase HPLC column
(10.times.150 mm) by gradient elution from 40% to 50% acetonitrile
in water containing 0.10% (v/v) trifluoroacetic acid at a flow rate
of 2.5 ml/min. The purity and structure of IF7C-GA was assessed by
ESI mass spectrometry. IF7C was also conjugated with Alexa fluor
488 C.sub.5-maleimide (Invitrogen, Carlsbad, Calif.) and purified
by HPLC in a similar manner as described above.
[0035] FIG. 3 shows surface plasmon resonance analysis of
IF-peptide on Anxa1 coated chip and control uncoated chip. IASys
(Affinity Sensors, Cambridge, UK) was used. Anxa1-His protein was
immobilized on the sensor chip by cross-linking to the aminosilane
surface using bis[sulfosuccinimidyl] substrate (Pierce). Binding of
IF7C dissolved in TBSC was recorded for 2 min, and dissociation of
IF7C was initiated by adding TBSC to the sensor chip and the arc
second was recorded for 5 min.
[0036] FIG. 4 shows isothermal colorimetry analysis of IF7K3C with
Anxa1-His protein. ITC was performed on a VP-ITC calorimeter from
Microcal (Northampton, Mass.). In this analysis IF7K3C peptide or
IFLLWQRKKKC (SEQ ID NO:12), in which IF7 was modified by inserted
with three lysine residues between IF7 and C to increase solubility
in water. Eight .mu.l aliquots of solution containing 1.0 or 1.5 mM
were injected into the cell containing 100 or 150 .mu.M Anxa1-His
protein. In each experiment 37 injections were made. The
experiments were performed at 23.degree. C. in buffer containing 20
mM Tris pH 7.9, 250 mM NaCl, 1 mM CaCl.sub.2 and 10%
dimethylsulfoxide. Experimental data were analyzed using Microcal
Origin software provided by the ITC manufacturer (Microcal,
Northampton, Mass.). An average (K.sub.d=11.+-.6 .mu.M, n=4) was
obtained using two different Anxa1-His preparations.
[0037] FIGS. 5A-5D shows in vivo targeting of IF7-A488 to tumors in
a dorsal skinfold chamber. (A) Lewis lung carcinoma (LLC) tumors
were allowed to vascularize for 3 days in dorsal skinfold chamber
window in nude mice. Mice were injected with 100 .mu.l of 50 .mu.M
IF7-A488 (row a) or RQ7-A488 (row b) in 5% glucose in water.
IF7-A488 was also injected to tumor-bearing mice pre-injected with
rabbit IgG (row c) or with rabbit anti-Anxa1 antibody (row d).
Fluorescence signals were monitored under a fluorescence microscope
up to 40 min post-injection. From left: bright field before
injection, and fluorescence at 0, 5, 20 min after injection. Far
right, the boundary of tumor and stroma at 20 min (Scale bar=500
.mu.m). (B) Quantitative analysis of fluorescence during the course
of the analysis shown in A. The line with the highest signal at 10
minutes represents IF&-A488. The line with the second highest
signal at 10 minutes represents the control IgG. The line with the
third highest signal at 10 minutes represents Anxa-1 antibody. The
line with the lowest signal at 10 minutes represents RQ7-A488.
Intensity of fluorescence was determined by Image J program. Error
bars represent SD (n=3). Asterisks show statistical significance or
p<0.0001, t test. (C) Fluorescence micrographs of tumor sections
20 min after injection with IF7-A488 and RQ7-A488. From left:
IF7-A488, DAPI, merged, and bright field. IF7-A488 signals (upper
row) were detected on endothelial cells of tumor vasculature (Scale
bar=50 .mu.m), while no RQ7-A488 signal was detected (lower row).
Experiments were repeated 3 times, and representative results are
shown. Arrows indicate fluorescence. (D) Fluorescence remained in
circulation in mice with or without subcutaneous B16 tumors.
IF7-A488 was injected intravenously through the tail vein, and
fluorescence remaining in circulation was measured. Upper line
represents "without tumor", middle line represents "with 3 days
tumor", and bottom line represents "with 6 days tumor." Error bars
represent SD (n=3).
[0038] FIGS. 6A and 6B show the effect of IF7-GA on melanoma, lung
carcinoma, prostate cancer, and breast cancer mouse models. A,
Effect of IF7-GA on tumor size. (a) Mouse melanoma B16F1 tumors
were grown subcutaneously in C57BL6 mice. On day 10, each mouse was
injected intravenously with either 100 .mu.l of 5% glucose or that
containing 0.13 .mu.moles of each IF7, GA, or IF7-GA. Injections
were administered every other day, for a total of three injections,
until day 14. Mice were euthanised on day 15 to measure tumor
weight. (b) Mouse Lewis lung carcinoma (LLC) tumors were grown
subcutaneously in C57BL6 mice. On day 7, each mouse was injected
intravenously with the compounds as in A-a, and injections
administered every other day, for a total of three injections,
until day 11. Mice were euthanised on day 13 and tumors weighed.
(c) Human prostate cancer PC3 tumors were grown orthotopically in
the prostate of SCID mice. On day 7, each mouse was injected
intravenously with the compounds as in A-a, and injections
administered every 4 days, for a total of four injections, until
day 22. Mice were euthanised on day 28 and tumors weighed. (d)
Human breast cancer MDA-MB-231 tumors were grown orthotopically in
fat pads of SCID mice. On day 7, each mouse was injected
intravenously with the compounds as in A-a, and injections
performed every 4 days, for a total of four injections, until day
22. Mice were euthanised on day 28 and tumors weighed. Asterisks
show statistical significance (Mann-Whitney's U test). B,
Histochemistry of tumors from the mice intravenously injected with
the compounds described in A. Apoptotic tumor cells along blood
vessels in transplanted tumors are detected by TUNEL assay. B16
melanoma (a), LLC lung carcinoma (b), PC3 prostate tumor (c) and
MDA-MB-231 breast tumor (d) from mice treated with each compound.
Note that perivascular cancer cells rarely show apoptosis in
control IF7, and GA groups, while perivascular tumor cells in the
IF7-GA groups show greater numbers of apoptotic cells (some areas
outlined in black) (Scale bar=50 .mu.m).
[0039] FIG. 7 shows Synthesis of IF7-SN38. SN-38 was synthesized by
Yakult (Tokyo, Japan). The conjugation was performed according to
the method described by Meyer-Losic et al., Clin Cancer Res 2008;
14: 2145-53, except followings.
Trans-4-(Aminomethyl)cyclohexanecarboxylic acid (Tranexamic acid,
500 mg) was dissolved in water (12.5 ml), and was added with
1M-phosphate buffer, pH 6.5 (1 ml). Both trans- and
cis-(Aminomethyl) cyclohexanecarboxylic acid were available;
however tranexamic acid was more effective. SMCC (1 g) was
dissolved in a mixture of acetonitrile (22 ml) and water (5.5 ml).
Tranexamic acid and SMCC were mixed, which was incubated at
45.degree. C. for 2 hours. The product
4-{4-[(N-maleimydomethyl)cyclohexanecarboxamido]methyl}cyclohexane-1-carb-
oxylic acid (BCH) was extracted to organic phase by dichloromethane
(60 ml) and 0.1 M NaCl in water (20 ml). Conjugation of BCH and
SN-38 was performed at room temperature for 4 hours, and
conjugation of IF7C and BCH-SN38 was performed by incubating them
in dimethylformamide at 45.degree. C. for 3 hours, following
addition of water and incubation at room temperature for 20 hours.
The product IF7-BCH-SN38 (IF7-SN38) was recovered from the
interphase of dichloromethane (30 ml) and 5M NaCl in water (2 ml).
Dried IF7-SN38 was dissolved in 50% acetonitrile in water, and
purified by reverse phase HPLC, by a gradient elution from 50% to
70% acetonitrile in water containing 0.10% trifluoroacetic acid.
The numbers in parenthesis are molecular weight of each compound,
which was verified by mass spectrometry.
[0040] FIGS. 8A-8F show the effect of IF7-GA and IF7-SN38 on
B16-tumor bearing mice. A. Survival of B16-tumor bearing mice
treated with IF7-GA or IF7-SN38. B16 cells were injected
subcutaneously into mice, and on day 14 each mouse was injected
intravenously with either 100 .mu.l of 5% glucose or that
containing 0.13 .mu.moles of each IF7-GA, RQ7-SN38 or IF7-SN38.
Injections were administered every other day, for a total of 7
injections, until day 26, as shown by arrows. Only IF7-SN38
exhibited high survival after 20 days; all of the others plummeted
prior to 20 days. B. Effect of IF7-SN38 on tumor size. Mouse
melanoma B16F1 tumors were grown subcutaneously in C57BL6 mice. On
day 13, each mouse was injected intravenously with either 100 .mu.l
of 5% glucose (darker line) or that containing 0.13 .mu.moles of
IF7-SN38 (lighter line). Injections were administered every other
day, for a total of five injections, until day 21. Size of tumors
was measured by using a caliper. Error bars represent SD (n=6). C.
Size of tumors was measured by using a caliper. Error bars
represent SD (n=6). Histology showed that a tumor from IF7-SN38
treated mouse is slightly smaller and contains more necrosis
compared to a tumor isolated from control mouse (FIG. 8C). D.
Survival of B16-tumor bearing mice treated with IF7-GA or IF7-SN38.
B16 cells were injected peritoneally into mice, and on day 1 each
mouse was injected intravenously with either 100 .mu.l of 5%
glucose or that containing 0.13 .mu.moles of each IF7-GA, RQ7-SN38
or IF7-SN38. Injections were administered every other day, for a
total of 6 injections, until day 12, as shown by arrows. Mice were
left until they showed a sign for weakness. IF7-SN38 exhibited high
survival after 22 days; IF7-GA exhibited high survival until 20
days; glucose and RQ7-SN38 plummeted prior to 20 days. E. Day 10
peritoneal B16 tumors from mice intravenously injected with 5%
glucose (a), IF7-GA (b), or IF7-SN38 (c). Scale bar represents 1
cm. F. Histology of peritoneal B16 tumors shown in E. Scale bars
represent 100 .mu.m.
[0041] FIG. 9 is a graph of tumor volume (in mm.sup.3) versus time
(in days) for B16 solid tumors in mice. Treatment with IF7-Dox
(doxorubicin), IF7-SN38, and a control (no treatment) are compared.
While both drugs suppressed tumor growth, anti-tumor activity by
IF7-SN38 was superior to IF7-Dox.
[0042] FIGS. 10A and 10B show images of HCT116-luc tumors
visualized by Xenogen IVIS imager. Drug IF7-SN38 6.5 .mu.moles/kg
was administered intravenously through tail vein.
[0043] FIGS. 11A and 11B show the effect of IF7-SN38 on HCT116-luc
tumors produced in nude mice. IF7-SN38 6.5 mmoles/kg was injected
daily to HCT116-luc tumor bearing mice. A. Tumor size monitored by
luciferase-based chemiluminescence. Photon numbers of 100% is the
order of e+07. B. Tumor size monitored by caliper measurement.
[0044] FIGS. 12A, 12B and 12C demonstrate the binding and
penetration of IF7C(RR)-conjugated FITC-poly-lysine to
Anxa1-expressing mouse endothelial cells. A. Binding of
IF7C(RR)-conjugated FITC-polylysine on the surface of mouse
endothelial F-2 cells cultured in vitro. B. Transport of
FITC-conjugated FITC-poly-L-lysine through apical surface to basal
space of F-2 cells monolayer. F-2 cells cultured on a filter of
trans well insert was added with IF7C(RR)-conjugated
FITC-poly-L-lysine to allow the reagent being bound to apical cell
surface of F-2 cells. After washing the monolayer with medium,
insert was placed in wells containing medium at 37.degree. C. or at
4.degree. C. Fluorescence moved to the lower chamber of insert was
measured. C. Targeting and penetration of intravenously injected
IF7C(RR)-conjugated FITC-poly-L-lysine in the B16 tumor in vivo in
the mouse. Tissue section was immunostained for endothelial cell
marker CD31. The fluorescence signals of FITC localize around and
often at basal side of the endothelial layer, suggesting the
penetration of IF7C(RR)-conjugated probe through endothelial
cells.
[0045] FIGS. 13A and 13B show (A) representative results of the
effect of IF7C(RR)-SN38 on a large HCT116-luc tumor (photon numbers
e+11 on day 0) produced in nude mice and (B) Dose of IF7C(RR)-SN38
for daily injection was 6.5 .mu.moles/kg, the same dose of IF7-SN38
shown in FIG. 11.
[0046] FIG. 14 shows the effect of low dosage IF7C(RR)-SN38 on
HCT116-luc tumors produced in nude mice. IF7C(RR)-SN38 used was one
eighth dose or 0.81 .mu.mole/kg/injection compared to IF7-SN38 used
at 6.5 .mu.moles/kg/injection (FIG. 11). Control RQ7C(RR)-SN38 was
prepared by conjugating SN-38 with reverse IF7C(RR), RQWLLFI-C-RR
peptide (SEQ ID NO:16).
[0047] FIGS. 15A, 15B and 15C show body weights (A), blood cell
counts (B), and blood chemistry (C) of the nude mice treated with
IF7C(RR)-SN38 and RQ7C(RR)-SN38 daily injection for 10 days. None
of the parameters showed significant differences to the control (no
drug injection) group.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The disclosed methods and compositions can be understood
more readily by reference to the following detailed description of
particular embodiments and the Example included therein and to the
Figures and their previous and following description.
[0049] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that they are not limited to specific synthetic methods
or specific recombinant biotechnology methods unless otherwise
specified, or to particular reagents unless otherwise specified, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to be limiting.
[0050] Chemotherapeutics administered to cancer patients
intravenously become diluted, therefore requiring high doses and
causing side effects, which could be circumvented by targeted drug
delivery (Ruoslahti E. et al. Annu Rev Immunol 2000; 18:813-27;
Neri, D. et al. Nat Rev Cancer 2005; 5:436-46; Bellone, M. et al.
Trends Immunol 2008; 29:235-41). Because malignancy is closely
associated with cancer cell surface carbohydrates, it was realized
that carbohydrate-based therapeutics are desirable. Such
therapeutics have previously not been explored. It was discovered
that one carbohydrate mimicry peptide, designated I-peptide, bound
to annexin 1 (Anxa1) (Hatakeyama, S. et al., Proc Natl Acad Sci
2009; 106: 3095-100). As Anxa1 specifically marks tumor vasculature
(Oh, P. et al. Nature 2004; 429:629-35), it has been discovered
that the I-peptide can serve as a tumor-targeting vehicle.
Investigating I-peptide related sequences (Fukuda, M. et al.,
Cancer Res 2000; 60:450-6; Zhang, J. et al. Cancer Res 2002;
62:4194-8; Fukuda, M. Methods Enzymol 2006; 416:51-60), lead to the
identification of a peptide designated IF7 that specifically homes
to tumors with high efficacy. Upon intravenous injection, IF7
conjugated with the potent anti-cancer drug SN-38 rescued terminal
stage mice harboring B16 tumors. IF7-SN38 also prolonged survival
of mice with peritoneal B16 tumors without side effects. These
results indicate that annexin 1-binding compounds can be used for
targeted chemotherapy.
[0051] Technical advances in genomics and proteomics together with
automated chemical synthesis of DNA and proteins have greatly
contributed to progress in biomedicine. By contrast, the use and
understanding of the role of carbohydrates have lagged behind due
to lack of advanced technologies. For example, recombinant or
amplifiable carbohydrates or chemically synthesized complex
carbohydrates cannot be produced automatically. Consequently,
carbohydrate-based drug discovery has been largely unexplored even
though cancer malignancy is closely associated with carbohydrate
structures found on the tumor cell surface (Hakomori, S. Proc Natl
Acad Sci, 2002; 99: 10231-3; Nakamori, S. et al. Cancer Res 1993;
53: 3632-7). Peptide-displaying phage technology can be used to
identify carbohydrate mimicry peptides (Fukuda, M. et al., Cancer
Res 2000; 60: 450-6; Fukuda, M. Methods Enzymol 2006; 416: 51-60;
Taki, T. et al. Biochim Biophys Acta 2008; 1780: 497-503; Scott, J.
et al. Proc Natl Acad Sci 1992; 89: 5398-402). For example,
I-peptide (IELLQAR; SEQ ID NO:13), was identified as a selectin
ligand mimic, and when injected intravenously into mice, synthetic
I-peptide inhibited carbohydrate-dependent cancer cell colonization
to the lung (Fukuda, M. et al., Cancer Res 2000; 60: 450-6; Zhang,
J. et al. Cancer Res 2002; 62: 4194-8).
[0052] Experiments designed to identify endothelial I-peptide
receptors, revealed that I-peptide binds to a fragment of annexin 1
(Anxa1) (Hatakeyama, S. et al., Proc Natl Acad Sci 2009; 106:
3095-100). The peptide sequence identified in an Anxa1 fragment
isolated from the rat lung by I-peptide affinity chromatography is
SEQ ID NO:11. The protein band at 15 kDa was digested with trypsin
and the peptide fragments were analyzed by mass-spectroscopy.
Unique peptide sequences that led to the identification of Anxa1
are shown by underlining.
TABLE-US-00001 (SEQ ID NO: 11)
MAMVSEFLKQARFLENQEQEYVQAVKSYKGGPGSAVSPYPS
FNVSSDVAALHKAIMVKGVDEATIIDILTKRTNAQRQQIKA
AYLQENGKPLDEVLRKALTGHLEEVVLAMLKTPAQFDADEL
RGAMKGLGTDEDTLIEILTTRSNEQIREINRVYREELKRDL
AKDITSDTSGDFRKALLALAKGDRCQDLSVNQDLADTDARA
LYEAGERRKGTDVNVFTTILTSRSFPHLRRVFQNYGKYSQH
DMNKALDLELKGDIEKCLTTIVKCATSTPAFFAEKLYEAMK
GAGTRHKALIRIMVSRSEIDMNEIKVFYQKKYGISLCQAIL DETKGDYEKILVALCGGN.
[0053] Anxa1 has been identified as a specific tumor endothelial
cell surface marker (Oh, P. et al. Nature 2004; 429: 629-35). When
B16 melanoma cells were injected subcutaneously in Anxa1 null
mutant mice, tumor growth was significantly reduced compared to
tumors produced in Anxa1 heterozygous mice (see FIGS. 1A and 1B).
Tumors produced in Anxa1 nulls were largely necrotic (see FIG. 1C).
Remarkably, no vasculature was found in tumors produced in Anxa1
null mice (see FIG. 1D). These findings indicate that Anxa1
expression on the endothelial cell surface (Oh, P. et al. Nature
2004; 429: 629-35) is essential for active tumor growth in the
mouse.
[0054] Disclosed are isolated peptides comprising an amino acid
sequence that can bind to a carbohydrate receptor on a cell. Also
disclosed are compositions comprising a moiety and a peptide
comprising an amino acid sequence that can bind to a carbohydrate
receptor on a cell. Also disclosed are isolated nucleic acids
comprising a nucleic acid sequence encoding a peptide comprising an
amino acid sequence that can bind to a carbohydrate receptor on a
cell.
[0055] Also disclosed are methods comprising administering to a
subject a composition comprising a moiety and a peptide comprising
an amino acid sequence that can bind to a carbohydrate receptor on
a cell. Also disclosed are methods of targeting a tumor cell in a
subject comprising administering to the subject a peptide
comprising an amino acid sequence that can bind to a carbohydrate
receptor on a cell. Also disclosed are methods of targeting a tumor
cell in a subject comprising administering to the subject a
composition comprising a moiety and a peptide comprising an amino
acid sequence that can bind to a carbohydrate receptor on a cell.
Also disclosed are methods comprising administering to the subject
a composition comprising a peptide comprising an amino acid
sequence that can bind to a carbohydrate receptor on a cell and
detecting the composition in the subject.
[0056] The carbohydrate receptor can be annexin 1. The amino acid
sequence can selectively bind the carbohydrate receptor. The
subject can comprise a cell. The cell can be a tumor cell.
[0057] The amino acid sequence can comprise SEQ ID NO:2 having one
or more conservative amino acid substitutions. The amino acid
sequence can have at least 55% sequence identity to SEQ ID NO:2,
wherein differences between the amino acid sequence and SEQ ID NO:2
consist of conservative amino acid substitutions. The amino acid
sequence can have at least 70% sequence identity to SEQ ID NO:2.
The amino acid sequence can have at least 80% sequence identity to
SEQ ID NO:2. The amino acid sequence can comprise SEQ ID NO:2. The
amino acid sequence can consist of SEQ ID NO:2. The amino acid
sequence can comprise at least 5 consecutive amino acids of SEQ ID
NO:2. The amino acid sequence can comprise at least 6 consecutive
amino acids of SEQ ID NO:2.
[0058] The peptide can comprise SEQ ID NO:2. The peptide can
comprise at least 6 amino acids. The peptide can comprise at least
7 amino acids. The peptide can comprise at least 8 amino acids. The
peptide can comprise at least 9 amino acids. The peptide can
further comprise a moiety peptide.
[0059] The moiety can be a small molecule, pharmaceutical drug,
toxin, fatty acid, detectable marker, conjugating tag, nanoshell,
or enzyme. The moiety can be covalently linked to the peptide. The
moiety can be linked to the amino terminal end of the peptide.
[0060] The moiety can be linked to the carboxy terminal end of the
peptide. The moiety can be linked to an amino acid within the
peptide. The moiety can be SN-38. The moiety can comprise a
detectable agent. The moiety can comprise a therapeutic agent.
[0061] The composition can further comprise a linker connecting the
moiety and the peptide. The composition can further comprise a
pharmaceutically acceptable carrier. The composition can further
comprise a detectable agent. The composition can further comprise a
therapeutic agent. The composition can further comprise an
anti-cancer agent.
[0062] Detecting the composition in the method can thereby detect a
tumor in the subject. Detecting the composition in the method can
thereby diagnose cancer in the subject. Detecting the composition
in the method can comprise detecting the level, amount,
concentration, or a combination of binding of the composition to
cancer tissue in the subject, wherein the level, amount,
concentration, or a combination of binding of the composition to
cancer tissue in the subject indicates the prognosis of the cancer
in the subject. The method can further comprise repeating the
administration and detection at a later time, wherein a change in
the level, amount, concentration, or a combination of binding of
the composition to cancer tissue in the subject indicates the
progress of the endometriosis in the subject. The method can
further comprise repeating the administration and detection
following treatment, wherein a change in the level, amount,
concentration, or a combination of binding of the composition to
cancer tissue in the subject indicates the progress the treatment
of the cancer in the subject.
[0063] Disclosed are methods of determining and/or assessing
annexin 1 level in a cell of a subject. The method can comprise
bringing into contact a cell of the subject and an annexin
1-binding composition comprising, for example, a detectable agent
linked to a composition comprising SEQ ID NO: 2; and detecting the
level of annexin 1-binding composition interacting with annexin 1,
thereby determining and/or assessing annexin 1 level in the
cell.
[0064] Disclosed herein are methods of identifying a subject having
a disease associated with annexin 1, the method comprising bringing
into contact a cell of the subject and an annexin 1-binding
composition, wherein the annexin 1-binding composition comprises,
for example, a moiety linked to a composition comprising SEQ ID
NO:2; and detecting interaction between annexin 1 and the annexin
1-binding composition, thereby detecting the presence or level of
annexin 1 on the cell, wherein the presence or level of annexin 1
receptor on the cell identifies the subject as having a disease
associated with annexin 1.
[0065] Disclosed herein are subjects having a disease associated
with annexin 1. By this is meant that the subject has an increased
level of annexin 1 or that annexin 1 can be effectively targeted to
treat or ameliorate the symptoms of a disease or disorder. By an
"increased level of annexin 1" is meant that the number of annexin
1 molecules on selected cells or tissues in the subject is
increased over normal, basal, or standard levels accepted by those
of skill in the art. It can also mean that the number of annexin 1
molecules present in a given cell are increased over a basal,
normal, or standard amount. One standard level that can be used for
this purpose is the level of annexin 1 on normal lung endothelial
cells. The standard level of annexin 1 can be determined using, for
example, normal lung endothelial cells of the same subject, a
different subject, or a group or population of individuals. One of
skill in the art would be able to determine annexin 1 levels in a
subject in cells and tissues, using the methods disclosed herein
and those known to those of skill in the art. Diseases associated
with the annexin 1 include some cancers, for example.
Definitions
[0066] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a pharmaceutical carrier" includes mixtures of two or
more such carriers, and the like.
[0067] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to" the value and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15. It is
also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0068] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0069] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
[0070] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon.
[0071] It is to be understood that the disclosed method and
compositions are not limited to specific synthetic methods,
specific analytical techniques, or to particular reagents unless
otherwise specified, and, as such, may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting.
[0072] By "treatment" and "treating" is meant the medical
management of a subject with the intent to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder. This term includes active treatment, that is, treatment
directed specifically toward the improvement of a disease,
pathological condition, or disorder, and also includes causal
treatment, that is, treatment directed toward removal of the cause
of the associated disease, pathological condition, or disorder. In
addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the
curing of the disease, pathological condition, or disorder;
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder. It is
understood that treatment, while intended to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder, need not actually result in the cure, ameliorization,
stabilization or prevention. The effects of treatment can be
measured or assessed as described herein and as known in the art as
is suitable for the disease, pathological condition, or disorder
involved. Such measurements and assessments can be made in
qualitative and/or quantitative terms. Thus, for example,
characteristics or features of a disease, pathological condition,
or disorder and/or symptoms of a disease, pathological condition,
or disorder can be reduced to any effect or to any amount.
[0073] The term "in need of treatment" as used herein refers to a
judgment made by a caregiver (e.g. physician, nurse, nurse
practitioner, or individual in the case of humans; veterinarian in
the case of animals, including non-human mammals) that a subject
requires or will benefit from treatment. This judgment is made
based on a variety of factors that are in the realm of a
caregiver's expertise, but that includes the knowledge that the
subject is ill, or will be ill, as the result of a condition that
is treatable by the compounds of the invention.
[0074] As used herein, "subject" includes, but is not limited to,
animals, plants, bacteria, viruses, parasites and any other
organism or entity. The subject can be a vertebrate, more
specifically a mammal (e.g., a human, horse, pig, rabbit, dog,
sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a
fish, a bird or a reptile or an amphibian. The subject can be an
invertebrate, more specifically an arthropod (e.g., insects and
crustaceans). The term does not denote a particular age or sex.
Thus, adult and newborn subjects, as well as fetuses, whether male
or female, are intended to be covered. A patient refers to a
subject afflicted with a disease or disorder. The term "patient"
includes human and veterinary subjects.
[0075] Annexins are a family closely related calcium- and
membrane-binding proteins expressed in most eukaryotic cell types.
Their diverse functions include vesicle trafficking, cell division,
apoptosis, calcium signaling, and growth regulation. Annexins are
linked to some of the most serious human diseases such as
cardiovascular disease and cancer. Annexin 1, a 37 kDa protein,
originally termed lipocortin, inhibits the inflammatory response
and participates in several cellular functions, including
phagocytosis, extravasation, mediator generation and neutrophil
recruitment. In addition, annexin 1 can affect cells relevant to
the inflammatory process, such as endothelial, epithelial, mast and
synovial cells.
Materials
[0076] Disclosed are the components to be used to prepare the
disclosed compositions as well as the compositions themselves to be
used within the methods disclosed herein. These and other materials
are disclosed herein, and it is understood that when combinations,
subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual
and collective combinations and permutation of these compounds may
not be explicitly disclosed, each is specifically contemplated and
described herein. For example, if a particular peptide is disclosed
and discussed and a number of modifications that can be made to a
number of molecules including the peptide are discussed,
specifically contemplated is each and every combination and
permutation of the peptides and the modifications that are possible
unless specifically indicated to the contrary. Thus, if a class of
molecules A, B, and C are disclosed as well as a class of molecules
D, E, and F and an example of a combination molecule, A-D is
disclosed, then even if each is not individually recited each is
individually and collectively contemplated meaning combinations,
A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered
disclosed. Likewise, any subset or combination of these is also
disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E
would be considered disclosed. This concept applies to all aspects
of this application including, but not limited to, steps in methods
of making and using the disclosed compositions. Thus, if there are
a variety of additional steps that can be performed it is
understood that each of these additional steps can be performed
with any specific embodiment or combination of embodiments of the
disclosed methods.
[0077] The disclosed amino acid sequences can bind to a
carbohydrate receptor on a cell. The disclosed peptides can
comprise an amino acid sequence that can bind to a carbohydrate
receptor on a cell. The disclosed compositions can comprise a
moiety and a peptide comprising an amino acid sequence that can
bind to a carbohydrate receptor on a cell. The disclosed are
isolated nucleic acids can comprise a nucleic acid sequence
encoding a peptide comprising an amino acid sequence that can bind
to a carbohydrate receptor on a cell.
[0078] The amino acid sequence can comprise SEQ ID NO:2 having one
or more conservative amino acid substitutions. The amino acid
sequence can have at least 55% sequence identity to SEQ ID NO:2,
wherein differences between the amino acid sequence and SEQ ID NO:2
consist of conservative amino acid substitutions. The amino acid
sequence can have at least 70% sequence identity to SEQ ID NO:2.
The amino acid sequence can have at least 80% sequence identity to
SEQ ID NO:2. The amino acid sequence can comprise SEQ ID NO:2. The
amino acid sequence can consist of SEQ ID NO:2. The amino acid
sequence can comprise at least 5 consecutive amino acids of SEQ ID
NO:2. The amino acid sequence can comprise at least 6 consecutive
amino acids of SEQ ID NO:2.
[0079] The peptide can comprise SEQ ID NO:2. The peptide can
comprise at least 6 amino acids. The peptide can comprise at least
7 amino acids. The peptide can comprise at least 8 amino acids. The
peptide can comprise at least 9 amino acids. The peptide can
further comprise a moiety peptide.
[0080] The composition can bind inside tumor blood vessels. The
composition can reduce tumor growth. The composition can comprise
at least 100 annexin 1-binding amino acid sequences. The
composition can comprise at least 1000 annexin 1-binding amino acid
sequences. The composition can comprise at least 10,000 annexin
1-binding amino acid sequences.
[0081] The composition can further comprise one or more moieties.
The moieties can be independently selected from the group
consisting of an anti-angiogenic agent, a pro-angiogenic agent, a
cancer chemotherapeutic agent, a cytotoxic agent, an
antiinflammatory agent, an anti-arthritic agent, a polypeptide, a
nucleic acid molecule, and a small molecule. At least one of the
moieties can be a therapeutic agent. The therapeutic agent can
comprise a compound or composition for treating cancer. The
therapeutic agent can comprise a compound or composition to induce
programmed cell death or apoptosis. The therapeutic agent can be
Abraxane. The therapeutic agent can be paclitaxel. The therapeutic
agent can be docetaxel. At least one of the moieties can be a
detectable agent. The detectable agent can be FAM.
[0082] The composition can selectively home to tumor vasculature.
The composition can have a therapeutic effect. The therapeutic
effect can be a slowing in the increase of or in a reduction of
tumor burden. The therapeutic effect can be a slowing of the
increase of or a reduction of tumor size. The therapeutic effect
can be a reduction or blocking of blood circulation in a tumor.
A. Annexin 1-Binding Compounds
[0083] The annexin 1-binding compound can be any compound with the
ability to interact with annexin 1. The annexin 1-binding compound
can be an annexin 1-binding amino acid sequence. The disclosed
amino acid sequences can be annexin 1-binding amino acid sequences.
Annexin 1-binding compounds can, for example, bind to annexin 1,
selectively bind to annexin 1, home to annexin 1-containing cells
and tissue, target annexin 1-containing cells and tissue, bind to
tumor vasculature, selectively bind to tumor vasculature,
accumulate at annexin 1-containing cells and tissue, and accumulate
in tumor vasculature. The disclosed annexin 1-binding compounds and
amino acid sequences can be homing molecules or homing
peptides.
[0084] The annexin 1-binding compound can comprise SEQ ID NO:2
having one or more conservative amino acid substitutions. The
annexin 1-binding compound can have at least 55% sequence identity
to SEQ ID NO:2, wherein differences between the annexin 1-binding
compound and SEQ ID NO:2 consist of conservative amino acid
substitutions. The annexin 1-binding compound can have at least 70%
sequence identity to SEQ ID NO:2. The annexin 1-binding compound
can have at least 80% sequence identity to SEQ ID NO:2. The annexin
1-binding compound can comprise SEQ ID NO:2. The annexin 1-binding
compound can consist of SEQ ID NO:2. The annexin 1-binding compound
can comprise at least 5 consecutive amino acids of SEQ ID NO:2. The
annexin 1-binding compound can comprise at least 6 consecutive
amino acids of SEQ ID NO:2. The annexin 1-binding compound can
comprises at least 6 amino acids. The annexin 1-binding compound
can comprise at least 7 amino acids. The peptide can comprise at
least 8 amino acids. The annexin 1-binding compound can comprise at
least 9 amino acids. The annexin 1-binding compound can comprise at
least 10 amino acids. The annexin 1-binding compound can comprise
at least 11 amino acids. The annexin 1-binding compound can
comprise at least 12 amino acids. The annexin 1-binding compound
can comprise at least 13 amino acids. The annexin 1-binding
compound can comprise at least 14 amino acids. The annexin
1-binding compound can comprise at least 15 amino acids.
[0085] The annexin 1-binding compound can comprise an amino acid
sequence comprising SEQ ID NO:2 having one or more conservative
amino acid substitutions. The annexin 1-binding compound can
comprise an amino acid sequence having at least 55% sequence
identity to SEQ ID NO:2, wherein differences between the amino acid
sequence and SEQ ID NO:2 consist of conservative amino acid
substitutions. The annexin 1-binding compound can comprise an amino
acid sequence having at least 70% sequence identity to SEQ ID NO:2.
The annexin 1-binding compound can comprise an amino acid sequence
having at least 80% sequence identity to SEQ ID NO:2. The annexin
1-binding compound can comprise an amino acid sequence comprising
SEQ ID NO:2. The annexin 1-binding compound can comprise an amino
acid sequence consisting of SEQ ID NO:2. The annexin 1-binding
compound can comprise an amino acid sequence comprising at least 5
consecutive amino acids of SEQ ID NO:2. The annexin 1-binding
compound can comprise an amino acid sequence comprising at least 6
consecutive amino acids of SEQ ID NO:2. The annexin 1-binding
compound can comprise a peptide comprising SEQ ID NO:2. The annexin
1-binding compound can comprise a peptide comprising at least 6
amino acids. The annexin 1-binding compound can comprise a peptide
comprising at least 7 amino acids. The annexin 1-binding compound
can comprise a peptide comprising at least 8 amino acids. The
annexin 1-binding compound can comprise a peptide comprising at
least 9 amino acids. The annexin 1-binding compound can comprise a
peptide comprising at least 10 amino acids. The annexin 1-binding
compound can comprise a peptide comprising at least 11 amino acids.
The annexin 1-binding compound can comprise a peptide comprising at
least 12 amino acids. The annexin 1-binding compound can comprise a
peptide comprising at least 13 amino acids. The annexin 1-binding
compound can comprise a peptide comprising at least 14 amino acids.
The annexin 1-binding compound can comprise a peptide comprising at
least 15 amino acids.
[0086] The annexin 1-binding compound can consist of an amino acid
sequence comprising SEQ ID NO:2 having one or more conservative
amino acid substitutions. The annexin 1-binding compound can
consist of an amino acid sequence having at least 55% sequence
identity to SEQ ID NO:2, wherein differences between the amino acid
sequence and SEQ ID NO:2 consist of conservative amino acid
substitutions. The annexin 1-binding compound can consist of an
amino acid sequence having at least 70% sequence identity to SEQ ID
NO:2. The annexin 1-binding compound can consist of an amino acid
sequence having at least 80% sequence identity to SEQ ID NO:2. The
annexin 1-binding compound can consist of an amino acid sequence
comprising SEQ ID NO:2. The annexin 1-binding compound can consist
of an amino acid sequence consisting of SEQ ID NO:2. The annexin
1-binding compound can consist of an amino acid sequence comprising
at least 5 consecutive amino acids of SEQ ID NO:2. The annexin
1-binding compound can consist of an amino acid sequence comprising
at least 6 consecutive amino acids of SEQ ID NO:2. The annexin
1-binding compound can consist of a peptide comprising SEQ ID NO:2.
The annexin 1-binding compound can consist of a peptide comprising
at least 6 amino acids. The annexin 1-binding compound can consist
of a peptide comprising at least 7 amino acids. The annexin
1-binding compound can consist of a peptide comprising at least 8
amino acids. The annexin 1-binding compound can consist of a
peptide comprising at least 9 amino acids. The annexin 1-binding
compound can consist of a peptide comprising at least 10 amino
acids. The annexin 1-binding compound can consist of a peptide
comprising at least 11 amino acids. The annexin 1-binding compound
can consist of a peptide comprising at least 12 amino acids. The
annexin 1-binding compound can consist of a peptide comprising at
least 13 amino acids. The annexin 1-binding compound can consist of
a peptide comprising at least 14 amino acids. The annexin 1-binding
compound can consist of a peptide comprising at least 15 amino
acids.
[0087] The annexin 1-binding compound can comprise IFLLWQRX (amino
acids 1 to 8 of SEQ ID NO:20), IFLLWQRXX (amino acids 1 to 9 of SEQ
ID NO:20), IFLLWQRXXX (amino acids 1 to 10 of SEQ ID NO:20),
IFLLWQRXXXX (amino acids 1 to 11 of SEQ ID NO:20), or IFLLWQRXXXXX
(SEQ ID NO:20), wherein each X is independently a polar or charged
amino acid. For example, each X can independently be selected from
all, any set of 10, any set of 9, any set of 8, any set of 7, any
set of 6, any set of 5, any set of 4, any set of 3, any set of 2,
or any 1 of the amino acids C, R, K, S, T, H, D, E, N, Q, and M.
For example, each X can independently be selected from the set of
three amino acids C, R, and K. As another example, each X can
independently be selected from the set of two amino acids C and R.
In some forms, a single one of the X can be C. In some forms, two
of the X can be C. In some forms, two of the X can be R. In some
forms, three of the X can be R. In some forms, four of the X can be
R. As an example, the annexin 1-binding compound can comprise
IFLLWQRCR (SEQ ID NO:17), IFLLWQRCRR (SEQ ID NO:19), IFLLWQRCRRR
(SEQ ID NO:18), or IFLLWQRCRRRR (SEQ ID NO:22).
[0088] The annexin 1-binding compound can comprise IFLLWQRX (amino
acids 1 to 8 of SEQ ID NO:20), IFLLWQRXX (amino acids 1 to 9 of SEQ
ID NO:20), IFLLWQRXXX (amino acids 1 to 10 of SEQ ID NO:20),
IFLLWQRXXXX (amino acids 1 to 11 of SEQ ID NO:20), or IFLLWQRXXXXX
(SEQ ID NO:20), wherein each X is independently a C, R, K, S, T, H,
D, E, N, Q, or M. In some forms, a single one of the X can be C. In
some forms, two of the X can be C. In some forms, two of the X can
be R. In some forms, three of the X can be R. In some forms, four
of the X can be R.
[0089] The annexin 1-binding compound can comprise IFLLWQRX (amino
acids 1 to 8 of SEQ ID NO:20), IFLLWQRXX (amino acids 1 to 9 of SEQ
ID NO:20), IFLLWQRXXX (amino acids 1 to 10 of SEQ ID NO:20),
IFLLWQRXXXX (amino acids 1 to 11 of SEQ ID NO:20), or IFLLWQRXXXXX
(SEQ ID NO:20), wherein each X is independently a C, R, or K. In
some forms, a single one of the X can be C. In some forms, two of
the X can be C. In some forms, two of the X can be R. In some
forms, three of the X can be R. In some forms, four of the X can be
R.
[0090] The annexin 1-binding compound can comprise IFLLWQRX (amino
acids 1 to 8 of SEQ ID NO:20), IFLLWQRXX (amino acids 1 to 9 of SEQ
ID NO:20), IFLLWQRXXX (amino acids 1 to 10 of SEQ ID NO:20),
IFLLWQRXXXX (amino acids 1 to 11 of SEQ ID NO:20), or IFLLWQRXXXXX
(SEQ ID NO:20), wherein each X is independently a C or R. In some
forms, a single one of the X can be C. In some forms, two of the X
can be C. In some forms, two of the X can be R. In some forms,
three of the X can be R. In some forms, four of the X can be R.
[0091] The annexin 1-binding compound can consist of IFLLWQRX
(amino acids 1 to 8 of SEQ ID NO:20), IFLLWQRXX (amino acids 1 to 9
of SEQ ID NO:20), IFLLWQRXXX (amino acids 1 to 10 of SEQ ID NO:20),
IFLLWQRXXXX (amino acids 1 to 11 of SEQ ID NO:20), or IFLLWQRXXXXX
(SEQ ID NO:20), wherein each X is independently a polar or charged
amino acid. For example, each X can independently be selected from
all, any set of 10, any set of 9, any set of 8, any set of 7, any
set of 6, any set of 5, any set of 4, any set of 3, any set of 2,
or any 1 of the amino acids C, R, K, S, T, H, D, E, N, Q, and M.
For example, each X can independently be selected from the set of
three amino acids C, R, and K. As another example, each X can
independently be selected from the set of two amino acids C and R.
In some forms, a single one of the X can be C. In some forms, two
of the X can be C. In some forms, two of the X can be R. In some
forms, three of the X can be R. In some forms, four of the X can be
R. As an example, the annexin 1-binding compound can consist of
IFLLWQRCR (SEQ ID NO:17), IFLLWQRCRR (SEQ ID NO:19), IFLLWQRCRRR
(SEQ ID NO:18), or IFLLWQRCRRRR (SEQ ID NO:22).
[0092] The annexin 1-binding compound can consist of IFLLWQRX
(amino acids 1 to 8 of SEQ ID NO:20), IFLLWQRXX (amino acids 1 to 9
of SEQ ID NO:20), IFLLWQRXXX (amino acids 1 to 10 of SEQ ID NO:20),
IFLLWQRXXXX (amino acids 1 to 11 of SEQ ID NO:20), or IFLLWQRXXXXX
(SEQ ID NO:20), wherein each X is independently a C, R, K, S, T, H,
D, E, N, Q, or M. In some forms, a single one of the X can be C. In
some forms, two of the X can be C. In some forms, two of the X can
be R. In some forms, three of the X can be R. In some forms, four
of the X can be R.
[0093] The annexin 1-binding compound can consist of IFLLWQRX
(amino acids 1 to 8 of SEQ ID NO:20), IFLLWQRXX (amino acids 1 to 9
of SEQ ID NO:20), IFLLWQRXXX (amino acids 1 to 10 of SEQ ID NO:20),
IFLLWQRXXXX (amino acids 1 to 11 of SEQ ID NO:20), or IFLLWQRXXXXX
(SEQ ID NO:20), wherein each X is independently a C, R, or K. In
some forms, a single one of the X can be C. In some forms, two of
the X can be C. In some forms, two of the X can be R. In some
forms, three of the X can be R. In some forms, four of the X can be
R.
[0094] The annexin 1-binding compound can consist of IFLLWQRX
(amino acids 1 to 8 of SEQ ID NO:20), IFLLWQRXX (amino acids 1 to 9
of SEQ ID NO:20), IFLLWQRXXX (amino acids 1 to 10 of SEQ ID NO:20),
IFLLWQRXXXX (amino acids 1 to 11 of SEQ ID NO:20), or IFLLWQRXXXXX
(SEQ ID NO:20), wherein each X is independently a C or R. In some
forms, a single one of the X can be C. In some forms, two of the X
can be C. In some forms, two of the X can be R. In some forms,
three of the X can be R. In some forms, four of the X can be R.
[0095] In some forms, the annexin 1-binding compound can have one
or more conservative amino acid substitutions in amino acids 1 to 7
of SEQ ID NO:20. In some forms, the portion of the annexin
1-binding compound corresponding to the sequence IFLLWQR (amino
acids 1 to 7 of SEQ ID NO:20) can have at least 55% sequence
identity to the sequence IFLLWQR (amino acids 1 to 7 of SEQ ID
NO:20), wherein differences between the annexin 1-binding compound
and IFLLWQR (amino acids 1 to 7 of SEQ ID NO:20) consist of
conservative amino acid substitutions. In some forms, the portion
of the annexin 1-binding compound corresponding to the sequence
IFLLWQR (amino acids 1 to 7 of SEQ ID NO:20) can have at least 70%
sequence identity to the sequence IFLLWQR (amino acids 1 to 7 of
SEQ ID NO:20). In some forms, the portion of the annexin 1-binding
compound corresponding to the sequence IFLLWQR (amino acids 1 to 7
of SEQ ID NO:20) can have at least 80% sequence identity to the
sequence IFLLWQR (amino acids 1 to 7 of SEQ ID NO:20). In some
forms, the annexin 1-binding compound has at least 5 consecutive
amino acids of the sequence IFLLWQR (amino acids 1 to 7 of SEQ ID
NO:20). In some forms, the annexin 1-binding compound has at least
6 consecutive amino acids of the sequence IFLLWQR (amino acids 1 to
7 of SEQ ID NO:20).
[0096] The annexin 1-binding compound can comprise IFLLWQRCX (amino
acids 1 to 9 of SEQ ID NO:21), IFLLWQRCXX (amino acids 1 to 10 of
SEQ ID NO:21), IFLLWQRCXXX (amino acids 1 to 11 of SEQ ID NO:21),
IFLLWQRCXXXX (SEQ ID NO:21), wherein each X is independently a
polar or charged amino acid. For example, each X can independently
be selected from all, any set of 10, any set of 9, any set of 8,
any set of 7, any set of 6, any set of 5, any set of 4, any set of
3, any set of 2, or any 1 of the amino acids C, R, K, S, T, H, D,
E, N, Q, and M. For example, each X can independently be selected
from the set of three amino acids C, R, and K. As another example,
each X can independently be selected from the set of two amino
acids C and R. In some forms, a single one of the X can be C. In
some forms, two of the X can be C. In some forms, two of the X can
be R. In some forms, three of the X can be R. In some forms, four
of the X can be R. As an example, the annexin 1-binding compound
can comprise IFLLWQRCR (SEQ ID NO:17), IFLLWQRCRR (SEQ ID NO:19),
IFLLWQRCRRR (SEQ ID NO:18), or IFLLWQRCRRRR (SEQ ID NO:22).
[0097] The annexin 1-binding compound can comprise IFLLWQRCX (amino
acids 1 to 9 of SEQ ID NO:21), IFLLWQRCXX (amino acids 1 to 10 of
SEQ ID NO:21), IFLLWQRCXXX (amino acids 1 to 11 of SEQ ID NO:21),
IFLLWQRCXXXX (SEQ ID NO:21), wherein each X is independently a C,
R, K, S, T, H, D, E, N, Q, or M. In some forms, a single one of the
X can be C. In some forms, two of the X can be C. In some forms,
two of the X can be R. In some forms, three of the X can be R. In
some forms, four of the X can be R.
[0098] The annexin 1-binding compound can comprise IFLLWQRCX (amino
acids 1 to 9 of SEQ ID NO:21), IFLLWQRCXX (amino acids 1 to 10 of
SEQ ID NO:21), IFLLWQRCXXX (amino acids 1 to 11 of SEQ ID NO:21),
IFLLWQRCXXXX (SEQ ID NO:21), wherein each X is independently a C,
R, or K. In some forms, a single one of the X can be C. In some
forms, two of the X can be C. In some forms, two of the X can be R.
In some forms, three of the X can be R. In some forms, four of the
X can be R.
[0099] The annexin 1-binding compound can comprise IFLLWQRCX (amino
acids 1 to 9 of SEQ ID NO:21), IFLLWQRCXX (amino acids 1 to 10 of
SEQ ID NO:21), IFLLWQRCXXX (amino acids 1 to 11 of SEQ ID NO:21),
IFLLWQRCXXXX (SEQ ID NO:21), wherein each X is independently a C or
R. In some forms, a single one of the X can be C. In some forms,
two of the X can be C. In some forms, two of the X can be R. In
some forms, three of the X can be R. In some forms, four of the X
can be R.
[0100] The annexin 1-binding compound can consist of IFLLWQRCX
(amino acids 1 to 9 of SEQ ID NO:21), IFLLWQRCXX (amino acids 1 to
10 of SEQ ID NO:21), IFLLWQRCXXX (amino acids 1 to 11 of SEQ ID
NO:21), IFLLWQRCXXXX (SEQ ID NO:21), wherein each X is
independently a polar or charged amino acid. For example, each X
can independently be selected from all, any set of 10, any set of
9, any set of 8, any set of 7, any set of 6, any set of 5, any set
of 4, any set of 3, any set of 2, or any 1 of the amino acids C, R,
K, S, T, H, D, E, N, Q, and M. For example, each X can
independently be selected from the set of three amino acids C, R,
and K. As another example, each X can independently be selected
from the set of two amino acids C and R. In some forms, a single
one of the X can be C. In some forms, two of the X can be C. In
some forms, two of the X can be R. In some forms, three of the X
can be R. In some forms, four of the X can be R. As an example, the
annexin 1-binding compound can consist of IFLLWQRCR (SEQ ID NO:17),
IFLLWQRCRR (SEQ ID NO:19), IFLLWQRCRRR (SEQ ID NO:18), or
IFLLWQRCRRRR (SEQ ID NO:22).
[0101] The annexin 1-binding compound can consist of IFLLWQRCX
(amino acids 1 to 9 of SEQ ID NO:21), IFLLWQRCXX (amino acids 1 to
10 of SEQ ID NO:21), IFLLWQRCXXX (amino acids 1 to 11 of SEQ ID
NO:21), IFLLWQRCXXXX (SEQ ID NO:21), wherein each X is
independently a C, R, K, S, T, H, D, E, N, Q, or M. In some forms,
a single one of the X can be C. In some forms, two of the X can be
C. In some forms, two of the X can be R. In some forms, three of
the X can be R. In some forms, four of the X can be R.
[0102] The annexin 1-binding compound can consist of IFLLWQRCX
(amino acids 1 to 9 of SEQ ID NO:21), IFLLWQRCXX (amino acids 1 to
10 of SEQ ID NO:21), IFLLWQRCXXX (amino acids 1 to 11 of SEQ ID
NO:21), IFLLWQRCXXXX (SEQ ID NO:21), wherein each X is
independently a C, R, or K. In some forms, a single one of the X
can be C. In some forms, two of the X can be C. In some forms, two
of the X can be R. In some forms, three of the X can be R. In some
forms, four of the X can be R.
[0103] The annexin 1-binding compound can consist of IFLLWQRCX
(amino acids 1 to 9 of SEQ ID NO:21), IFLLWQRCXX (amino acids 1 to
10 of SEQ ID NO:21), IFLLWQRCXXX (amino acids 1 to 11 of SEQ ID
NO:21), IFLLWQRCXXXX (SEQ ID NO:21), wherein each X is
independently a C or R. In some forms, a single one of the X can be
C. In some forms, two of the X can be C. In some forms, two of the
X can be R. In some forms, three of the X can be R. In some forms,
four of the X can be R.
[0104] In some forms, the portion of the annexin 1-binding compound
corresponding to the sequence IFLLWQRC (amino acids 1 to 8 of SEQ
ID NO:21) can have one or more conservative amino acid
substitutions in amino acids 1 to 8 of SEQ ID NO:21. In some forms,
the portion of the annexin 1-binding compound corresponding to the
sequence IFLLWQRC (amino acids 1 to 8 of SEQ ID NO:21) can have at
least 55% sequence identity to the sequence IFLLWQRC (amino acids 1
to 8 of SEQ ID NO:21), wherein differences between the annexin
1-binding compound and IFLLWQRC (amino acids 1 to 8 of SEQ ID
NO:21) consist of conservative amino acid substitutions. In some
forms, the portion of the annexin 1-binding compound corresponding
to the sequence IFLLWQRC (amino acids 1 to 8 of SEQ ID NO:21) can
have at least 70% sequence identity to the sequence IFLLWQRC (amino
acids 1 to 8 of SEQ ID NO:21). In some forms, the portion of the
annexin 1-binding compound corresponding to the sequence IFLLWQRC
(amino acids 1 to 8 of SEQ ID NO:21) can have at least 80% sequence
identity to the sequence IFLLWQRC (amino acids 1 to 8 of SEQ ID
NO:21). In some forms, the annexin 1-binding compound has at least
5 consecutive amino acids of the sequence IFLLWQRC (amino acids 1
to 8 of SEQ ID NO:21). In some forms, the annexin 1-binding
compound has at least 6 consecutive amino acids of the sequence
IFLLWQRCR (SEQ ID NO:17), IFLLWQRCRR (SEQ ID NO:19), IFLLWQRCRRR
(SEQ ID NO:18), or IFLLWQRCRRRR (SEQ ID NO:22).
[0105] The annexin 1-binding compound can comprise amino acids 1 to
8, 1 to 9, 1 to 10, 1 to 11, or 1 to 12 of SEQ ID NO:20 having
none, one, or more conservative amino acid substitutions in amino
acids 1 to 7. The annexin 1-binding compound can comprise amino
acids 1 to 8, 1 to 9, 1 to 10, 1 to 11, or 1 to 12 of SEQ ID NO:20
and the portion of the annexin 1-binding compound corresponding to
the sequence IFLLWQR (amino acids 1 to 7 of SEQ ID NO:20) can have
at least 55% sequence identity to amino acids 1 to 7 of SEQ ID
NO:20, wherein differences between the annexin 1-binding compound
and amino acids 1 to 7 of SEQ ID NO:20 consist of conservative
amino acid substitutions. The annexin 1-binding compound can
comprise amino acids 1 to 8, 1 to 9, 1 to 10, 1 to 11, or 1 to 12
of SEQ ID NO:20 and the portion of the annexin 1-binding compound
corresponding to the sequence IFLLWQR (amino acids 1 to 7 of SEQ ID
NO:20) can have at least 70% sequence identity to amino acids 1 to
7 of SEQ ID NO:20. The annexin 1-binding compound can comprise
amino acids 1 to 8, 1 to 9, 1 to 10, 1 to 11, or 1 to 12 of SEQ ID
NO:20 and the portion of the annexin 1-binding compound
corresponding to the sequence IFLLWQR (amino acids 1 to 7 of SEQ ID
NO:20) can have at least 80% sequence identity to amino acids 1 to
7 of SEQ ID NO:20. The annexin 1-binding compound can comprise
amino acids 1 to 8, 1 to 9, 1 to 10, 1 to 11, or 1 to 12 of SEQ ID
NO:20. The annexin 1-binding compound can consist of amino acids 1
to 8, 1 to 9, 1 to 10, 1 to 11, or 1 to 12 of SEQ ID NO:20. The
annexin 1-binding compound can comprise amino acid 8 or amino acids
8 to 9, 8 to 10, 8 to 11, or 8 to 12 of SEQ ID NO:20 and at least 5
consecutive amino acids of amino acids 1 to 7 of SEQ ID NO:20. The
annexin 1-binding compound can comprise amino acids 1 to 8, 1 to 9,
1 to 10, 1 to 11, or 1 to 12 of SEQ ID NO:20 and at least 6
consecutive amino acids of amino acids 1 to 7 of SEQ ID NO:20.
[0106] The annexin 1-binding compound can comprise amino acids 1 to
9, 1 to 10, 1 to 11, or 1 to 12 of SEQ ID NO:21 having none, one,
or more conservative amino acid substitutions in amino acids 1 to
8. The annexin 1-binding compound can comprise amino acids 1 to 9,
1 to 10, 1 to 11, or 1 to 12 of SEQ ID NO:21 and the portion of the
annexin 1-binding compound corresponding to the sequence IFLLWQRC
(amino acids 1 to 8 of SEQ ID NO:21) can have at least 55% sequence
identity to amino acids 1 to 8 of SEQ ID NO:21, wherein differences
between the annexin 1-binding compound and amino acids 1 to 8 of
SEQ ID NO:21 consist of conservative amino acid substitutions. The
annexin 1-binding compound can comprise amino acids 1 to 9, 1 to
10, 1 to 11, or 1 to 12 of SEQ ID NO:21 and the portion of the
annexin 1-binding compound corresponding to the sequence IFLLWQRC
(amino acids 1 to 8 of SEQ ID NO:21) can have at least 70% sequence
identity to amino acids 1 to 7 of SEQ ID NO:21. The annexin
1-binding compound can comprise amino acids 1 to 9, 1 to 10, 1 to
11, or 1 to 12 of SEQ ID NO:21 and the portion of the annexin
1-binding compound corresponding to the sequence IFLLWQRC (amino
acids 1 to 8 of SEQ ID NO:21) can have at least 80% sequence
identity to amino acids 1 to 8 of SEQ ID NO:21. The annexin
1-binding compound can comprise amino acids 1 to 9, 1 to 10, 1 to
11, or 1 to 12 of SEQ ID NO:21. The annexin 1-binding compound can
consist of amino acids 1 to 9, 1 to 10, 1 to 11, or 1 to 12 of SEQ
ID NO:21. The annexin 1-binding compound can comprise amino acid 9
or amino acids 9 to 10, 9 to 11, or 9 to 12 of SEQ ID NO:21 and at
least 5 consecutive amino acids of amino acids 1 to 8 of SEQ ID
NO:21. The annexin 1-binding compound can comprise amino acid 9 or
amino acids 9 to 10, 9 to 11, or 9 to 12 of SEQ ID NO:21 and at
least 6 consecutive amino acids of amino acids 1 to 8 of SEQ ID
NO:21.
[0107] The disclosed peptides and compositions can comprise any
number of annexin 1-binding amino acid sequences. By way of
example, the composition can comprise at least 1, 5, 10, 15, 20,
25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,
375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,
700, 625, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000,
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250,
2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000,
30,000, 35,000, 40,000, 45,000, 50,000, 75,000, or 100,000, or more
annexin 1-binding amino acid sequences. The peptide or composition
can also comprise any number in between those numbers listed
above.
[0108] The term "homing molecule" as used herein, means any
molecule that selectively homes in vivo to specified target sites
or tissues in preference to other tissue or normal tissue.
Similarly, the term "homing peptide" or "homing peptidomimetic"
means a peptide that selectively homes in vivo to specified target
sites or tissues in preference to other tissue or normal tissue. It
is understood that a homing molecule that selectively homes in vivo
to, for example, tumors can home to all tumors or can exhibit
preferential homing to one or a subset of tumor types.
[0109] By "selectively homes" it is meant that, in vivo, the homing
molecule binds preferentially to the target as compared to
non-target. For example, the homing molecule can bind
preferentially to tumor vasculature, as compared to non-tumoral
tissue or non-vascular tissue. Such a homing molecule can
selectively home, for example, to tumors. Selective homing to, for
example, tumors generally is characterized by at least a two-fold
greater localization within tumors (or other target), as compared
to several tissue types of non-tumor tissue. A homing molecule can
be characterized by 5-fold, 10-fold, 20-fold or more preferential
localization to tumors (or other target) as compared to several or
many tissue types of non-tumoral tissue, or as compared to-most or
all non-tumoral tissue. Thus, it is understood that, in some cases,
a homing molecule homes, in part, to one or more normal organs in
addition to homing to the target tissue. Selective homing can also
be referred to as targeting.
[0110] Many homing molecules and homing peptides home to the
vasculature of the target tissue. However, for the sake of
convenience homing is referred to in some places herein as homing
to the tissue or cells associated with the vasculature to which the
homing molecule or homing peptide may actually home. Thus, for
example, a homing peptide that homes to tumor vasculature can be
referred to herein as homing to tumor tissue or to tumor cells. By
including or associating a homing molecule or homing peptide with,
for example, a protein, peptide, amino acid sequence, or
composition the protein, peptide, amino acid sequence, or
composition can be targeted or can home to the target of the homing
molecule or homing peptide. In this way, the protein, peptide,
amino acid sequence, or composition can be said to home to the
target of the homing molecule or homing peptide. For convenience
and unless otherwise indicated, reference to homing of a protein,
peptide, amino acid sequence, composition, etc. is intended to
indicate that the protein, peptide, amino acid sequence,
composition, etc. includes or is associated with an appropriate
homing molecule or homing peptide.
[0111] The composition, peptide, or amino acid sequence can
selectively home to a tumor. The composition, peptide, or amino
acid sequence can selectively home to tumor vasculature. The
composition, peptide, or amino acid sequence can selectively home
to one or more particular types of tumor. The composition, peptide,
or amino acid sequence can selectively home to the vasculature of
one or more particular types of tumor. The composition, peptide, or
amino acid sequence can selectively home to one or more particular
stages of a tumor or cancer. The composition, peptide, or amino
acid sequence can selectively home to the vasculature of one or
more particular stages of a tumor or cancer. The composition,
peptide, or amino acid sequence can selectively home to one or more
particular stages of one or more particular types of tumor. The
composition, peptide, or amino acid sequence can selectively home
to the vasculature of one or more different stages of one or more
particular types of tumor.
[0112] The disclosed annexin 1-binding compounds can include
modified forms of annexin 1-binding compounds. The annexin
1-binding compounds can have any useful modification. For example,
some modifications can stabilize the annexin 1-binding compound.
For example, the disclosed annexin 1-binding amino acid sequences
include methylated annexin 1-binding amino acid sequences.
[0113] As used herein, a "methylated derivative" of a protein,
peptide, amino acid segment, amino acid sequence, etc. refers to a
form of the protein, peptide, amino acid segment, amino acid
sequence, etc. that is methylated. Unless the context indicates
otherwise, reference to a methylated derivative of a protein,
peptide, amino acid segment, amino acid sequence, etc. does not
include any modification to the base protein, peptide, amino acid
segment, amino acid sequence, etc. other than methylation.
Methylated derivatives can also have other modifications, but such
modifications generally will be noted. For example, conservative
variants of an amino acid sequence would include conservative amino
acid substitutions of the based amino acid sequence. Thus,
reference to, for example, a "methylated derivative" of a specific
amino acid sequence "and conservative variants thereof" would
include methylated forms of the specific amino acid sequence and
methylated forms of the conservative variants of the specific amino
acid sequence, but not any other modifications of derivations. As
another example, reference to a methylated derivative of an amino
acid segment that includes amino acid substitutions would include
methylated forms of the amino acid sequence and methylated forms of
the amino acid sequence that include amino acid substitutions.
[0114] Peptides can have a variety of modifications. Modifications
can be used to change or improve the properties of the peptides.
For example, the disclosed peptides can be N-methylated,
O-methylated, S-methylated, C-methylated, or a combination at one
or more amino acids.
[0115] The amino and/or carboxy termini of the disclosed peptides
can be modified. Amino terminus modifications include methylation
(e.g., --NHCH.sub.3 or --N(CH.sub.3).sub.2), acetylation (e.g.,
with acetic acid or a halogenated derivative thereof such as
.alpha.-chloroacetic acid, .alpha.-bromoacetic acid, or
.alpha.-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group,
or blocking the amino terminus with any blocking group containing a
carboxylate functionality defined by RCOO-- or sulfonyl
functionality defined by R--SO.sub.2--, where R is selected from
the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and
the like, and similar groups. One can also incorporate a desamino
acid at the N-terminus (so that there is no N-terminal amino group)
to decrease susceptibility to proteases or to restrict the
conformation of the peptide compound. In preferred embodiments, the
N-terminus is acetylated with acetic acid or acetic anhydride.
[0116] Carboxy terminus modifications include replacing the free
acid with a carboxamide group or forming a cyclic lactam at the
carboxy terminus to introduce structural constraints. One can also
cyclize the disclosed peptides, or incorporate a desamino or
descarboxy residue at the termini of the peptide, so that there is
no terminal amino or carboxyl group, to decrease susceptibility to
proteases or to restrict the conformation of the peptide.
C-terminal functional groups of the disclosed peptides include
amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,
hydroxy, and carboxy, and the lower ester derivatives thereof, and
the pharmaceutically acceptable salts thereof.
[0117] One can replace the naturally occurring side chains of the
genetically encoded amino acids (or the stereoisomeric D amino
acids) with other side chains, for instance with groups such as
alkyl, lower (C.sub.1-6) alkyl, cyclic 4-, 5-, 6-, to 7-membered
alkyl, amide, amide lower alkyl amide di(lower alkyl), lower
alkoxy, hydroxy, carboxy and the lower ester derivatives thereof,
and with 4-, 5-, 6-, to 7-membered heterocyclic. In particular,
proline analogues in which the ring size of the proline residue is
changed from 5 members to 4, 6, or 7 members can be employed.
Cyclic groups can be saturated or unsaturated, and if unsaturated,
can be aromatic or non-aromatic. Heterocyclic groups preferably
contain one or more nitrogen, oxygen, and/or sulfur heteroatoms.
Examples of such groups include the furazanyl, furyl,
imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl,
morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.,
1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl,
pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,
pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl),
pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl,
thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These
heterocyclic groups can be substituted or unsubstituted. Where a
group is substituted, the substituent can be alkyl, alkoxy,
halogen, oxygen, or substituted or unsubstituted phenyl.
[0118] One can also readily modify peptides by phosphorylation, and
other methods [e.g., as described in Hruby, et al. (1990) Biochem
J. 268:249-262].
[0119] The disclosed peptides also serve as structural models for
non-peptidic compounds with similar biological activity. Those of
skill in the art recognize that a variety of techniques are
available for constructing compounds with the same or similar
desired biological activity as the lead peptide compound, but with
more favorable activity than the lead with respect to solubility,
stability, and susceptibility to hydrolysis and proteolysis [See,
Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243-252]. These
techniques include, but are not limited to, replacing the peptide
backbone with a backbone composed of phosphonates, amidates,
carbamates, sulfonamides, secondary amines, and N-methylamino
acids.
[0120] All of the disclosed annexin 1-binding compounds, peptides,
amino acid sequences, moieties, therapeutic agents, etc. can be
used as described herein. Every generic or particular annexin
1-binding compounds, peptides, amino acid sequences, moieties,
therapeutic agents, etc. described herein can be specifically
included or excluded, either individually or in groups, in or from
any set of annexin 1-binding compounds, peptides, amino acid
sequences, moieties, therapeutic agents, etc. and/or in or from any
use. For example, the I-peptide and/or sequence variants of the
I-peptide can be specifically excluded from any set of, for
example, annexin 1-binding compounds, peptides, amino acid
sequences, etc. and/or from any use. As another example,
geldanamycin can be specifically excluded from any set of, for
example, moieties, therapeutic agents, etc. and/or from any use. As
another example, IF7 (SEQ ID NO:2) and/or sequence variants of IF7
can be specifically excluded from any set of, for example, annexin
1-binding compounds, peptides, amino acid sequences, etc. and/or
from any use. Every generic or particular annexin 1-binding
compounds, peptides, amino acid sequences, moieties, therapeutic
agents, etc. described herein can be specifically included or
excluded, either individually or in groups, in or from any set of
annexin 1-binding compounds, peptides, amino acid sequences,
moieties, therapeutic agents, etc. as described herein and/or in or
from any use as described herein.
B. Peptides and Amino Acid Sequences
[0121] In some forms, the disclosed peptides and amino acid
sequences can be or include a peptide, peptidomimetic, and/or amino
acid segment. Unless the context indicates otherwise, reference
herein to "peptide" is intended to refer also to amino acid
sequences, which can form a part of, or constitute an entire,
peptide. The disclosed peptides can be in isolated form. As used
herein in reference to the disclosed peptides, the term "isolated"
means a peptide that is in a form that is relatively free from
material such as contaminating polypeptides, lipids, nucleic acids
and other cellular material that normally is associated with the
peptide in a cell or that is associated with the peptide in a
library or in a crude preparation.
[0122] The disclosed peptides and amino acid sequences can have any
suitable length. The disclosed peptides can have, for example, a
relatively short length of less than six, seven, eight, nine, ten,
12, 15, 20, 25, 30, 35 or 40 residues. The disclosed peptides also
can be useful in the context of a significantly longer sequence.
Thus, the peptides can have, for example, a length of up to 50,
100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In
particular embodiments, a peptide can have a length of at least 10,
20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further
embodiments, a peptide can have a length of 5 to 200 residues, 5 to
100 residues, 5 to 90 residues, 5 to 80 residues, 5 to 70 residues,
5 to 60 residues, 5 to 50 residues, 5 to 40 residues, 5 to 30
residues, 5 to 20 residues, 5 to 15 residues, 5 to 10 residues, 10
to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80
residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues,
10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 20 to 200
residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues,
20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40
residues or 20 to 30 residues. As used herein, the term "residue"
refers to an amino acid or amino acid analog.
[0123] The disclosed amino acid sequences can have, for example, a
relatively short length of less than six, seven, eight, nine, ten,
12, 15, 20, 25, 30, 35 or 40 residues. The disclosed amino acid
sequences also can be useful in the context of a significantly
longer sequence. Thus, the amino acid sequences can have, for
example, a length of up to 50, 100, 150, 200, 250, 300, 400, 500,
1000 or 2000 residues. In particular embodiments, an amino acid
sequence can have a length of at least 10, 20, 30, 40, 50, 60, 70,
80, 90, 100 or 200 residues. In further embodiments, an amino acid
sequence can have a length of 5 to 200 residues, 5 to 100 residues,
5 to 90 residues, 5 to 80 residues, 5 to 70 residues, 5 to 60
residues, 5 to 50 residues, 5 to 40 residues, 5 to 30 residues, 5
to 20 residues, 5 to 15 residues, 5 to 10 residues, 10 to 200
residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues,
10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40
residues, 10 to 30 residues, 10 to 20 residues, 20 to 200 residues,
20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70
residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues
or 20 to 30 residues. As used herein, the term "residue" refers to
an amino acid or amino acid analog.
[0124] As this specification discusses various proteins, protein
sequences, peptides, peptides sequences, and amino acid sequences,
it is understood that the nucleic acids that can encode those
sequences are also disclosed. This would include all degenerate
sequences related to a specific protein sequence, i.e. all nucleic
acids having a sequence that encodes one particular protein
sequence as well as all nucleic acids, including degenerate nucleic
acids, encoding the disclosed variants and derivatives of the
protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence.
[0125] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include
--CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH-- (cis and trans), --COCH.sub.2 --, --CH(OH)CH.sub.2--,
and --CH.sub.2SO-- (These and others can be found in Spatola, A. F.
in Chemistry and Biochemistry of Amino Acids, Peptides, and
Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267
(1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,
Peptide Backbone Modifications (general review); Morley, Trends
Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot
Res 14:177-185 (1979) (--CH.sub.2NH--, --CH.sub.2CH.sub.2--);
Spatola et al. Life Sci 38:1243-1249 (1986) (--CH.sub.2S--); Hann
J. Chem. Soc Perkin Trans. I 307-314 (1982) (--CH.dbd.CH--, cis and
trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980)
(--COCH.sub.2--); Jennings-White et al. Tetrahedron Lett 23:2533
(1982) (--COCH.sub.2--); Szelke et al. European Appln, EP 45665 CA
(1982): 97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay et al.
Tetrahedron. Lett 24:4401-4404 (1983) (--CH(OH)CH.sub.2--); and
Hruby Life Sci 31:189-199 (1982) (--CH.sub.2--S--); each of which
is incorporated herein by reference. A particularly preferred
non-peptide linkage is --CH.sub.2NH--. It is understood that
peptide analogs can have more than one atom between the bond atoms,
such as .beta.-alanine, .gamma.-aminobutyric acid, and the
like.
[0126] Also disclosed are bifunctional peptides, which contain, for
example, an annexin1-binding peptide fused to a second peptide
having a separate function. Such bifunctional peptides have at
least two functions conferred by different portions of the
full-length molecule and can, for example, display anti-angiogenic
activity or pro-apoptotic activity in addition to tumor homing.
[0127] Also disclosed are isolated multivalent peptides that
include at least two subsequences each independently containing a
peptide or amino acid sequence (for example, the amino acid
sequence SEQ ID NO: 2, or a conservative variant or peptidomimetic
thereof). The multivalent peptide can have, for example, at least
three, at least five or at least ten of such subsequences each
independently containing a peptide. In particular embodiments, the
multivalent peptide can have two, three, four, five, six, seven,
eight, nine, ten, fifteen or twenty identical or non-identical
subsequences. This is in addition to the multiple annexin 1-binding
amino acid sequences that can comprise the disclosed compositions.
In a further embodiment, the multivalent peptide can contain
identical subsequences, such as repeats of SEQ ID NO: 2. In a
further embodiment, the multivalent peptide contains contiguous
identical or non-identical subsequences, which are not separated by
any intervening amino acids.
[0128] As used herein, the term "peptide" is used broadly to mean
peptides, proteins, fragments of proteins and the like. The term
"peptidomimetic," as used herein, means a peptide-like molecule
that has the activity of the peptide upon which it is structurally
based. Such peptidomimetics include chemically modified peptides,
peptide-like molecules containing non-naturally occurring amino
acids, and peptoids and have an activity such as selective
interaction with a target of the peptide upon which the
peptidomimetic is derived (see, for example, Goodman and Ro,
Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry
and Drug Discovery" Vol. 1 (ed. M. E. Wolff; John Wiley & Sons
1995), pages 803-861).
[0129] A variety of peptidomimetics are known in the art including,
for example, peptide-like molecules which contain a constrained
amino acid, a non-peptide component that mimics peptide secondary
structure, or an amide bond isostere. A peptidomimetic that
contains a constrained, non-naturally occurring amino acid can
include, for example, an .alpha.-methylated amino acid;
.alpha.,.alpha.-dialkylglycine or .alpha.-aminocycloalkane
carboxylic acid; an N.sup..alpha.--C.sup..alpha. cyclized amino
acid; an N.sup..alpha.-methylated amino acid; a .beta.- or
.gamma.-amino cycloalkane carboxylic acid; an
.alpha.,.beta.-unsaturated amino acid; a .beta.,.beta.-dimethyl or
.beta.-methyl amino acid; a .beta.-substituted-2,3-methano amino
acid; an N--C.sup..epsilon. or C.sup..alpha.--C.sup..DELTA.
cyclized amino acid; a substituted proline or another amino acid
mimetic. A peptidomimetic which mimics peptide secondary structure
can contain, for example, a non-peptidic .beta.-turn mimic;
.gamma.-turn mimic; mimic of .beta.-sheet structure; or mimic of
helical structure, each of which is well known in the art. A
peptidomimetic also can be a peptide-like molecule which contains,
for example, an amide bond isostere such as a retro-inverse
modification; reduced amide bond; methylenethioether or
methylene-sulfoxide bond; methylene ether bond; ethylene bond;
thioamide bond; trans-olefin or fluoroolefin bond;
1,5-disubstituted tetrazole ring; ketomethylene or
fluoroketomethylene bond or another amide isostere. One skilled in
the art understands that these and other peptidomimetics are
encompassed within the meaning of the term "peptidomimetic" as used
herein.
[0130] 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. As an example, the
Cambridge Structural Database contains a collection of greater than
300,000 compounds that have known crystal structures (Allen et al.,
Acta Crystalloqr. 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 disclosed peptide, as well
as potential geometrical and chemical complementarity to a target
molecule. Where no crystal structure of a peptide or a target
molecule that binds the peptide is available, a structure can be
generated using, for example, the program CONCORD (Rusinko et al.,
J. Chem. Inf. Comput. Sci. 29:251 (1989)). Another database, the
Available Chemicals Directory (Molecular Design Limited,
Information Systems; San Leandro Calif.), contains about 100,000
compounds that are commercially available and also can be searched
to identify potential peptidomimetics of a peptide, for example,
with activity in selectively interacting with cancerous cells.
C. Moieties
[0131] The compositions disclosed herein can comprise one or more
moieties. For example, moieties can be molecules, conjugates,
associations, compositions, and mixtures. Examples of moieties
include, but are not limited to, anti-angiogenic agents,
pro-angiogenic agents, cancer chemotherapeutic agents, cytotoxic
agents, anti-inflammatory agents, anti-arthritic agents,
polypeptides, nucleic acid molecules, small molecules,
nanoparticles, and microparticles. At least one of the moieties can
be a therapeutic agent. Examples of therapeutic agents are
paclitaxel and docetaxel. At least one of the moieties can be a
detectable agent. Moieties that are peptides or amino acid
sequences can be referred to as moiety peptides or moiety amino
acid sequences, respectively.
[0132] As used herein, the term "moiety" is used broadly to mean a
physical, chemical, or biological material that generally imparts a
biologically useful function to a linked or conjugated molecule. As
disclosed herein, the properties of the moiety can also be found in
a peptide or amino acid sequence, or both the peptide or amino acid
sequence and the moiety can share one of the traits disclosed
herein. For example, the peptide or amino acid sequence can
comprise a detectable agent, while the moiety can comprise a
therapeutic agent. This also applies for the annexin 1-binding
compound, which can also comprise one or more of the properties of
moieties as disclosed herein. The description of therapeutic and
detectable agents which follows is intended to apply to any of
moieties, peptides, amino acid sequences, or annexin 1-binding
compounds. Thus, for example, moieties can be conjugated to,
coupled to, or can be part of the disclosed peptides, amino acid
sequences, annexin 1-binding compounds, compositions, or conjugates
of peptides, amino acid sequences and annexin 1-binding
compounds.
[0133] A moiety can be any natural or nonnatural material
including, without limitation, a biological material, such as a
cell, phage or other virus; an organic chemical such as a small
molecule; a nanoparticle, a radionuclide; a nucleic acid molecule
or oligonucleotide; a polypeptide; or a peptide. For example,
moieties can affect the target, such as moieties with therapeutic
effect, or can facilitate detection, visualization or imaging of
the target, such as fluorescent molecule or radionuclides. Useful
moieties include, but are not limited to, therapeutic agents such
as cancer chemotherapeutic agents, cytotoxic agents, pro-apoptotic
agents, and anti-angiogenic agents; detectable labels and imaging
agents; and tags or other insoluble supports. Useful moieties
further include, without limitation, phage and other viruses,
cells, liposomes, polymeric matrices, non-polymeric matrices or
particles such as gold particles, microdevices and nanodevices, and
nano-scale semiconductor materials. These and other moieties known
in the art can be components of a composition.
[0134] Components of the disclosed compositions can be combined,
linked and/or coupled in any suitable manner. For example, moieties
and other molecules can be associated covalently or non-covalently,
directly or indirectly, with or without a linker moiety.
[0135] 1. Therapeutic Agents
[0136] The moiety can be a therapeutic agent. As used herein, the
term "therapeutic agent" means a molecule which has one or more
biological activities in a normal or pathologic tissue. A variety
of therapeutic agents can be used as a moiety. The therapeutic
agent can comprise a compound or composition for treating cancer.
The therapeutic agent can comprise a compound or composition to
induce programmed cell death or apoptosis. For example, the
therapeutic agent can be (KLAKLAK).sub.2 or .sub.D(KLAKLAK).sub.2
(SEQ ID NO:24).
[0137] In some embodiments, the therapeutic agent can be a cancer
chemotherapeutic agent. As used herein, a "cancer chemotherapeutic
agent" is a chemical agent that inhibits the proliferation, growth,
life-span or metastatic activity of cancer cells. Such a cancer
chemotherapeutic agent can be, without limitation, a taxane such as
docetaxel; an anthracyclin such as doxorubicin; an alkylating
agent; a vinca alkaloid; an anti-metabolite such as methotrexate; a
platinum agent such as cisplatin, carboplatin, or oxaliplatin; a
steroid; an antibiotic such as adriamycin; a ifosfamide; or a
selective estrogen receptor modulator; an antibody such as
trastuzumab; paclitaxel such as Abraxane.
[0138] Taxanes are chemotherapeutic agents useful with the
compositions disclosed herein. Useful taxanes include, without
limitation, docetaxel (Taxotere; sanofi-aventis; Parsippany, N.J.)
and paclitaxel (Taxol; Bristol-Myers Squibb; Princeton, N.J.). See,
for example, Chan et al., J. Clin. Oncol. 17:2341-2354 (1999), and
Paridaens et al., J. Clin. Oncol. 18:724 (2000).
[0139] A cancer chemotherapeutic agent useful with the compositions
disclosed herein also can be an anthracyclin such as doxorubicin,
idarubicin or daunorubicin. Doxorubicin is a commonly used cancer
chemotherapeutic agent and can be useful, for example, for treating
breast cancer (Stewart and Ratain, In: "Cancer: Principles and
practice of oncology" 5th ed., chap. 19 (eds. DeVita, Jr., et al.;
J. P. Lippincott 1997); Harris et al., In "Cancer: Principles and
practice of oncology," supra, 1997). In addition, doxorubicin has
anti-angiogenic activity (Folkman, Nature Biotechnology 15:510
(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.
[0140] An alkylating agent such as melphalan, ifosfamide, or
chlorambucil also can be a useful cancer chemotherapeutic agent.
Similarly, a vinca alkaloid such as vindesine, vinblastine or
vinorelbine; or an antimetabolite such as 5-fluorouracil,
5-fluorouridine, methotrexate, or a derivative thereof can be a
useful cancer chemotherapeutic agent.
[0141] A platinum agent also can be a useful cancer
chemotherapeutic agent. Such a platinum agent can be, for example,
cisplatin, carboplatin, or oxaliplatin as described, for example,
in Crown, Seminars in Oncol. 28:28-37 (2001). Other useful cancer
chemotherapeutic agents include, without limitation, mitomycin-C,
adriamycin (doxorubicin), and ansamycins.
[0142] A cancer chemotherapeutic agent useful for treatment of
breast cancer and other hormonally-dependent cancers also can be an
agent that antagonizes the effect of estrogen, such as a selective
estrogen receptor modulator or an anti-estrogen. The selective
estrogen receptor modulator, tamoxifen, is a cancer
chemotherapeutic agent that can be used in a composition for
treatment of breast cancer (Fisher et al., J. Natl. Cancer Instit.
90:1371-1388 (1998)).
[0143] The therapeutic agent can be an antibody such as a humanized
monoclonal antibody. As an example, the anti-epidermal growth
factor receptor 2 (HER2) antibody, trastuzumab (Herceptin;
Genentech, South San Francisco, Calif.) can be a therapeutic agent
useful for treating HER2/neu overexpressing breast cancers (White
et al., Annu. Rev. Med. 52:125-141 (2001)).
[0144] Useful therapeutic agents also can be a cytotoxic agent,
which, as used herein, can be any molecule that directly or
indirectly promotes cell death. Useful cytotoxic agents include,
without limitation, small molecules, polypeptides, peptides,
peptidomimetics, nucleic acid-molecules, cells and viruses. As
non-limiting examples, useful cytotoxic agents include cytotoxic
small molecules such as doxorubicin, docetaxel or trastuzumab;
antimicrobial peptides such as those described further below;
pro-apoptotic polypeptides such as caspases and toxins, for
example, caspase-8; diphtheria toxin A chain, Pseudomonas exotoxin
A, cholera toxin, ligand fusion toxins such as DAB389EGF, Ricinus
communis toxin (ricin); and cytotoxic cells such as cytotoxic T
cells. See, for example, Martin et al., Cancer Res. 60:3218-3224
(2000); Kreitman and Pastan, Blood 90:252-259 (1997); Allam et al.,
Cancer Res. 57:2615-2618 (1997); and Osborne and Coronado-Heinsohn,
Cancer J. Sci. Am. 2:175 (1996). One skilled in the art understands
that these and additional cytotoxic agents described herein or
known in the art can be useful in the disclosed compositions and
methods.
[0145] In some forms, a therapeutic agent can be a therapeutic
polypeptide. As used herein, a therapeutic polypeptide can be any
polypeptide with a biologically useful function. Useful therapeutic
polypeptides encompass, without limitation, cytokines, antibodies,
cytotoxic polypeptides; pro-apoptotic polypeptides; and
anti-angiogenic polypeptides. As non-limiting examples, useful
therapeutic polypeptides can be a cytokine such as tumor necrosis
factor-.alpha. (TNF-.alpha.), tumor necrosis factor-.beta.
(TNF-.beta.), granulocyte macrophage colony stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF),
interferon-.alpha. (IFN-.alpha.); interferon-.gamma. (IFN-.gamma.),
interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3),
interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7),
interleukin-10 (IL-10), interleukin-12 (IL-12), lymphotactin (LTN)
or dendritic cell chemokine 1 (DC-CK1); an anti-HER2 antibody or
fragment thereof; a cytotoxic polypeptide including a toxin or
caspase, for example, diphtheria toxin A chain, Pseudomonas
exotoxin A, cholera toxin, a ligand fusion toxin such as DAB389EGF
or ricin; or an anti-angiogenic polypeptide such as angiostatin,
endostatin, thrombospondin, platelet factor 4; anastellin; or one
of those described further herein or known in the art. It is
understood that these and other polypeptides with biological
activity can be a "therapeutic polypeptide." Examples of
pro-apoptotic therapeutic agents are (KLAKLAK).sub.2 or
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:24) (del Rio, 2001; Ellerby,
1999). .sub.D(KLAKLAK).sub.2 (SEQ ID NO:24) refers to the sequence
KLAKLAKKLAKLAK (SEQ ID NO:24) made with D amino acids. These
peptides can be used in any of the disclosed compositions and
combined with any of the disclosed peptides or annexin 1-binding
compounds. Examples of such compositions include
IFLLWQR-KLAKLAKKLAKLAK (SEQ ID NOs:2 and 24),
IFLLWQRC-KLAKLAKKLAKLAK (SEQ ID NOs:14 and 24),
IFLLWQRCR-KLAKLAKKLAKLAK (SEQ ID NOs:17 and 24),
IFLLWQRCRR-KLAKLAKKLAKLAK (SEQ ID NOs:19 and 24),
IFLLWQRCRRR-KLAKLAKKLAKLAK (SEQ ID NOs:18 and 24), or
IFLLWQRCRRRR-KLAKLAKKLAKLAK (SEQ ID NOs:22 and 24).
[0146] A therapeutic agent can also be an anti-angiogenic agent. As
used herein, the term "anti-angiogenic agent" means a molecule that
reduces or prevents angiogenesis, which is the growth and
development of blood vessels. A variety of anti-angiogenic agents
can be prepared by routine methods. Such anti-angiogenic agents
include, without limitation, small molecules; proteins such as
dominant negative forms of angiogenic factors, transcription
factors and antibodies; peptides; and nucleic acid molecules
including ribozymes, antisense oligonucleotides, and nucleic acid
molecules encoding, for example, dominant negative forms of
angiogenic factors and receptors, transcription factors, and
antibodies and antigen-binding fragments thereof. See, for example,
Hagedom and Bikfalvi, Crit. Rev. Oncol. Hematol. 34:89-110 (2000),
and Kirsch et al., J. Neurooncol. 50:149-163 (2000).
[0147] Vascular endothelial growth factor (VEGF) has been shown to
be important for angiogenesis in many types of cancer, including
breast cancer angiogenesis in vivo (Borgstrom et al., Anticancer
Res. 19:4213-4214 (1999)). The biological effects of VEGF include
stimulation of endothelial cell proliferation, survival, migration
and tube formation, and regulation of vascular permeability. An
anti-angiogenic agent can be, for example, an inhibitor or
neutralizing antibody that reduces the expression or signaling of
VEGF or another angiogenic factor, for example, an anti-VEGF
neutralizing monoclonal antibody (Borgstrom et al., supra, 1999).
An anti-angiogenic agent also can inhibit another angiogenic factor
such as a member of the fibroblast growth factor family such as
FGF-1 (acidic), FGF-2 (basic), FGF-4 or FGF-5 (Slavin et al., Cell
Biol. 19:431-444 (1995); Folkman and Shing, J. Biol. Chem.
267:10931-10934 (1992)) or an angiogenic factor such as
angiopoietin-1, a factor that signals through the endothelial
cell-specific Tie2 receptor tyrosine kinase (Davis et al., Cell
87:1161-1169 (1996); and Suri et al., Cell 87:1171-1180 (1996)), or
the receptor of one of these angiogenic factors. It is understood
that a variety of mechanisms can act to inhibit activity of an
angiogenic factor including, without limitation, direct inhibition
of receptor binding, indirect inhibition by reducing secretion of
the angiogenic factor into the extracellular space, or inhibition
of expression, function or signaling of the angiogenic factor.
[0148] A variety of other molecules also can function as
anti-angiogenic agents including, without limitation, angiostatin;
a kringle peptide of angiostatin; endostatin; anastellin,
heparin-binding fragments of fibronectin; modified forms of
antithrombin; collagenase inhibitors; basement membrane turnover
inhibitors; angiostatic steroids; platelet factor 4 and fragments
and peptides thereof, thrombospondin and fragments and peptides
thereof, and doxorubicin (O'Reilly et al., Cell 79:315-328 (1994));
O'Reilly et al., Cell 88:277-285 (1997); Homandberg et al., Am. J.
Path. 120:327-332 (1985); Homandberg et-al., Biochim. Biophys. Acta
874:61-71 (1986); and O'Reilly et al., Science 285:1926-1928
(1999)). Commercially available anti-angiogenic agents include, for
example, angiostatin, endostatin, metastatin and 2ME2 (EntreMed;
Rockville, Md.); anti-VEGF antibodies such as Avastin (Genentech;
South San Francisco, Calif.); and VEGFR-2 inhibitors such as
SU5416, a small molecule inhibitor of VEGFR-2 (SUGEN; South San
Francisco, Calif.) and SU6668 (SUGEN), a small molecule inhibitor
of VEGFR-2, platelet derived growth factor and fibroblast growth
factor I receptor. It is understood that these and other
anti-angiogenic agents can be prepared by routine methods and are
encompassed by the term "anti-angiogenic agent" as used herein.
[0149] Some other examples of useful therapeutic agents include
nitrogen mustards, nitrosoureas, ethyleneimine, alkane sulfonates,
tetrazine, platinum compounds, pyrimidine analogs, purine analogs,
antimetabolites, folate analogs, anthracyclines, taxanes, vinca
alkaloids, topoisomerase inhibitors and hormonal agents. Exemplary
chemotherapy drugs are Actinomycin-D, Alkeran, Ara-C, Anastrozole,
Asparaginase, BiCNU, Bicalutamide, Bleomycin, Busulfan,
Capecitabine, Carboplatin, Carboplatinum, Carmustine, CCNU,
Chlorambucil, Chlomaphazine, Cisplatin, Cladribine, CPT-11,
Cyclophosphamide, Cytarabine, Cytosine arabinoside, Cytoxan,
Dacarbazine, Dactinomycin, Daunorubicin, Dexrazoxane, Docetaxel
(TAXOTERE.RTM., Rhone-Poulenc Rorer, Antony, France), Doxil,
Doxorubicin, DTIC, Epirubicin, Estramustine, Ethyleneimine,
Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide,
Fotemustine, Gemcitabine, Herceptin, Hexamethylamine, Hydroxyurea,
Idarubicin, Ifosfamide, Irinotecan, Lomustine, Mechlorethamine,
mechlorethamine oxide hydrochloride, Melphalan, Mercaptopurine,
Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Novembiehin,
Oxaliplatin, Paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology,
Princeton, N.J.), Pamidronate, Pentostatin, Phenesterine,
Plicamycin, Prednimustine, Procarbazine, Rituximab, Steroids,
STI-571, Streptozocin, Tamoxifen, Temozolomide, Teniposide,
Tetrazine, Thioguanine, Thiotepa, Tomudex, Topotecan, Treosulphan,
Trimetrexate, Trofosfamide, Vinblastine, Vincristine, Vindesine,
Vinorelbine, VP-16, and Xeloda. Alkylating agents such as Thiotepa
and; alkyl sulfonates such as Busulfan, Improsulfan and Piposulfan;
aziridines such as Benzodopa, Carboquone, Meturedopa, and Uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitroureas
such as Cannustine, Chlorozotocin, Fotemustine, Lomustine,
Nimustine, and Ranimustine; antibiotics such as Aclacinomysins,
Actinomycin, Authramycin, Azaserine, Bleomycins, Cactinomycin,
Calicheamicin, Carabicin, Caminomycin, Carzinophilin,
Chromoinycins, Dactinomycin, Daunorubicin, Detorubicin,
6-diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Esorubicin,
Idambicin, Marcellomycin, Mitomycins, mycophenolic acid,
Nogalamycin, Olivomycins, Peplomycin, Potfiromycin, Puromycin,
Quelamycin, Rodorubicin, Streptonigrin, Streptozocin, Tubercidin,
Ubenimex, Zinostatin, and Zorubicin; anti-metabolites such as
Methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as Denopterin, Methotrexate, Pteropterin, and Trimetrexate; purine
analogs such as Fludarabine, 6-mercaptopurine, Thiamiprine, and
Thioguanine; pyrimidine analogs such as Ancitabine, Azacitidine,
6-azauridine, Carmofur, Cytarabine, Dideoxyuridine, Doxifluridine,
Enocitabine, Floxuridine, and 5-FU; androgens such as Calusterone,
Dromostanolone Propionate, Epitiostanol, Rnepitiostane, and
Testolactone; anti-adrenals such as aminoglutethimide, Mitotane,
and Trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine;
Demecolcine; Diaziquone; Elfomithine; elliptinium acetate;
Etoglucid; gallium nitrate; hydroxyurea; Lentinan; Lonidamine;
Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin;
Phenamet; Pirarubicin; podophyllinic acid; 2-ethylhydrazide;
Procarbazine; PSK; Razoxane; Sizofrran; Spirogermanium; tenuazonic
acid; triaziquone; 2,2',2''-trichlorotriethylamine; Urethan;
Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol;
Pipobroman; Gacytosine; Arabinoside ("Ara-C"); cyclophosphamide;
thiotepa; taxoids, e.g., Paclitaxel and Doxetaxel; Gemcitabine;
6-thioguanine; Mercaptopurine; Methotrexate; platinum analogs such
as Cisplatin, Carboplatin, and Oxaliplatin; etoposide (VP-16);
Ifosfamide; Mitomycin C; Mitoxantrone; Vinblastine; Vincristine;
Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin;
Aminopterin; Xeloda; Ibandronate; CPT-11; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
Esperamicins; Capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included are
anti-hormonal agents that act to regulate or inhibit hormone action
on tumors such as anti-estrogens including for example Tamoxifen,
Raloxifene, aromatase inhibiting 4(5)-imidazoles, 4
Hydroxytamoxifen, Trioxifene, Keoxifene, Onapristone, And
Toremifene (Fareston); and anti-androgens such as Flutamide,
Nilutamide, Bicalutamide, Leuprolide, and Goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. Useful therapeutic agents include, for example,
doxorubicin, Herceptin, and liposomal doxorubicin.
[0150] The therapeutic agents can also comprise a boron containing
compound. Boron containing compounds have received increasing
attention as therapeutic agents over the past few years as
technology in organic synthesis has expanded to include this atom
(Boron Therapeutics on the horizon, Groziak, M. P.; American
Journal of Therapeutics (2001) 8, 321-328). The most notable boron
containing therapeutic is the boronic acid bortezomib which was
recently launched for the treatment of multiple myeloma. This
breakthrough demonstrates the feasibility of using boron containing
compounds as pharmaceutical agents. Boron containing compounds have
been shown to have various biological activities including
herbicides (Organic boron compounds as herbicides. Barnsley, G. E.;
Eaton, J. K.; Airs, R. S.; (1957), DE 1016978 19571003), boron
neutron capture therapy (Molecular Design and Synthesis of B-10
Carriers for Neutron Capture Therapy. Yamamoto, Y.; Pure Appl.
Chem., (1991) 63, 423-426), serine protease inhibition (Borinic
acid inhibitors as probes of the factors involved in binding at the
active sites of subtilisin Carlsberg and .alpha.-chymotrypsin.
Simpelkamp, J.; Jones, J. B.; Bioorganic & Medicinal Chemistry
Letters, (1992), 2(11), 1391-4; Design, Synthesis and Biological
Evaluation of Selective Boron-containing Thrombin Inhibitors.
Weinand, A.; Ehrhardt, C.; Metternich, R.; Tapparelli, C.;
Bioorganic and Medicinal Chemistry, (1999), 7, 1295-1307),
acetylcholinesterase inhibition (New, specific and reversible
bifunctional alkylborinic acid inhibitor of acetylcholinesterase.
Koehler, K. A.; Hess, G. P.; Biochemistry (1974), 13, 5345-50) and
as antibacterial agents (Boron-Containing Antibacterial Agents:
Effects on Growth and Morphology of Bacteria Under Various Culture
Conditions. Bailey, P. J.; Cousins, G.; Snow, G. A.; and White, A.
J.; Antimicrobial Agents and Chemotherapy, (1980), 17, 549-553).
The boron containing compounds with antibacterial activity can be
sub-divided into two main classes, the diazaborinines, which have
been known since the 1960's, and dithienylborinic acid complexes.
This latter class has been expanded to include many different
diarylborinic acid complexes with potent antibacterial activity
(Preparation of diarylborinic acid esters as DNA methyl transferase
inhibitors. Benkovic, S. J.; Shapiro, L.; Baker, S. J.; Wahnon, D.
C.; Wall, M.; Shier, V. K.; Scott, C. P.; Baboval, J.; PCT Int.
Appl. (2002), WO 2002044184).
[0151] It is understood by one skilled in the art of medicinal
oncology that these and other agents are useful therapeutic agents,
which can be used separately or together in the disclosed
compositions and methods. Thus, it is understood that the
compositions disclosed herein can contain one or more of such
therapeutic agents and that additional components can be included
as part of the composition, if desired. As a non-limiting example,
it can be desirable in some cases to utilize an oligopeptide spacer
between the disclosed peptides, amino acid sequences, and annexin
1-binding compounds and the therapeutic agent (Fitzpatrick and
Gamett, Anticancer Drug Des. 10:1-9 (1995)).
[0152] 2. Detectable Agents
[0153] The moiety in the disclosed compositions can also be a
detectable agent. A variety of detectable agents are useful in the
disclosed methods. As used herein, the term "detectable agent"
refers to any molecule which can be detected. Useful detectable
agents include compounds and molecules that can be administered in
vivo and subsequently detected. Detectable agents useful in the
disclosed compositions and methods include yet are not limited to
radiolabels and fluorescent molecules. The detectable agent can be,
for example, any moiety or molecule that facilitates detection,
either directly or indirectly, preferably by a non-invasive and/or
in vivo visualization technique. For example, a detectable agent
can be detectable by any known imaging techniques, including, for
example, a radiological technique, a magnetic resonance technique,
or an ultrasound technique. Detectable agents can include, for
example, a contrast agent. The contrast agent can be, for example,
Feridex. The contrasting agent can be, for example, ionic or
non-ionic. In some embodiments, for instance, the detectable agent
comprises a tantalum compound and/or a barium compound, e.g.,
barium sulfate. In some embodiments, the detectable agent comprises
iodine, such as radioactive iodine. In some embodiments, for
instance, the detectable agent comprises an organic iodo acid, such
as iodo carboxylic acid, triiodophenol, iodoform, and/or
tetraiodoethylene. In some embodiments, the detectable agent
comprises a non-radioactive detectable agent, e.g., a
non-radioactive isotope. For example, iron oxide and Gd can be used
as a non-radioactive detectable agent in certain embodiments.
Detectable agents can also include radioactive isotopes, enzymes,
fluorophores, and quantum dots (Qdot.RTM.). For example, the
detection moiety can be an enzyme, biotin, metal, or epitope tag.
Other known or newly discovered detectable markers are contemplated
for use with the provided compositions. In some embodiments, for
instance, the detectable agent comprises a barium compound, e.g.,
barium sulfate.
[0154] The detectable agent can be one or more imaging agents.
Examples of imaging agents include radiologic contrast agent, such
as diatrizoic acid sodium salt dihydrate, iodine, and barium
sulfate, a fluorescing imaging agent, such as Lissamine Rhodamine
PE, a fluorescent or non-fluorescent stain or dye, for example,
that can impart a visible color or that reflects a characteristic
spectrum of electromagnetic radiation at visible or other
wavelengths, for example, infrared or ultraviolet, such as
Rhodamine, a radioisotope, a positron-emitting isotope, such as
.sup.18F or .sup.124I (although the short half-life of a
positron-emitting isotope may impose some limitations), a metal, a
ferromagnetic compound, a paramagnetic compound, such as
gadolinium, a superparamagnetic compound, such as iron oxide, and a
diamagnetic compound, such as barium sulfate. Imaging agents can be
selected to optimize the usefulness of an image produced by a
chosen imaging technology. For example, the imaging agent can be
selected to enhance the contrast between a feature of interest,
such as a gastrointestinal polyp, and normal gastrointestinal
tissue. Imaging can be accomplished using any suitable imaging
techniques such as X-Ray, computed tomography (CT), MRI, Positron
Emission Tomography (PET) or SPECT. In some forms, the disclosed
components, compounds, and compositions can be coupled to a nuclear
medicine imaging agent such as Indium-III or Technetium-99, to PET
imaging agents, or to MRI imaging agents such as nanoparticles.
[0155] Examples of imaging techniques include magnetic resonance
imaging (MRI), computerized tomography (CT), single photon emission
computerized tomography (SPECT), and positron emission tomography
(PET). Imaging agents generally can be classified as either being
diagnostic or therapeutic in their application. Because of
radiation's damaging effect on tissues, it is useful to target the
biodistribution of radiopharmaceuticals as accurately as possible.
PET can use imaging agents labeled with, for example, the
positron-emitters such as .sup.18F, .sup.11C, .sup.13N and
.sup.15O, .sup.75Br, .sup.76Br and .sup.124I. SPECT can use imaging
agents labeled with, for example, the single-photon-emitters such
as .sup.201Tl, .sup.99Tc, .sup.123I and .sup.131I.
[0156] Glucose-based and amino acid-based compounds can be used as
imaging agents. Amino acid-based compounds are more useful in
analyzing tumor cells, due to their faster uptake and incorporation
into protein synthesis. Of the amino acid-based compounds,
.sup.11C- and .sup.18F-containing compounds have been used with
success. .sup.11C-containing radiolabeled amino acids suitable for
imaging include, for example, L-[1-.sup.11C]leucine (Keen et al. J.
Cereb. Blood Flow Metab. 1989 (9):429-45), L-[1-.sup.11C]tyrosine
(Wiesel et al. J. Nucl. Med. 1991 (32):2041-49),
L-[methyl-.sup.11C]methionine (Comar et al. Eur. J. Nucl. Med. 1976
(1):11-14) and L-[1-.sup.11C]methionine (Bolster et al. Appl.
Radiat. Isot. 1986 (37):1069-70).
[0157] PET involves the detection of gamma rays in the form of
annihilation photons from short-lived positron emitting radioactive
isotopes including, but not limited to, .sup.18F with a half-life
of approximately 110 minutes, .sup.11C with a half-life of
approximately 20 minutes, .sup.13N with a half-life of
approximately 10 minutes and .sup.15O with a half-life of
approximately 2 minutes, using the coincidence method. For PET
imaging studies, compounds such as [.sup.11C]meta-hydroxyephedrine
(HED) and 2-[.sup.18F]fluoro-2-deoxy-D-glucose (FDG) can be used.
SPECT can use longer-lived isotopes including, but not limited to,
.sup.99mTc with a half-life of approximately 6 hours and .sup.201Tl
with a half-life of approximately 74 hours. Radio-iodinated
meta-iodobenzylguanidine (MIBG) is a radiotracing agent that can be
used in nuclear medicine imaging studies.
[0158] Other examples of detectable agents include molecules which
emit or can be caused to emit detectable radiation (e.g.,
fluorescence excitation, radioactive decay, spin resonance
excitation, etc.), molecules which affect local electromagnetic
fields (e.g., magnetic, ferromagnetic, ferromagnetic, paramagnetic,
and/or superparamagnetic species), molecules which absorb or
scatter radiation energy (e.g., chromophores and/or fluorophores),
quantum dots, heavy elements and/or compounds thereof. See, e.g.,
detectable agents described in U.S. Publication No. 2004/0009122.
Other examples of detectable agents include proton-emitting
molecules, radiopaque molecules, and/or radioactive molecules, such
as a radionuclide like Tc-99m and/or Xe-13. Such molecules can be
used as a radiopharmaceutical. In still other embodiments, the
disclosed compositions can comprise one or more different types of
detectable agents, including any combination of the detectable
agents disclosed herein.
[0159] Useful fluorescent moieties include fluorescein
isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red,
nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,
rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin,
BODIPY.RTM., Cascade Blue.RTM.regon Green.RTM., pyrene, lissamine,
xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates
of lanthanide ions such as quantum Dye.TM., fluorescent energy
transfer dyes, such as thiazole orange-ethidium heterodimer, and
the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Examples of other
specific fluorescent labels include 3-Hydroxypyrene 5,8,10-Tri
Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin
Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl
Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon
Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine,
Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine
Sulphate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV,
Bodipy F1, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green,
Calcofluor RW Solution, Calcofluor White, Calcophor White ABT
Solution, Calcophor White Standard Solution, Carbostyryl, Cascade
Yellow, Catecholamine, Chinacrine, Coriphosphine O,
Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl Amino
Naphaline 5 Sulphonic Acid), Dansa (Diamino Naphtyl Sulphonic
Acid), Dansyl NH--CH.sub.3, Diamino Phenyl Oxydiazole (DAO),
Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride,
Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC,
Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange,
Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl
Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF,
Gloxalic Acid, Granular Blue, Haematoporphyrin, Indo-1, Intrawhite
Cf Liquid, Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine
Rhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS,
Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon
Brilliant Flavin 8 GFF, MPS (Methyl Green Pyronine Stilbene),
Mithramycin, NBD Amine, Nitrobenzoxadidole, Noradrenaline, Nuclear
Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin E8G, Oxadiazole,
Pacific Blue, Pararosaniline (Feulgen), Phorwite AR Solution,
Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R,
Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue
Black, Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B,
Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123,
Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200,
Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine WT,
Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron
Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS (Primuline),
SITS (Stilbene Isothiosulphonic acid), Stilbene, Snarf 1, sulpho
Rhodamine B Can C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine
Red R, Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte,
Thiozol Orange, Tinopol CBS, True Blue, Ultralite, Uranine B,
Uvitex SFC, Xylene Orange, and XRITC.
[0160] Particularly useful fluorescent labels include fluorescein
(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine
(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5,
Cy5.5 and Cy7. The absorption and emission maxima, respectively,
for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm),
Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703
nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous
detection. Other examples of fluorescein dyes include
6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein
(TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX),
2',7'-dimethoxy-4', 5'-dichloro-6-carboxyrhodamine (JOE),
2'-chloro-5'-fluoro-7',8'-fused
phenyl-1,4-dichloro-6-carboxyfluorescein (NED), and
2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).
Fluorescent labels can be obtained from a variety of commercial
sources, including Amersham Pharmacia Biotech, Piscataway, N.J.;
Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland,
Ohio. Fluorescent probes and there use are also described in
Handbook of Fluorescent Probes and Research Products by Richard P.
Haugland.
[0161] Further examples of radioactive detectable agents include
gamma emitters, e.g., the gamma emitters In-111, I-125 and I-131,
Rhenium-186 and 188, and Br-77 (see. e.g., Thakur, M. L. et al.,
Throm Res. Vol. 9 pg. 345 (1976); Powers et al., Neurology Vol. 32
pg. 938 (1982); and U.S. Pat. No. 5,011,686); positron emitters,
such as Cu-64, C-11, and O-15, as well as Co-57, Cu-67, Ga-67,
Ga-68, Ru-97, Tc-99m, In-113m, Hg-197, Au-198, and Pb-203. Other
radioactive detectable agents can include, for example tritium,
C-14 and/or thallium, as well as Rh-105, 1-123, Nd-147, Pm-151,
Sm-153, Gd-159, Tb-161, Er-171 and/or Tl-201.
[0162] The use of Technitium-99m (Tc-99m) is preferable and has
been described in other applications, for example, see U.S. Pat.
Nos. 4,418,052 and 5,024,829. Tc-99m is a gamma emitter with single
photon energy of 140 keV and a half-life of about 6 hours, and can
readily be obtained from a Mo-99/Tc-99 generator.
[0163] In some embodiments, compositions comprising a radioactive
detectable agent can be prepared by coupling radioisotopes suitable
for detection to the disclosed components and compositions.
Coupling can be, for example, via a chelating agent such as
diethylenetriaminepentaacetic acid (DTPA),
4,7,10-tetraazacyclododecane-N--,N',N'',N'''-tetraacetic acid
(DOTA) and/or metallothionein, any of which can be covalently
attached to the disclosed components, compounds, and compositions.
In some embodiments, an aqueous mixture of technetium-99m, a
reducing agent, and a water-soluble ligand can be prepared and then
allowed to react with a disclosed component, compound, or
composition. Such methods are known in the art, see e.g.,
International Publication No. WO 99/64446. In some embodiments,
compositions comprising radioactive iodine, can be prepared using
an exchange reaction. For example, exchange of hot iodine for cold
iodine is well known in the art. Alternatively, a radio-iodine
labeled compound can be prepared from the corresponding bromo
compound via a tributylstannyl intermediate.
[0164] Magnetic detectable agents include paramagnetic contrasting
agents, e.g., gadolinium diethylenetriaminepentaacetic acid, e.g.,
used with magnetic resonance imaging (MRI) (see, e.g., De Roos, A.
et al., Int. J. Card. Imaging Vol. 7 pg. 133 (1991)). Some
preferred embodiments use as the detectable agent paramagnetic
atoms that are divalent or trivalent ions of elements with an
atomic number 21, 22, 23, 24, 25, 26, 27, 28, 29, 42, 44, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. Suitable ions
include, but are not limited to, chromium(III), manganese(II),
iron(II), iron(III), cobalt(II), nickel(II), copper(II),
praseodymium(III), neodymium(III), samarium(III) and
ytterbium(III), as well as gadolinium(III), terbiurn(III),
dysoprosium(III), holmium(III), and erbium(III). Some preferred
embodiments use atoms with strong magnetic moments, e.g.,
gadolinium(III).
[0165] In some embodiments, compositions comprising magnetic
detectable agents can be prepared by coupling the disclosed
components, compounds, and compositions with a paramagnetic atom.
For example, the metal oxide or a metal salt, such as a nitrate,
chloride or sulfate salt, of a suitable paramagnetic atom can be
dissolved or suspended in a water/alcohol medium, such as methyl,
ethyl, and/or isopropyl alcohol. The mixture can be added to a
solution of an equimolar amount of the disclosed components,
compounds, and compositions in a similar water/alcohol medium and
stirred. The mixture can be heated moderately until the reaction is
complete or nearly complete. Insoluble compositions formed can be
obtained by filtering, while soluble compositions can be obtained
by evaporating the solvent. If acid groups on the chelating
moieties remain in the disclosed compositions, inorganic bases
(e.g., hydroxides, carbonates and/or bicarbonates of sodium,
potassium and/or lithium), organic bases, and/or basic amino acids
can be used to neutralize acidic groups, e.g., to facilitate
isolation or purification of the composition.
[0166] In preferred embodiments, the detectable agent can be
coupled to the composition in such a way so as not to interfere
with the ability of the disclosed compositions, peptides, amino
acid sequences, and annexin 1-binding compounds to interact with
annexin 1. In some embodiments, the detectable agent can be
chemically bound to the composition, peptide, amino acid sequence,
or annexin 1-binding compound. In some embodiments, the detectable
agent can be chemically bound to a moiety that is itself chemically
bound to the composition, peptide, amino acid sequence, or annexin
1-binding compound, indirectly linking the imaging and the
disclosed components, compounds, and compositions.
[0167] The moiety can also include poly-L-lysine and related
molecules. For example, moieties can include any of the moieties
disclosed herein with the addition of poly-L-lysine conjugated or
coupled to the moiety. For example, the moiety can be
FITC-poly-L-lysine or Alexa488-poly-L-lysine. Examples of
compositions with such moieties include IF7C(RR)-conjugated
FITC-poly-L-lysine and IF7C(RR)-conjugated
Alexa488-poly-L-lysine.
D. Linkages, Linkers, and Cleavable Bonds
[0168] The disclosed annexin 1-binding compounds (such as the
disclosed peptides and amino acid sequences) and moieties (or other
components of the disclosed compositions) can be linked in any
useful way. For example, annexin 1-binding compounds and moieties
can be covalently coupled (directly or indirectly), noncovalently
coupled (directly or indirectly), or both. Covalent coupling is
useful. Direct coupling can be via a covalent bond between the
annexin 1-binding compound and the moiety. The covalent bond in
such cases can be considered the linkage between the annexin
1-binding compound and the moiety. Indirect coupling can be via one
or more intervening molecules or components. Useful indirect
coupling can be via a linker. The linker, any bond in the linker
that couples the annexin 1-binding compound and the moiety, the
bond between the annexi 1-binding compound and the linker, and/or
the bond between the moiety and the linker can be considered a
linkage. Any suitable linker can be used. For example, the linker
can be an oligomer, such as a peptide or peptide mimetic.
[0169] The linker can contain or linkage can be a cleavable bond. A
cleavable bond can be useful for freeing the moiety at the site of
targeting, for example. The cleavable bond can be cleaved in any
suitable way. For example, the cleavable bond can be cleaved
enzymatically or non-enzymatically. For enzymatic cleavage, the
cleaving enzyme can be supplied or can be present at a site where
the composition is delivered, homes, travels or accumulates. For
example, the enzyme can be present in proximity to a cell to which
the composition is delivered, homes, travels, or accumulates. For
non-enzymatic cleavage, the composition can be brought into contact
with a cleaving agent, can be placed in cleaving conditions, or
both. A cleaving agent is any substance that can mediate or
stimulate cleavage of the cleavable bond. A non-enzymatic cleaving
agent is any cleaving agent except enzymes. Cleaving conditions can
be any solution or environmental conditions that can mediate or
stimulate cleavage of the cleavable bond. For example, some labile
bonds can be cleaved in acid conditions, alkaline conditions, in
the presence of a reactive group, etc. Non-enzymatic cleaving
conditions are any cleaving conditions except the presence of
enzymes. Non-agent cleaving conditions are any cleaving conditions
except the presence of cleaving agents.
[0170] A "protease-cleavable bond" refers to a cleavable bond that
can be cleaved by a protease. Useful proteases include proteases
that may be present at the location where the disclosed
compositions are delivered, target, home, etc. Examples of useful
proteases include, for example, serine proteases (including, for
example, plasmin and pasminogen activators), proprotein convertases
(see, for example, Duckert et al., Prediction of proprotein
convertase cleavage sites Protein engineering Design and Selection
17(1):107-112 (2004)), furins, and carboxypeptidases. Serine
proteases are particularly useful for compositions targeted to
cancer cells and tumors. Examples of enzymes that cleave on the C
terminal side of basic residues include Arg-C protease (which
cleaves on the C terminal side of arginine residues; Keil,
Specificity of Proteolysis (Springer-Verlag, Berlin-Heidelberg-New
York (1992)), clostripain (which cleaves on the C terminal side of
arginine residues; Keil, 1992), enterokinase (which cleaves after
the sequence -Asp-Asp-Asp-Asp-Lys-; SEQ ID NO:23), Factor Xa (which
cleaves after the sequence -Gly-Arg-; Fujikawa et al., Activation
of bovine factor X (Stuart factor): conversion of factor Xa alpha
to factor Xa beta, Proc. Natl. Acad. Sci. 72: 3359-3363 (1975)),
Lys-C (which cleaves on the C terminal side of lysine residues;
Keil, 1992), thrombin (which cleaves on the C terminal side of
arginine residues; Keil, 1992), trypsin (which cleaves on the C
terminal side of arginine and lysine residues; Keil, 1992), serine
proteases, proprotein convertases (such as PC1, PC2, PC3, PC4, PC5,
PC6, PC7, PC8, furin, Pace, PACE4, Site 1 protease, SIP, SKI,
NARC-1, PCSK1, PCSK2, PCSK3, PCSK4, PCSK5, PCSK6, PCSK7, PCSK8, and
PCSK9), plasmin, and plasminogen activators. Examples of enzymes
that recognize sequence on the C terminal side of their cleavage
site include Asp-N endopeptidase (which cleaves on the N terminal
side of aspartic acid; Keil, 1992) and carboxypeptidases such as
carboxypeptidase A (which cleaves C-terminal residues except
proline, lysine and arginine).
[0171] Examples of proteases are also described in Hook,
Proteolytic and cellular mechanisms in prohormone and proprotein
processing, RG Landes Company, Austin, Tex., USA (1998); Hooper et
al., Biochem. J. 321: 265-279 (1997); Werb, Cell 91: 439-442
(1997); Wolfsberg et al., J. Cell Biol. 131: 275-278 (1995);
Murakami and Etlinger, Biochem. Biophys. Res. Comm. 146: 1249-1259
(1987); Berg et al., Biochem. J. 307: 313-326 (1995); Smyth and
Trapani, Immunology Today 16: 202-206 (1995); Talanian et al., J.
Biol. Chem. 272: 9677-9682 (1997); and Thomberry et al., J. Biol.
Chem. 272: 17907-17911 (1997).
[0172] An "esterase-cleavable bond" refers to a cleavable bond that
can be cleaved by a protease. Useful esterases include esterases
that may be present at the location where the disclosed
compositions are delivered, target, home, etc.
E. Pharmaceutical Compositions and Carriers
[0173] The disclosed compositions can be administered in vivo
either alone or in a pharmaceutically acceptable carrier. By
"pharmaceutically acceptable" is meant a material that is not
biologically or otherwise undesirable, i.e., the material can be
administered to a subject, along with the composition disclosed
herein, without causing any undesirable biological effects or
interacting in a deleterious manner with any of the other
components of the pharmaceutical composition in which it is
contained. The carrier would naturally be selected to minimize any
degradation of the active ingredient and to minimize any adverse
side effects in the subject, as would be well known to one of skill
in the art. The materials can be in solution, suspension (for
example, incorporated into microparticles, liposomes, or
cells).
[0174] 1. Pharmaceutically Acceptable Carriers
[0175] The compositions disclosed herein can be used
therapeutically in combination with a pharmaceutically acceptable
carrier.
[0176] Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.
R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically,
an appropriate amount of a pharmaceutically-acceptable salt is used
in the formulation to render the formulation isotonic. Examples of
the pharmaceutically-acceptable carrier include, but are not
limited to, saline, Ringer's solution and dextrose solution. The pH
of the solution is preferably from about 5 to about 8, and more
preferably from about 7 to about 7.5. Further carriers include
sustained release preparations such as semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, liposomes or
microparticles. It will be apparent to those persons skilled in the
art that certain carriers can be more preferable depending upon,
for instance, the route of administration and concentration of
composition being administered.
[0177] Pharmaceutical carriers are known to those skilled in the
art. These most typically would be standard carriers for
administration of drugs to humans, including solutions such as
sterile water, saline, and buffered solutions at physiological pH.
The compositions can be administered intramuscularly or
subcutaneously. Other compounds will be administered according to
standard procedures used by those skilled in the art.
[0178] Pharmaceutical compositions can include carriers,
thickeners, diluents, buffers, preservatives, surface active agents
and the like in addition to the molecule of choice. Pharmaceutical
compositions can also include one or more active ingredients such
as antimicrobial agents, antiinflammatory agents, anesthetics, and
the like.
[0179] The pharmaceutical composition can be administered in a
number of ways depending on whether local or systemic treatment is
desired, and on the area to be treated. Administration can be
topically (including ophthalmically, vaginally, rectally,
intranasally), orally, by inhalation, or parenterally, for example
by intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection. The disclosed antibodies can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, or transdermally.
[0180] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives can also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0181] Formulations for topical administration can include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0182] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0183] Some of the compositions can be administered as a
pharmaceutically acceptable acid- or base-addition salt, formed by
reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0184] 2. Nanoparticles, Microparticles, and Microbubbles
[0185] The term "nanoparticle" refers to a nanoscale particle with
a size that is measured in nanometers, for example, a nanoscopic
particle that has at least one dimension of less than about 100 nm.
Examples of nanoparticles include paramagnetic nanoparticles,
superparamagnetic nanoparticles, metal nanoparticles, nanoworms,
fullerene-like materials, inorganic nanotubes, dendrimers (such as
with covalently attached metal chelates), nanofibers, nanohoms,
nano-onions, nanorods, nanoropes and quantum dots.
[0186] Microspheres (or microbubbles) can also be used with the
methods disclosed herein. Microspheres containing chromophores have
been utilized in an extensive variety of applications. The
monodispersity of the microspheres can be important.
[0187] Nanoparticles, such as, for example, metal nanoparticles,
metal oxide nanoparticles, or semiconductor nanocrystals can be
incorporated into microspheres. The nanoparticle can be, for
example, a heat generating nanoshell. As used herein, "nanoshell"
is a nanoparticle having a discrete dielectric or semi-conducting
core section surrounded by one or more conducting shell layers.
U.S. Pat. No. 6,530,944 is hereby incorporated by reference herein
in its entirety for its teaching of the methods of making and using
metal nanoshells. Nanoshells can be formed with a core of a
dielectric or inert material such as silicon, coated with a
material such as a highly conductive metal which can be excited
using radiation such as near infrared light (approximately 800 to
1300 nm). Upon excitation, the nanoshells emit heat. The resulting
hyperthermia can kill the surrounding cell(s) or tissue. The
combined diameter of the shell and core of the nanoshells ranges
from the tens to the hundreds of nanometers. Near infrared light is
advantageous for its ability to penetrate tissue. Other types of
radiation can also be used, depending on the selection of the
nanoparticle coating and targeted cells. Examples include x-rays,
magnetic fields, electric fields, and ultrasound.
[0188] The nanoparticle can be a metal nanoparticle, a metal oxide
nanoparticle, or a semiconductor nanocrystal. The metal of the
metal nanoparticle or the metal oxide nanoparticle can include
titanium, zirconium, hafnium, vanadium, niobium, tantalum,
chromium, molybdenum, tungsten, manganese, technetium, rhenium,
iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel,
palladium, platinum, copper, silver, gold, zinc, cadmium, scandium,
yttrium, lanthanum, a lanthanide series or actinide series element
(e.g., cerium, praseodymium, neodymium, promethium, samarium,
europium, gadolinium, terbium, dysprosium, holmium, erbium,
thulium, ytterbium, lutetium, thorium, protactinium, and uranium),
boron, aluminum, gallium, indium, thallium, silicon, germanium,
tin, lead, antimony, bismuth, polonium, magnesium, calcium,
strontium, and barium. In certain embodiments, the metal can be
iron, ruthenium, cobalt, rhodium, nickel, palladium, platinum,
silver, gold, cerium or samarium. The metal oxide can be an oxide
of any of these materials or combination of materials. For example,
the metal can be gold, or the metal oxide can be an iron oxide, a
cobalt oxide, a zinc oxide, a cerium oxide, or a titanium oxide.
Preparation of metal and metal oxide nanoparticles is described,
for example, in U.S. Pat. Nos. 5,897,945 and 6,759,199, each of
which is incorporated by reference in its entirety.
[0189] 3. Liposomes
[0190] "Liposome" as the term is used herein refers to a structure
comprising an outer lipid bi- or multi-layer membrane surrounding
an internal aqueous space. Liposomes can be used to package any
biologically active agent for delivery to cells.
[0191] Materials and procedures for forming liposomes are
well-known to those skilled in the art. Upon dispersion in an
appropriate medium, a wide variety of phospholipids swell, hydrate
and form multilamellar concentric bilayer vesicles with layers of
aqueous media separating the lipid bilayers. These systems are
referred to as multilamellar liposomes or multilamellar lipid
vesicles ("MLVs") and have diameters within the range of 10 nm to
100 .mu.m. These MLVs were first described by Bangham, et al., J
Mol. Biol. 13:238-252 (1965). In general, lipids or lipophilic
substances are dissolved in an organic solvent. When the solvent is
removed, such as under vacuum by rotary evaporation, the lipid
residue forms a film on the wall of the container. An aqueous
solution that typically contains electrolytes or hydrophilic
biologically active materials is then added to the film. Large MLVs
are produced upon agitation. When smaller MLVs are desired, the
larger vesicles are subjected to sonication, sequential filtration
through filters with decreasing pore size or reduced by other forms
of mechanical shearing. There are also techniques by which MLVs can
be reduced both in size and in number of lamellae, for example, by
pressurized extrusion (Barenholz, et al., FEBS Lett. 99:210-214
(1979)).
[0192] Liposomes can also take the form of unilamnellar vesicles,
which are prepared by more extensive sonication of MLVs, and
consist of a single spherical lipid bilayer surrounding an aqueous
solution. Unilamellar vesicles ("ULVs") can be small, having
diameters within the range of 20 to 200 nm, while larger ULVs can
have diameters within the range of 200 nm to 2 .mu.m. There are
several well-known techniques for making unilamellar vesicles. In
Papahadjopoulos, et al., Biochim et Biophys Acta 135:624-238
(1968), sonication of an aqueous dispersion of phospholipids
produces small ULVs having a lipid bilayer surrounding an aqueous
solution. Schneider, U.S. Pat. No. 4,089,801 describes the
formation of liposome precursors by ultrasonication, followed by
the addition of an aqueous medium containing amphiphilic compounds
and centrifugation to form a biomolecular lipid layer system.
[0193] Small ULVs can also be prepared by the ethanol injection
technique described by Batzri, et al., Biochim et Biophys Acta
298:1015-1019 (1973) and the ether injection technique of Deamer,
et al., Biochim et Biophys Acta 443:629-634 (1976). These methods
involve the rapid injection of an organic solution of lipids into a
buffer solution, which results in the rapid formation of
unilamellar liposomes. Another technique for making ULVs is taught
by Weder, et al. in "Liposome Technology", ed. G. Gregoriadis, CRC
Press Inc., Boca Raton, Fla., Vol. I, Chapter 7, pg. 79-107 (1984).
This detergent removal method involves solubilizing the lipids and
additives with detergents by agitation or sonication to produce the
desired vesicles.
[0194] Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes
the preparation of large ULVs by a reverse phase evaporation
technique that involves the formation of a water-in-oil emulsion of
lipids in an organic solvent and the drug to be encapsulated in an
aqueous buffer solution. The organic solvent is removed under
pressure to yield a mixture which, upon agitation or dispersion in
an aqueous media, is converted to large ULVs. Suzuki et al., U.S.
Pat. No. 4,016,100, describes another method of encapsulating
agents in unilamellar vesicles by freezing/thawing an aqueous
phospholipid dispersion of the agent and lipids.
[0195] In addition to the MLVs and ULVs, liposomes can also be
multivesicular. Described in Kim, et al., Biochim et Biophys Acta
728:339-348 (1983), these multivesicular liposomes are spherical
and contain internal granular structures. The outer membrane is a
lipid bilayer and the internal region contains small compartments
separated by bilayer septum. Still yet another type of liposomes is
oligolamellar vesicles ("OLVs"), which have a large center
compartment surrounded by several peripheral lipid layers. These
vesicles, having a diameter of 2-15 .mu.m, are described in Callo,
et al., Cryobiology 22(3):251-267 (1985).
[0196] Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also
describe methods of preparing lipid vesicles. More recently, Hsu,
U.S. Pat. No. 5,653,996 describes a method of preparing liposomes
utilizing aerosolization and Yiournas, et al., U.S. Pat. No.
5,013,497 describes a method for preparing liposomes utilizing a
high velocity-shear mixing chamber. Methods are also described that
use specific starting materials to produce ULVs (Wallach, et al.,
U.S. Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848
and 5,628,936).
[0197] A comprehensive review of all the aforementioned lipid
vesicles and methods for their preparation are described in
"Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca
Raton, Fla., Vol. I, II & III (1984). This and the
aforementioned references describing various lipid vesicles
suitable for use in the invention are incorporated herein by
reference.
F. Computer Assisted Drug Design
[0198] The disclosed compositions can be used as targets for any
molecular modeling technique to identify either the structure of
the disclosed compositions or to identify potential or actual
molecules, such as small molecules, which interact in a desired way
with the disclosed compositions.
[0199] It is understood that when using the disclosed compositions
in modeling techniques, molecules, such as macromolecular
molecules, will be identified that have particular desired
properties such as inhibition or stimulation or the target
molecule's function. The molecules identified and isolated when
using the disclosed compositions, peptides, etc., are also
disclosed. Thus, the products produced using the molecular modeling
approaches that involve the disclosed compositions are also
considered herein disclosed.
[0200] Thus, one way to isolate molecules that bind a molecule of
choice is through rational design. This can be achieved through
structural information and computer modeling. Computer modeling
technology allows visualization of the three-dimensional atomic
structure of a selected molecule and the rational design of new
compounds that will interact with the molecule. The
three-dimensional construct typically depends on data from x-ray
crystallographic analyses or NMR imaging of the selected molecule.
The molecular dynamics require force field data. The computer
graphics systems enable prediction of how a new compound will link
to the target molecule and allow experimental manipulation of the
structures of the compound and target molecule to perfect binding
specificity. Prediction of what the molecule-compound interaction
will be when small changes are made in one or both requires
molecular mechanics software and computationally intensive
computers, usually coupled with user-friendly, menu-driven
interfaces between the molecular design program and the user.
[0201] Examples of molecular modeling systems are the CHARMm and
QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm
performs the energy minimization and molecular dynamics functions.
QUANTA performs the construction, graphic modeling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules with each other.
[0202] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen, et al., 1988
Acta Pharmaceutica Fennica 97, 159-166; Ripka, New Scientist 54-57
(Jun. 16, 1988); McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol.
Toxiciol. 29, 111-122; Perry and Davies, QSAR: Quantitative
Structure--Activity Relationships in Drug Design pp. 189-193 (Alan
R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. 236,
125-140 and 141-162; and, with respect to a model enzyme for
nucleic acid components, Askew, et al., 1989 J Am. Chem. Soc. 111,
1082-1090. Other computer programs that screen and graphically
depict chemicals are available from companies such as BioDesign,
Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada,
and Hypercube, Inc., Cambridge, Ontario. Although these are
primarily designed for application to drugs specific to particular
proteins, they can be adapted to design of molecules specifically
interacting with specific regions of DNA or RNA, once that region
is identified.
[0203] Although described above with reference to design and
generation of compounds which could alter binding, one could also
screen libraries of known compounds, including natural products or
synthetic chemicals, and biologically active materials, including
proteins, for compounds which alter substrate binding or enzymatic
activity.
G. Compositions with Similar Functions
[0204] It is understood that the compositions disclosed herein have
certain functions, such as binding to annexin 1 or homing to tumor
vasculature. Disclosed herein are certain structural requirements
for performing the disclosed functions, and it is understood that
there are a variety of structures which can perform the same
function which are related to the disclosed structures, and that
these structures will ultimately achieve the same result, for
example stimulation or inhibition.
H. Kits
[0205] Disclosed herein are kits that are drawn to reagents that
can be used in practicing the methods disclosed herein. The kits
can include any reagent or combination of reagent discussed herein
or that would be understood to be required or beneficial in the
practice of the disclosed methods. For example, the kits can
include the compositions disclosed herein.
I. Mixtures
[0206] Whenever the method involves mixing or bringing into contact
compositions or components or reagents, performing the method
creates a number of different mixtures. For example, if the method
includes 3 mixing steps, after each one of these steps a unique
mixture is formed if the steps are performed separately. In
addition, a mixture is formed at the completion of all of the steps
regardless of how the steps were performed. The present disclosure
contemplates these mixtures, obtained by the performance of the
disclosed methods as well as mixtures containing any disclosed
reagent, composition, or component, for example, disclosed
herein.
J. Systems
[0207] Disclosed are systems useful for performing, or aiding in
the performance of, the disclosed method. Systems generally
comprise combinations of articles of manufacture such as
structures, machines, devices, and the like, and compositions,
compounds, materials, and the like. Such combinations that are
disclosed or that are apparent from the disclosure are
contemplated.
K. Computer Readable Media
[0208] It is understood that the disclosed nucleic acids and
proteins can be represented as a sequence consisting of the
nucleotides of amino acids. There are a variety of ways to display
these sequences, for example the nucleotide guanosine can be
represented by G or g. Likewise the amino acid valine can be
represented by Val or V. Those of skill in the art understand how
to display and express any nucleic acid or protein sequence in any
of the variety of ways that exist, each of which is considered
herein disclosed. Specifically contemplated herein is the display
of these sequences on computer readable mediums, such as,
commercially available floppy disks, tapes, chips, hard drives,
compact disks, and video disks, or other computer readable mediums.
Also disclosed are the binary code representations of the disclosed
sequences. Those of skill in the art understand what computer
readable mediums. Thus, computer readable mediums on which the
nucleic acids or protein sequences are recorded, stored, or
saved.
L. Peptide Synthesis
[0209] The compositions disclosed herein and the compositions
necessary to perform the disclosed methods can be made using any
method known to those of skill in the art for that particular
reagent or compound unless otherwise specifically noted.
[0210] One method of producing the disclosed proteins, such as SEQ
ID NO:2, is to link two or more peptides or polypeptides together
by protein chemistry techniques. For example, peptides or
polypeptides can be chemically synthesized using currently
available laboratory equipment using either Fmoc
(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)
chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One
skilled in the art can readily appreciate that a peptide or
polypeptide corresponding to the disclosed proteins, for example,
can be synthesized by standard chemical reactions. For example, a
peptide or polypeptide can be synthesized and not cleaved from its
synthesis resin whereas the other fragment of a peptide or protein
can be synthesized and subsequently cleaved from the resin, thereby
exposing a terminal group which is functionally blocked on the
other fragment. By peptide condensation reactions, these two
fragments can be covalently joined via a peptide bond at their
carboxyl and amino termini, respectively, to form an antibody, or
fragment thereof (Grant GA (1992) Synthetic Peptides: A User Guide.
W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed.
(1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY
(which is herein incorporated by reference at least for material
related to peptide synthesis). Alternatively, the peptide or
polypeptide is independently synthesized in vivo as described
herein. Once isolated, these independent peptides or polypeptides
can be linked to form a peptide or fragment thereof via similar
peptide condensation reactions.
[0211] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide--thioester with another unprotected peptide
segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site (Baggiolini M et al.
(1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J. Biol. Chem.,
269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128
(1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
[0212] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
Methods
[0213] Disclosed herein are methods comprising administering to a
subject the disclosed compositions, annexin 1-binding compounds,
annexin 1-binding amino acid sequences, peptides, or amino acid
sequences. The compositions, annexin 1-binding compounds, annexin
1-binding amino acid sequences, peptides, and amino acid sequences
can selectively home to tumor vasculature. The composition can
accumulate in tumor vasculature. Some forms of the method comprise
administering to a subject the composition, annexin 1-binding
compound, annexin 1-binding amino acid sequence, peptide, or amino
acid sequence disclosed herein, wherein the composition, annexin
1-binding compound, annexin 1-binding amino acid sequence, peptide,
or amino acid sequence selectively homes to tumor vasculature,
wherein the composition, annexin 1-binding compound, annexin
1-binding amino acid sequence, peptide, or amino acid sequence
accumulates in tumor vasculature. The composition, annexin
1-binding compound, annexin 1-binding amino acid sequence, peptide,
or amino acid sequence can selectively home to tumor
vasculature.
[0214] Disclosed are methods comprising administering to a subject
a composition comprising a moiety and a peptide comprising an amino
acid sequence that can bind to a carbohydrate receptor on a cell.
Also disclosed are methods of targeting a tumor cell in a subject
comprising administering to the subject a peptide comprising an
amino acid sequence that can bind to a carbohydrate receptor on a
cell. Also disclosed are methods of targeting a tumor cell in a
subject comprising administering to the subject a composition
comprising a moiety and a peptide comprising an amino acid sequence
that can bind to a carbohydrate receptor on a cell. Also disclosed
are methods comprising administering to the subject a composition
comprising a peptide comprising an amino acid sequence that can
bind to a carbohydrate receptor on a cell and detecting the
composition in the subject.
[0215] In one example, the composition can have a therapeutic
effect. This effect can be enhanced by the delivery of a
therapeutic agent to the site of the tumor.
[0216] The therapeutic effect can be a slowing in the increase of
or a reduction of tumor burden. This slowing in the increase of, or
reduction in the tumor burden, can be 1%, 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or
1000% or more improvement in the increase of, or reduction in the
tumor burden of, compared with a non-treated tumor, or a tumor
treated by a different method.
[0217] The therapeutic effect can also be a reduction or blocking
of blood circulation in a tumor. This reduction or blocking of
blood circulation in a tumor, can be 1%, 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or
1000% or more improvement in effective blocking of blood
circulation in a tumor, compared with a non-treated tumor, or a
tumor treated by a different method.
[0218] The disclosed compositions can be used to treat any disease
where annexin 1 is present in higher than normal amounts such as
cancers. A non-limiting list of different types of cancers that can
be treated includes lymphomas (Hodgkins and non-Hodgkins),
carcinomas, carcinomas of solid tissues, squamous cell carcinomas,
adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas,
neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas,
hypoxic tumors, myelomas, AIDS-related lymphomas or sarcomas,
metastatic cancers, or cancers in general.
[0219] A representative but non-limiting list of cancers that the
disclosed compositions can be used to treat is the following:
lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides,
Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer,
nervous system cancer, head and neck cancer, squamous cell
carcinoma of head and neck, kidney cancer, lung cancers such as
small cell lung cancer and non-small cell lung cancer,
neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,
prostate cancer, skin cancer, liver cancer, melanoma, squamous cell
carcinomas of the mouth, throat, larynx, and lung, colon cancer,
cervical cancer, cervical carcinoma, breast cancer, and epithelial
cancer, renal cancer, genitourinary cancer, pulmonary cancer,
esophageal carcinoma, gastric cancer, head and neck carcinoma,
large bowel cancer, hematopoietic cancers; testicular cancer; colon
and rectal cancers, prostatic cancer, or pancreatic cancer.
[0220] The disclosed compositions can also be administered
following decoy particle pretreatment to reduce uptake of the
compositions by reticuloendothelial system (RES) tissues. Such
decoy particle pretreatment can prolong the blood half-life of the
particles and increases tumor targeting.
[0221] The method can further comprise, following administering,
detecting the disclosed peptides and compositions. The disclosed
peptides and compositions can be detected by fluorescence, CT scan,
PET or MRI. The disclosed peptides and compositions can be detected
by fluorescence. The disclosed peptides and compositions can
conjugate with tumor vasculature or a tumor in a subject.
EXAMPLES
[0222] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
disclosure. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
A. Example 1: Highly Efficient Drug Delivery Targeted to Malignant
Tumors by Carbohydrate Mimicry Peptide IF7
[0223] 1. Introduction
[0224] Technical advances in genomics and proteomics together with
automated chemical synthesis of DNA and proteins have greatly
contributed to progress in biomedicine. By contrast, understanding
the role of carbohydrates has lagged behind due to lack of advanced
technologies: currently, recombinant or amplifiable carbohydrates
can not be produced nor can complex carbohydrates automatically be
chemically synthesized. Consequently, carbohydrate-based drug
discovery has been largely unexplored despite the fact that cancer
malignancy is closely associated with carbohydrate structures found
on the tumor cell surface (S. Hakomori. Glycosylation defining
cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci
USA 99:10231-10233 (2002); S. Nakamori, et al. Increased expression
of sialyl Lewisx antigen correlates with poor survival in patients
with colorectal carcinoma: clinicopathological and
immunohistochemical study. Cancer Res 53:3632-3637 (1993)). To move
beyond this dilemma, peptide-displaying phage technology and
identified carbohydrate mimicry peptides have been employed (M. N.
Fukuda, et al. A peptide mimic of E-selectin ligand inhibits sialyl
Lewis X-dependent lung colonization of tumor cells. Cancer Res
60:450-456 (2000); M. N. Fukuda. Screening of peptide-displaying
phage libraries to identify short peptides mimicking carbohydrates.
Methods Enzymol 416:51-60 (2006); T. Taki, et al. A new approach
for drug discovery from glycobiology and phage-displayed peptide
library technology. Biochim Biophys Acta 1780:497-503 (2008); J. K.
Scott, D. Loganathan, R. B. Easley, X. Gong, I. J. Goldstein. A
family of concanavalin A-binding peptides from a hexapeptide
epitope library. Proc Natl Acad Sci USA 89:5398-5402 (1992)). For
example, I-peptide, or IELLQAR, was identified as a selectin ligand
mimic, and when injected intravenously into mice, synthetic
I-peptide inhibited carbohydrate-dependent cancer cell colonization
to the lung (M. N. Fukuda, et al. A peptide mimic of E-selectin
ligand inhibits sialyl Lewis X-dependent lung colonization of tumor
cells. Cancer Res 60:450-456 (2000); J. Zhang, et al. Sialyl Lewis
X-dependent lung colonization of B16 melanoma cells through a
selectin-like endothelial receptor distinct from E- or P-selectin.
Cancer Res 62:4194-4198 (2002)).
[0225] When I-peptide was loaded into apoptosis-inducing liposomes
(R. De Maria, et al. Requirement for GD3 ganglioside in CD95- and
ceramide-induced apoptosis. Science 277:1652-1655 (1997)) and
injected intravenously into mice without tumors, I-peptide targeted
those liposomes to lung endothelial cells, which display potential
carbohydrate-dependent sites allowing cancer cell colonization (S.
Hatakeyama et al. Identification of mRNA splicing factors as the
endothelial receptor for carbohydrate-dependent lung colonization
of cancer cells. Proc Natl Acad Sci USA 106:3095-3100 (2009)). Mice
treated with I-peptide-loaded liposomes did not show
carbohydrate-dependent cancer colonization of the lung.
Significantly, when I-peptide-loaded liposomes were injected into
mice bearing subcutaneously produced B16 tumors, the size of
primary tumor was reduced. Anti-tumor activity promoted by
I-peptide can be mediated by annexin 1 (Anxa1), as I-peptide bound
to an annexin 1 (Anxa1) fragment (S. Hatakeyama et al.
Identification of mRNA splicing factors as the endothelial receptor
for carbohydrate-dependent lung colonization of cancer cells. Proc
Natl Acad Sci USA 106:3095-3100 (2009)). Extensive subtractive
proteomics identified Anxa1 as a specific tumor endothelial cell
surface marker (Oh et al. Subtractive proteomic mapping of the
endothelial surface in lung and solid tumours for tissue-specific
therapy. Nature 429:629-635 (2004)). These preliminary observations
together with carbohydrate binding activity by annexin family
proteins (R. Hannon et al. Aberrant inflammation and resistance to
glucocorticoids in annexin 1-/- mouse. Faseb J 17:253-255 (2003);
H. A. Lehr et al. Dorsal skinfold chamber technique for intravital
microscopy in nude mice. Am J Pathol 143:1055-1062 (1993)) prompted
development of a tumor vasculature-specific targeting vehicle
utilizing a carbohydrate-mimicry peptide. Herein, Anxa1-binding
carbohydrate mimicry peptide, designated IF7, has been identified
as a highly efficient tumor-targeting vehicle for anti-cancer
drugs.
[0226] 2. Results
[0227] i. Relevance of Anxa1 as a tumor vasculature marker
[0228] When B16 melanoma cells were injected subcutaneously in
Anxa1 null mutant mice (R. Hannon et al. Aberrant inflammation and
resistance to glucocorticoids in annexin 1-/- mouse. Faseb J
17:253-255, 2003) completely backcrossed to C57BL/6, tumor growth
was significantly reduced compared to tumors produced in Anxa1
heterozygous mice (FIG. 1A, B). Tumors produced in Anxa1 nulls were
largely necrotic (FIG. 1C). Remarkably, no vasculature was found in
tumors produced in Anxa1 null mice (FIG. 1D). These findings
suggest that Anxa1 expression on the endothelial cell surface (P.
Oh et al. Subtractive proteomic mapping of the endothelial surface
in lung and solid tumours for tissue-specific therapy. Nature
429:629-635, 2004) is essential for active tumor growth in the
mouse.
[0229] ii. Identification of IF7: Tumor-Targeting and Anxa1-Binding
Peptide
[0230] I-peptide (IELLQAR; SEQ ID NO:13), was identified as a
selectin ligand mimicry, and when injected intravenously into mice,
synthetic I-peptide inhibited carbohydrate-dependent cancer cell
colonization to the lung (Fukuda et al., Cancer Res 2000; 60:
450-6; Zhang et al. Cancer Res 2002; 62:4194-8). I-peptide binding
activity to Anxa1 can be used as a tumor-targeting vehicle.
However, as previously shown the I-peptide also targets to normal
lung through pre-mRNA splicing factors (Hatakeyama, S. et al., Proc
Natl Acad Sci 2009; 106:3095-100). Thus, a peptide sequence
specifically targeting tumor but not normal lung vasculature is
desirable. Organ targeting of phage clones intravenously injected
into tumor-bearing mice indicated that IFLLWQR, designated IF7,
targets tumor but not lung tissue (FIG. 1E, clone #2). Furthermore,
tumor targeting by IF7 phage was completely inhibited by anti-Anxa1
antibody (FIG. 1F), showing that IF7 binds to tumor vasculature
through Anxa1 expressed on the endothelial cell surface (Oh et al.
Nature 2004; 429:629-35). Although low levels of Anxa1 was detected
in lung by biotinylation (Hatakeyama et al., Proc Natl Acad Sci
2009; 106: 3095-100), IF7 targeting to the lung in tumor-bearing
mice could not be detected (FIG. 1F), showing that Anxa1 levels
expressed on the lung endothelial cell surface are significantly
lower than those expressed on the tumor vasculature.
[0231] Chemically synthesized IF7 conjugated with fluorescent Alexa
488, the conjugate, designated IF7-A488 (FIG. 2), binds to
recombinant Anxa1-His.sub.6 protein in a plate assay, whereas
RQ7-A488 (Alexa 488 conjugated with RQWLLFI (SEQ ID NO:15) or
reverse IF7) did not bind to Anxa1 (FIG. 1G). IF7 binding to Anxa1
was confirmed by surface plasmon resonance and isothermal titration
assays (FIGS. 3 and 4). Fucosylated carbohydrates bound to Anxa1
(FIG. 4), and that binding was inhibited by IF7 but not by RQ7
(FIG. 1H).
[0232] iii. In Vivo Tumor Targeting Activity by IF7
[0233] To test in vivo tumor vasculature targeting activity by IF7,
tumors were produced in a dorsal skinfold chamber installed on nude
mice (Lehr et al., Am J Pathol 1993; 143: 1055-62). IF7-A488 was
injected through the tail vein and tumor fluorescence was monitored
microscopically, fluorescence signals appeared in the tumor within
one minute, reached a plateau in 9 min, and remained high for 40
min or until the experiment was terminated (FIG. 5A-a, FIG. 5B). By
contrast, control peptide RQ7-A488 signals were either not
detectable or remained at background levels (FIG. 5A-b, FIG. 5B).
When anti-Anxa1 antibody was injected prior to IF7-A488 injection,
fluorescence signals in tumors were significantly reduced (FIG.
5A-d, 5B). On the other hand, an irrelevant rabbit IgG antibody did
not inhibit IF7-A488 tumor targeting (FIG. 5A-c, 5B). Tissue
sections prepared from the tumor 20 min after injection showed
vascular staining by IF7-A488 (FIG. 5C-a) but not RQ7-A488 (FIG.
5C-b). These results indicate that IF7 targets the tumors through
Anxa1 expressed on the endothelial cell surface.
[0234] IF7-A488 levels remaining in circulation of tumor-bearing
mice can indicate the tumor targeting efficacy by IF7 (FIG. 5D).
When IF7-A488 was injected intravenously into control mice without
tumors, IF7-A488 remained in circulation after an initial surge.
However, when IF7-A488 was injected into B16-tumor-bearing mice,
the initial surge of fluorescent signals was significantly reduced,
followed by their complete disappearance from the circulation,
indicating extremely high tumor-targeting efficacy of IF7.
[0235] iv. Tumor-Specific Delivery by an IF7-Conjugated Drug
[0236] An anti-cancer drug conjugated to IF7 can deliver the drug
to the tumor and suppress tumor growth in vivo. IF7 was conjugated
with a geldanamycin analogue 17-AAG, an apoptosis-inducing drug
(Vasilevskaya, I. et al., Cancer Res 2003; 63: 3241-6; Mandler, R.
et al., J Natl Cancer Inst 2000; 92: 1573-81) (FIG. 2). When the
IF7-GA conjugate was injected intravenously into B16 tumor-bearing
mice, tumor growth was suppressed: tumors from IF7-GA-treated mice
were significantly smaller than those from control mice (FIG.
6A-a). It should be noted that the dose of 17-AAG used as IF7-GA
was 5 mg/kg, whereas 50-75 mg/kg 17-AAG has been used in previous
studies of mouse tumor models (Eiseman et al., Cancer Chemother
Pharmacol 2005; 55:21-32; Solit et al., Clin Cancer Res 2002;
8:986-93; Mitsiades, C. et al., Blood 2006; 107:1092-100). While
B16 tumors from control mice showed active growth around blood
vessels (FIG. 6B-a), B16 cells in tumors from IF7-GA-treated mice
showed clear signs of apoptosis along vessels and morphologically
apparent necrosis of blood vessels, indicative of a GA effect
against tumor and tumor endothelial cells (Vasilevskaya et al.,
Cancer Res 2003; 63: 3241-6; Solit et al., Cancer Res 2003;
63:2139-44). Histological analysis of major organs from all mice
used showed no apparent abnormalities. Blood tests from all mice
showed no abnormalities in liver function, kidney function and
blood cell count. However, tumor-bearing mice treated by IF7-GA did
not survive longer than control mice.
[0237] Similar results were obtained using a lung carcinoma tumor
model produced by subcutaneous injection of Lewis lung carcinoma
(LLC) cells into BL6 mice (FIG. 6A-b, FIG. 6B-b), and in human
prostate and breast cancer mouse models produced by respective
orthotopic injection of human prostate cancer PC3 cells and breast
cancer MDA-MB-231 cells into immunodeficient mice (FIGS. 6A-c,
6A-d, 6B-c and 6B-d).
[0238] The IF7-GA dosage used herein can be considered to be
optimal, as increasing doses did not improve survival of
tumor-bearing mice or reduce tumor size. The failure to rescue
IF7-GA-treated mice is due to modest activity of GA, which induces
apoptosis by inhibiting Hsp-90 (Clarke et al., Oncogene 2000;
19:4125-33; Panaretou et al., Mol Cell 2002; 10:1307-18). Therefore
IF7 was conjugated to SN-38, a highly potent anti cancer drug
(Meyer-Losic et al., Clin Cancer Res 2008; 14:2145-53) (FIG. 7)
using an esterase-cleavable cross-linker. Remarkably, when IF7-SN38
was injected intravenously into mice with large B16 solid tumors,
those mice were rescued (FIG. 8A): tumor-bearing mice survived as
long as IF7-SN38 injections continued (FIG. 8A). Thus, IF7-SN38 was
effective in rescuing mice at near terminal stages, whereas IF7-GA
was not. IF7-SN38 slowed tumor cell proliferation immediately
following administration, although tumor size gradually increased
(FIG. 8B). Despite large tumor sizes, mice showed no signs of
weakness during extended days of survival mediated by IF7-SN38.
Tumors from IF7-SN38-injected mice occasionally showed edema, while
tumors from control mice did not. These observations indicate that
fluid accumulation contributed to increased tumor size seen in
IF7-SN38 injected mice. To determine the effect of IF7-SN38 on
early stage of tumors, IF7-SN38 was injected on day 8, and tumors
were isolated two days later. Tumors from IF7-SN38-injected mice
are smaller and contain more necrosis compared to those from
control mice (FIG. 8C). The IF7-SN38 dosage used in this study was
at 7.5 mg/kg, whereas SN-38 conjugated with a peptide without tumor
vasculature targeting activity was used at 95 mg/kg in a previous
study (Meyer-Losic et al., Clin Cancer Res 2008; 14:2145-53).
[0239] To further analyze the effect of IF7-SN38, mice with
peritoneally injected B16 tumors were tested. In peritoneal B16
tumors, cancer cells grew rapidly and mouse survival was limited.
In these experiments, IF7-SN38 lengthened the survival time of B16
tumor-bearing mice (FIG. 8D). IF7-GA was also effective in
lengthening the survival of B16 tumor-bearing mice. Tumors isolated
from IF7-GA treated and IF7-SN38 treated mice were smaller than
those from control mice (FIG. 8E). In control tumor-bearing mice,
many small foci resulting from micrometastasis were seen on the
peritoneal wall, whereas micrometastasis was not detected in
IF7-GA-treated and IF7-SN38-treated mice. Histology showed that
tumors from IF7-GA and IF7-SN38 treated mice show more fat cells
than tumor from control mouse (FIG. 8F), indicating that both
IF7-GA and IF7-SN38 suppressed proliferation of cancer cells. Note
that the effect shown in FIGS. 8E and 8F of IF7-SN38 is superior to
the effect of IF7-GA. Blood tests showed no abnormalities in liver
function, kidney function and blood cell count.
[0240] In above described experiments, tumor size was measured
using a caliper. However, this method is not the most accurate, in
particular if necrosis causes edema or accumulation of lymphatic
fluid in the tumor. In order to monitor the effect of IF7-SN38 in
vivo in the mouse, a luciferase expressing stable cell line,
HCT116-luc, was produced and photon numbers produced by live
HCT116-luc cells were measured. When IF7-SN38 (0.68 .mu.moles) was
injected to HCT116-luc tumor bearing mice, numbers of live
HCT116-luc cells reduced in the next day, whereas without IF7-SN38
injection these cells increased 2 days later (FIG. 10). This
indicated that IF7-SN38 should be administered daily to suppress
tumor growth. When IF7-SN38 was injected intravenously every day to
HCT116-luc tumor-bearing mice, growth the tumors was completely
suppressed, which was demonstrated by both photon numbers and
caliper measurements (FIG. 11).
[0241] v. Targeting and Penetration of IF7C(RR) Peptide to the
Tumor Vasculature
[0242] Since IF7-SN38 is highly hydrophobic, there was a concern
regarding a possibility that IF7-SN38 becomes insoluble after
intravenous injection. Such may reduce the activity of IF7-SN38 in
vivo. To increase the solubility of IF7-SN38 in aqueous
environment, two arginine residues were added to IF7 after the
cysteine residue. Thus IFLLWQR-C-RR (SEQ ID NO:17) or IF7C(RR) was
synthesized. When IF7C(RR) was conjugated to FITC-poly-L-lysine,
IF7C(RR)-conjugated FITC-poly-L-lysine bound to the surface of
Anxa1-expressing mouse endothelial F-2 cells (FIG. 12A). The
cytoplasmic and nuclear FITC signals are consistent with the
localization of Anxa1 in the cytoplasm and nucleus (Gerke, 2005
#5299). FITC-poly-L-lysine without IF7C(RR) did not bind to F-2
cells (data not shown). When IF7C(RR)-conjugated FITC-poly-L-lysine
was added to F-2 cells grown on a filter of the insert for trans
well chamber, washed and then incubated in a medium at 37.degree.
C. or at 4.degree. C., fluorescence migrated to the lower chamber
from cells incubated at 37.degree. C. but not at 4.degree. C. (FIG.
12B). This indicates that IF7C(RR) binding and transport is
mediated by an active transport mechanism through cells. To
determine if the binding and transport of IF7C(RR) occur in vivo in
the tumor vasculature, IF7C(RR)-conjugated FITC-poly-L-lysine was
injected intravenously into a B16 tumor-bearing mouse. The tumor
tissue sections showed green fluorescence signals around the
abluminal area of the vasculatures (FIG. 12C), indicating the
binding of IF7C(RR) to the tumor vasculature and transport of this
peptide from luminal to abluminal surface through endothelial
cells. Small molecules such as SN-38 conjugated with IF7C(RR) can
penetrate to the tumor deeper and faster than the large molecule
such as poly-L-lysine.
[0243] vi. Effect of IF7C(RR) Conjugated SN-38 on HCT116-Luc
Tumors
[0244] The activity of IF7C(RR)-SN38 was tested using HCT116-luc
tumors described above. A nude mouse with large HCT116-luc tumor
was injected with IF7C(RR)-SN38 by daily injection and tumor size
was monitored by luciferase-based chemiluminescence (FIG. 13). It
appears that IF7C(RR)-SN38 had stronger anti-tumor activity than
1F7-SN38 shown in FIG. 11. IF7C(RR)-SN38 not only suppressed the
growth but also reduce the size significantly, while IF7-SN38 did
not substantially reduce the tumor size below the pre-drug
injection level.
[0245] To determine further the anti-tumor activity of
IF7C(RR)-SN38, the effect of this drug was tested at low doses.
Reduced dosage experiments showed that IF7C(RR)-SN38 effectively
suppressed HCT116-luc tumor growth as low as at 0.81 .mu.moles/kg
(FIG. 14). We anticipated that IF7C(RR)-SN38 injected at these
dosages have no side effects. This was confirmed by a series of
blood tests (FIG. 15).
[0246] 3. Discussion
[0247] Anxa1 localizes to the tumor endothelial cell surface in
endothelial caveolae and is internalized through endocytosis
(Schnitzer et al., J Biol Chem 1995; 270:14399-404). A recent study
indicates that a ligand bound to endothelial caveoli protein at the
apical cell surface is efficiently transported to the basal surface
and released to the stroma below (Schnitzer Adv Drug Deliv Rev
2001; 49:265-80). Therefore, IF7-conjugated drug captured by
endothelial cells at the luminal surface can be released to the
stroma where cancer cells could be exposed to drug at high
concentration. During these processes, the peptide moiety of the
anti-cancer drug conjugate would likely be digested by proteases
allowing drug to penetrate tumor cells. This hypothesis is
consistent with histological observations showing that cancer cells
located around the vasculature underwent apoptosis in tumor-bearing
mice injected with the IF7-conjugated apoptosis-inducing drug
IF7-GA (FIG. 6B). Free SN-38 can be produced through the action of
serum or tissue esterases. This allows SN-38 to enter cells and
have its effect.
[0248] An IF7-conjugated anti-cancer drug improves chemotherapy
efficacy through binding of IF7 to Anxa1 (FIG. 1F-H) and by
expression of Anxa1 on tumor vasculature (FIG. 1F, 5A-C) (P. Oh et
al. Subtractive proteomic mapping of the endothelial surface in
lung and solid tumours for tissue-specific therapy. Nature
429:629-635 (2004)). These features lead to extremely high
specificity and efficacy in delivering an IF7-conjugated compound
to the tumor (FIG. 5). Given that it may take at least 15-30
minutes for an antibody to bind its antigen, the efficacy of IF7
binding to the tumor vasculature exceeds the levels of any
tumor-targeting reagent known so far. Since it is a 7-mer peptide,
production and quality control of IF7 is much easier than that used
to produce humanized monoclonal antibodies for clinical trials (S.
Izumoto et al. Phase II clinical trial of Wilms tumor 1 peptide
vaccination for patients with recurrent glioblastoma multiforme. J
Neurosurg 108:963-971 (2008)). Furthermore, peptides are readily
degradable, and therefore concerns regarding human and
environmental toxicity should be minimal. A short 7-mer peptide
such as IF7 likely does not function as an antigen, and therefore
concerns regarding immune reactions in patients injected by IF7
should be minimal.
[0249] In general, peptide-based drugs have been considered
unstable as they are susceptible to proteases in vivo (L. Otvos,
Jr. Peptide-based drug design: here and now. Methods Mol Biol
494:1-8 (2008), L. A. Landon et al. Is phage display technology on
target for developing peptide-based cancer drugs? Curr Drug Discov
Technol 1:113-132 (2004)). However, the extremely high tumor
vasculature-targeting achieved by IF7 can overcome potential
problems caused by proteolysis: IF7 functions as a vehicle for
anti-cancer drug, and most IF7-conjugated drug can be delivered
before IF7 undergoes proteolysis. Short peptides have been tested
successfully for tumor vasculature targeting in vivo in the mouse
(W. Arap et al. Cancer treatment by targeted drug delivery to tumor
vasculature in a mouse model. Science 279:377-380 (1998); E. A.
Murphy et al. Nanoparticle-mediated drug delivery to tumor
vasculature suppresses metastasis. Proc Natl Acad Sci USA
105:9343-9348 (2008); N. Oku et al. Anti-neovascular therapy using
novel peptides homing to angiogenic vessels. Oncogene 21:2662-2669
(2002); F. Donate et al. Pharmacology of the novel antiangiogenic
peptide ATN-161 (Ac--PHSCN-NH2): observation of a U-shaped
dose-response curve in several preclinical models of angiogenesis
and tumor growth. Clin Cancer Res 14:2137-2144 (2008)). However,
clinical trials have not yet yielded promising outcomes. IF7 can
have advantages over previously known tumor vasculature-targeting
peptides because it targets Anxa1, which was identified after
rigorous comparisons of normal and tumor vasculature (P. Oh et al.
Subtractive proteomic mapping of the endothelial surface in lung
and solid tumours for tissue-specific therapy. Nature 429:629-635
(2004)).
[0250] Successful targeting of drug delivery to the tumor
vasculature has not been achieved in humans (Ruoslahti E. et al.
Annu Rev Immunol 2000; 18:813-27; Neri, D. et al. Nat Rev Cancer
2005; 5:436-46; Bellone, M. et al. Trends Immunol 2008; 29:235-41).
The carbohydrate mimicry peptide IF7 can serve as a vehicle for
such delivery because IF7 targets Anxa1, which is specifically
expressed on tumor vasculature (Oh et al. Nature 2004; 429:629-35).
Peptide-based therapeutics are advantageous, as large quantities of
short, highly purified peptides can be synthesized at low cost for
clinical trials (Izumoto et al., J Neurosurg 2008; 108:963-71).
Furthermore, peptides are readily degradable, concerns regarding
human and environmental toxicity should be minimal. Although no
peptide-based anti-cancer drugs have been established (Arap et al.,
Science 1998; 279:377-80; Murphy, E. et al., Proc Natl Acad Sci
2008; 105: 9343-8; Oku, N. et al., Oncogene 2002; 21:2662-9;
Donate, F., et al., Clin Cancer Res 2008; 14:2137-44), clear
anti-cancer activity by IF7-GA and IF7-SN38 indicates that the
rapid delivery of 1F7-conjugated drug overcomes potential problems
of IF7 proteolytic degradation.
[0251] Effective chemotherapy should rescue patients with malignant
tumors not only in early but in advanced stages, and further
suppress recurrence of malignancy by eradicating cancer stem cells.
Highly efficient targeted drug delivery by IF7 would allow multiple
chemotherapies by different anti-cancer drugs each with a distinct
activity. Nonetheless, the efficacy of 1F7-conjugated anti-cancer
drugs remains to be evaluated clinically in cancer patients.
[0252] 4. Materials and Methods.
[0253] i. Materials.
[0254] Peptides were synthesized by GenScript (Piscataway, N. J.).
Rabbit anti-annexin 1 antibody (H-65) was from Santa Cruz
Biotechnologies (Santa Cruz, Calif.). Phage clones each displaying
I-peptide and IF7 have been described (Fukuda, M. et al., Cancer
Res 2000; 60:450-6).
[0255] ii. Use of Vertebrate Animals.
[0256] Mouse protocols were approved by Institutional Review
Committees at Burnham Institute for Medical Research.
[0257] iii. In Vivo Phage Targeting.
[0258] Mouse melanoma B16F1 cells (2.times.10.sup.5 cells/100 .mu.l
PBS) were injected subcutaneously into the dorsal flank of C57BL/6
female mice (8-10 weeks old). Ten days later, I-peptide displaying
phage clones or each clone displaying I-peptide related sequence
(1.times.10.sup.5 pfu) in 100 .mu.l PBS was injected intravenously.
In a separate set of experiments, rabbit anti-Anxa1 antibody (H-65,
Santa Cruz) or rabbit IgG (20 .mu.g IgG) was injected 15 minutes
prior to phage injection. The mouse was perfused with TBS
containing 1 mM CaCl.sub.2) (TBSC), and tumor and lung tissue was
isolated. Tissue homogenates (100 mg protein) were incubated with
competent K91 bacteria, and plated on LB agar containing
tetracycline (10 .mu.g/ml) and Kanamycin (100 .mu.g/ml). Colonies
appearing on an agar plate after culturing at 37.degree. C. for 20
hours were counted.
[0259] iv. Binding of IF7-A488 to IF7-His.sub.6 Protein.
[0260] Full-length cDNA encoding Anxa1 was obtained from Invitrogen
(Carlsbad, Calif.) and subcloned into pET29a vector (Novagen) to
produce an IF7-His.sub.6 fusion protein. Recombinant proteins were
purified by Ni.sup.+ affinity chromatography. Wells of a black
384-well plate (Greiner bio-one) were coated with recombinant
IF7-His.sub.6 protein (10 .mu.g/well). IF7-A488 (4 .mu.g/ml)
dissolved in 10 mM Tris-HCl buffer, pH7.4, containing 1 mM
CaCl.sub.2) and 0.05% Tween 20, was added. After washing the plate,
fluorescence was measured by a Molecular Devices Analyst HT plate
reader. Analysis of inhibition of binding of Lewis A
oligosaccharide to Anxa1-His.sub.6 by IF7 and control RQ7 peptide
was carried out using FITC-conjugated polyacrylamide-LeA
(Glycotech) as described above.
[0261] v. In Vivo Imaging of IF7-A488 in Dorsal Skinfold Chamber
Window
[0262] A Lewis lung carcinoma (LLC) tumor was produced in a donor
nude mouse by subcutaneous injection, and small piece of tumor
(less than 1 mm.sup.3) was transplanted to a dorsal skinfold
chamber in a recipient nude mouse (8-10 weeks female Balb/c nude)
as described (Lehr et al., Am J Pathol 1993; 143:1055-62; Oh et
al., Nat Biotechnol 2007; 25:327-37). Three days later, the mouse
was anesthetized by peritoneal injection of 1.25%
2,2,2-Tribromoethanol (25 .mu.l/g). IF7-A488 or RQ7-A488 (100
.mu.l; 50 mM in 5% glucose solution) was injected through the tail
vein. Intravital Alexa 488 signals in the tumor were detected and
recorded by a Zeiss Axioplan fluorescence microscope and a digital
camera system (DP70 and DP controller, Olympus). For inhibition
assays, rabbit anti-Anxa1 antibody (H-65, Santa Cruz) or rabbit IgG
(20 .mu.g IgG) was injected 15 minutes prior to IF7-A488 injection.
Signal intensity in the tumor from 0 min to 40 min was measured by
Image J (NIH, Maryland). After 10 min, irradiation of specimens by
a UV lamp was limited only to times when photos were taken to avoid
fluorescence bleaching. The tumor was isolated from the dorsal skin
folder chamber, fixed with 4% paraformaldehyde at room temperature
for 15 min, immersed in O. C.T compound, and cryosections were
made. Frozen sections were overlaid with Vectashield containing
DAPI (Vector laboratories) and examined under a Zeiss Axioplan
fluorescence microscope.
[0263] vi. Tumor Models and IF7-GA or IF7-SN38 Treatment.
[0264] Mouse melanoma B16F1 cells (2.times.10.sup.5 cells/100 .mu.l
serum-free DMEM) were injected subcutaneously into the dorsal flank
of C57BL/6 female mice (8-10 weeks old). Ten days later, mice were
divided randomly into 4 groups, which received (1) 100 .mu.l 5%
glucose or the same amount of 5% glucose containing (2) IF7, (3) GA
or (4) IF7-GA at 1.3 mM each on days 10, 12, and 14. On day 15,
mice were sacrificed and tumor weights determined. Mouse Lewis lung
carcinoma (LLC) cells (4.times.10.sup.5 cells/100 .mu.l serum-free
DMEM) were injected subcutaneously into the dorsal flank of C57BL/6
female mice (8-10 weeks old). Seven days later, mice were divided
randomly into 4 groups and received (1) 100 .mu.l 5% glucose or 5%
glucose containing (2) IF7, (3) GA or (4) IF7-GA at 1.3 mM on days
7, 9, and 11. On day 13, mice were sacrificed and tumor weights
determined. For the prostate cancer model, SCID/C. B-17 male mice
(6-8 weeks old) were anesthetized by peritoneal injection of 1.25%
2,2,2-Tribromoethanol. Human prostate cancer PC3 cells
(1.times.10.sup.6 cells/20 .mu.l serum-free DMEM) were injected
orthotopically into the mouse prostate. On day 7, mice were divided
into 4 groups and treated as above on days 7, 12, 17 and 22. On day
28, mice were sacrificed and prostate tumor weights determined. For
the breast cancer model, SCID/C. B-17 female mice (8-10 weeks old)
were injected with human breast cancer MDA-MB-231 cells
(1.times.10.sup.6 cells in 50 .mu.l of Hanks' Balanced Salt
Solution) together with 50 .mu.l of matrigel (Becton Dickinson, San
Jose, Calif.) into mammary fat. Intravenous injection of IF7-GA and
control reagents followed schedules described for the prostate
cancer model.
[0265] For the results shown in FIG. 9, mouse melanoma B16F1 cells
(2.times.10.sup.5 cells/100 ul serum-free DMEM) were injected
subcutaneously into the dorsal flank of C57BL/6 female mice (8-10
weeks old). Ten days later, mice were divided randomly into 3
groups, which received 100 .mu.l 5% glucose containing 0.082
.mu.moles of 1F7-Dox or IF7-SN38 each on days 10, 12, 14 and 16.
Tumor sizes were measure using a caliper.
[0266] vii. Statistical Analysis.
[0267] Statistical analyses were performed using SPSS (Chicago,
Ill.) and Microsoft Excel (Redmond, Wash.) programs. All values in
figures and text are expressed as means.+-.standard deviation (SD)
of n observations, where n is the number of animals analyzed. Data
sets were compared with Student's unpaired t-test (two tailed) or
Mann-Whitney's U test. A p value .ltoreq.0.05 was considered
significant.
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TABLE-US-00002 [0300] Sequences SEQ ID NO: 1 IELLQAR SEQ ID NO: 2
IFLLWQR SEQ ID NO: 3 IILLQAR SEQ ID NO: 4 IDLMQAR SEQ ID NO: 5
ISLLQAR SEQ ID NO: 6 FSLLDAR SEQ ID NO: 7 ISLLGAR SEQ ID NO: 8
PLWRPSR SEQ ID NO: 9 LLLMQLR SEQ ID NO: 10 LYLQRLR SEQ ID NO: 11
MAMVSEFLKQARFLENQEQEYVQAVKSYKGGPGSAVSPYPSFN
VSSDVAALHKAIMVKGVDEATIIDILTKRTNAQRQQIKAAYLQ
ENGKPLDEVLRKALTGHLEEVVLAMLKTPAQFDADELRGAMKG
LGTDEDTLIEILTTRSNEQIREINRVYREELKRDLAKDITSDT
SGDFRKALLALAKGDRCQDLSVNQDLADTDARALYEAGERRKG
TDVNVFTTILTSRSFPHLRRVFQNYGKYSQHDMNKALDLELKG
DIEKCLTTIVKCATSTPAFFAEKLYEAMKGAGTRHKALIRIMV
SRSEIDMNEIKVFYQKKYGISLCQAILDETKGDYEKILVALCG GN SEQ ID NO: 12
IFLLWQRKKKC SEQ ID NO: 13 IELLQAR SEQ ID NO: 14 IFLLWQRC SEQ ID NO:
15 RQWLLFI SEQ ID NO: 16 RQWLLFICRR SEQ ID NO: 17 IFLLWQRCR SEQ ID
NO: 18 IFLLWQRCRRR SEQ ID NO: 19 IFLLWQRCRR SEQ ID NO: 20
IFLLWQRXXXX (X can be any amino acid) SEQ ID NO: 21 IFLLWQRCXXXX (X
can be any amino acid) SEQ ID NO: 22 IFLLWQRCRRRR SEQ ID NO: 23
DDDDK SEQ ID NO: 24 KLAKLAKKLAKLAK
Sequence CWU 1
1
2417PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 1Ile Glu Leu Leu Gln Ala Arg1
527PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 2Ile Phe Leu Leu Trp Gln Arg1
537PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 3Ile Ile Leu Leu Gln Ala Arg1
547PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 4Ile Asp Leu Met Gln Ala Arg1
557PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 5Ile Ser Leu Leu Gln Ala Arg1
567PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 6Phe Ser Leu Leu Asp Ala Arg1
577PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 7Ile Ser Leu Leu Gly Ala Arg1
587PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 8Pro Leu Trp Arg Pro Ser Arg1
597PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 9Leu Leu Leu Met Gln Leu Arg1
5107PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(7) 10Leu Tyr Leu Gln Arg Leu Arg1
511346PRTRattus norvegicusPEPTIDE(1)..(346)Anxa1 fragment 11Met Ala
Met Val Ser Glu Phe Leu Lys Gln Ala Arg Phe Leu Glu Asn1 5 10 15Gln
Glu Gln Glu Tyr Val Gln Ala Val Lys Ser Tyr Lys Gly Gly Pro 20 25
30Gly Ser Ala Val Ser Pro Tyr Pro Ser Phe Asn Val Ser Ser Asp Val
35 40 45Ala Ala Leu His Lys Ala Ile Met Val Lys Gly Val Asp Glu Ala
Thr 50 55 60Ile Ile Asp Ile Leu Thr Lys Arg Thr Asn Ala Gln Arg Gln
Gln Ile65 70 75 80Lys Ala Ala Tyr Leu Gln Glu Asn Gly Lys Pro Leu
Asp Glu Val Leu 85 90 95Arg Lys Ala Leu Thr Gly His Leu Glu Glu Val
Val Leu Ala Met Leu 100 105 110Lys Thr Pro Ala Gln Phe Asp Ala Asp
Glu Leu Arg Gly Ala Met Lys 115 120 125Gly Leu Gly Thr Asp Glu Asp
Thr Leu Ile Glu Ile Leu Thr Thr Arg 130 135 140Ser Asn Glu Gln Ile
Arg Glu Ile Asn Arg Val Tyr Arg Glu Glu Leu145 150 155 160Lys Arg
Asp Leu Ala Lys Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe 165 170
175Arg Lys Ala Leu Leu Ala Leu Ala Lys Gly Asp Arg Cys Gln Asp Leu
180 185 190Ser Val Asn Gln Asp Leu Ala Asp Thr Asp Ala Arg Ala Leu
Tyr Glu 195 200 205Ala Gly Glu Arg Arg Lys Gly Thr Asp Val Asn Val
Phe Thr Thr Ile 210 215 220Leu Thr Ser Arg Ser Phe Pro His Leu Arg
Arg Val Phe Gln Asn Tyr225 230 235 240Gly Lys Tyr Ser Gln His Asp
Met Asn Lys Ala Leu Asp Leu Glu Leu 245 250 255Lys Gly Asp Ile Glu
Lys Cys Leu Thr Thr Ile Val Lys Cys Ala Thr 260 265 270Ser Thr Pro
Ala Phe Phe Ala Glu Lys Leu Tyr Glu Ala Met Lys Gly 275 280 285Ala
Gly Thr Arg His Lys Ala Leu Ile Arg Ile Met Val Ser Arg Ser 290 295
300Glu Ile Asp Met Asn Glu Ile Lys Val Phe Tyr Gln Lys Lys Tyr
Gly305 310 315 320Ile Ser Leu Cys Gln Ala Ile Leu Asp Glu Thr Lys
Gly Asp Tyr Glu 325 330 335Lys Ile Leu Val Ala Leu Cys Gly Gly Asn
340 3451211PRTArtificial Sequencechemically synthesized; annexin-1
binding compoundpeptide(1)..(11) 12Ile Phe Leu Leu Trp Gln Arg Lys
Lys Lys Cys1 5 10137PRTArtificial Sequencechemically synthesized;
selectin ligand mimicPEPTIDE(1)..(7) 13Ile Glu Leu Leu Gln Ala Arg1
5148PRTArtificial Sequencechemically synthesized; annexin-1 binding
compoundpeptide(1)..(8) 14Ile Phe Leu Leu Trp Gln Arg Cys1
5157PRTArtificial Sequencechemically synthesized; control
peptidepeptide(1)..(7) 15Arg Gln Trp Leu Leu Phe Ile1
51610PRTArtificial Sequencechemically synthesized; control
peptidepeptide(1)..(10) 16Arg Gln Trp Leu Leu Phe Ile Cys Arg Arg1
5 10179PRTArtificial Sequencechemically synthesized; annexin-1
binding compoundpeptide(1)..(9) 17Ile Phe Leu Leu Trp Gln Arg Cys
Arg1 51811PRTArtificial Sequencechemically synthesized; annexin-1
binding compoundPEPTIDE(1)..(11) 18Ile Phe Leu Leu Trp Gln Arg Cys
Arg Arg Arg1 5 101910PRTArtificial Sequencechemically synthesized;
annexin-1 binding compoundpeptide(1)..(10) 19Ile Phe Leu Leu Trp
Gln Arg Cys Arg Arg1 5 102011PRTArtificial Sequencechemically
synthesized; annexin-1 binding compoundpeptide(1)..(11)Xaa can be
any amino acid 20Ile Phe Leu Leu Trp Gln Arg Xaa Xaa Xaa Xaa1 5
102112PRTArtificial Sequencechemically synthesized; annexin-1
binding compoundpeptide(1)..(12)Xaa can be any amino acid 21Ile Phe
Leu Leu Trp Gln Arg Cys Xaa Xaa Xaa Xaa1 5 102212PRTArtificial
Sequencechemically synthesized; annexin-1 binding
peptidepeptide(1)..(12) 22Ile Phe Leu Leu Trp Gln Arg Cys Arg Arg
Arg Arg1 5 10235PRTArtificial Sequencechemically synthesized;
cleavage site for enterokinasepeptide(1)..(5) 23Asp Asp Asp Asp
Lys1 52414PRTArtificial Sequencechemically synthesized; therapeutic
agentpeptide(1)..(14) 24Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys
Leu Ala Lys1 5 10
* * * * *