U.S. patent application number 11/437095 was filed with the patent office on 2007-02-22 for peptides whose uptake by cells is controllable.
Invention is credited to Todd Aguilera, Tao Jiang, Quyen Nguyen, Emilia S. Olsen, Roger Tsien, Michael Whitney, Edmund Wong.
Application Number | 20070041904 11/437095 |
Document ID | / |
Family ID | 37431596 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070041904 |
Kind Code |
A1 |
Jiang; Tao ; et al. |
February 22, 2007 |
Peptides whose uptake by cells is controllable
Abstract
A generic structure for the peptides of the present invention
includes A-X-B-C, where C is a cargo moiety, the B portion includes
basic amino acids, X is a cleavable linker sequence, and the A
portion includes acidic amino acids. The intact structure is not
significantly taken up by cells; however, upon extracellular
cleavage of X, the B-C portion is taken up, delivering the cargo to
targeted cells. Cargo may be, for example, a contrast agent for
diagnostic imaging, a chemotherapeutic drug, or a
radiation-sensitizer for therapy. X may be cleaved extracellularly
or intracellularly. The molecules of the present invention may be
linear, cyclic, branched, or have a mixed structure.
Inventors: |
Jiang; Tao; (San Diego,
CA) ; Olsen; Emilia S.; (La Jolla, CA) ;
Whitney; Michael; (San Diego, CA) ; Aguilera;
Todd; (US) ; Nguyen; Quyen; (US) ;
Wong; Edmund; (San Diego, CA) ; Tsien; Roger;
(La Jolla, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
37431596 |
Appl. No.: |
11/437095 |
Filed: |
May 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11133804 |
May 19, 2005 |
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11437095 |
May 19, 2006 |
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10699562 |
Oct 31, 2003 |
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11133804 |
May 19, 2005 |
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Current U.S.
Class: |
424/1.69 ;
424/9.322; 435/456; 530/317 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 49/14 20130101; A61K 47/645 20170801; A61K 49/0056 20130101;
A61K 41/0095 20130101; A61K 49/085 20130101; C07K 14/4728 20130101;
A61K 49/0043 20130101; A61K 38/00 20130101; A61K 51/088 20130101;
C07K 14/00 20130101; A61K 49/0032 20130101; A61K 47/65 20170801;
A61K 49/146 20130101; C07K 7/08 20130101; A61K 47/64 20170801 |
Class at
Publication: |
424/001.69 ;
424/009.322; 530/317; 435/456 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 49/10 20070101 A61K049/10; C07K 7/64 20070101
C07K007/64; C12N 15/86 20060101 C12N015/86 |
Goverment Interests
STATEMENT OF FEDERALLY-SPONSORED RESEARCH
[0002] This work was supported in part by grants from the
Department of Energy, DE-FG03-01ER63276 and from the National
Institutes of Health Grants DK54441, GM54038 and NS27177 (NINCDS).
The government may have certain rights in this invention.
Claims
1. A cyclic molecule of the structure A-X1-B-X2, wherein B is a
peptide portion of about 5 to about 20 basic amino acid residues,
which is suitable for cellular uptake, A is a peptide portion of
about 2 to about 20 acidic amino acid residues, which when linked
with portion B is effective to inhibit or prevent cellular uptake
of portion B; and A and B are linked together and linked via
linkers X1 and X2 to form a cyclic compound; where X1 and X2 are
each a cleavable linker of about 2 to about 100 atoms joining A
with B to form a cyclic compound where each of X1 and X2 link to
both A and B.
2. The molecule of claim 1, wherein at least one of cleavable
linkers X1 or X2 is configured for cleavage exterior to a cell.
3. The molecule of claim 1, wherein at least one of cleavable
linkers X1 or X2 is configured for cleavage interior to a cell.
4. The molecule of claim 2, wherein at least one of cleavable
linkers X1 or X2 is configured for cleavage by an enzyme.
5. The molecule of claim 4, wherein said enzyme is a matrix
metalloprotease.
6. The molecule of claim 5 wherein at least one of cleavable
linkers X1 or X2 comprises the amino acid sequence PLGLAG (SEQ ID
NO:1).
7. The molecule of claim 5 wherein at least one of cleavable
linkers X1 or X2 comprises the amino acid sequence EDDDDKA (SEQ ID
NO:2).
8. The molecule of claim 1 wherein at least one of cleavable
linkers X1 or X2 comprises a S-S linkage.
9. The molecule of claim 1, further comprising a cargo moiety C
linked to peptide portion B.
10. The molecule of claim 9, wherein said cargo moiety C comprises
a label.
11. The molecule of claim 10, wherein said label comprises a label
selected from the group consisting of fluorescent labels,
luminescent labels, radioactive labels, contrast agents, radiation
sensitizers, antibodies, and antigens.
12. The molecule of claim 9, wherein said cargo moiety C comprises
a phage.
13. The molecule of claim 11, wherein said cargo moiety C comprises
a radioactive technetium label.
14. The molecule of claim 9, wherein said cargo moiety C comprises
a contrast agent.
15. The molecule of claim 14, wherein said cargo moiety C comprises
a gadolinium contrast agent.
16. A pharmaceutical composition comprising: A cyclic molecule of
the structure A-X1-B-X2, wherein B is a peptide portion of about 5
to about 20 basic amino acid residues, which is suitable for
cellular uptake, A is a peptide portion of about 2 to about 20
acidic amino acid residues, which when linked with portion B is
effective to inhibit or prevent cellular uptake of portion B; and A
and B are linked together and linked via linkers X1 and X2 to form
a cyclic compound; where X1 and X2 are each a cleavable linker of
about 2 to about 100 atoms joining A with B to form a cyclic
compound where each of X1 and X2 link to both A and B, and a
pharmaceutically acceptable carrier.
17. The pharmaceutical composition of claim 16, further comprising
a cargo moiety C linked to peptide portion B.
18. The pharmaceutical composition of claim 17, wherein said cargo
moiety C comprises a therapeutic agent.
19. The pharmaceutical composition of claim 17, wherein said cargo
moiety C comprises a radioactive agent.
20. A method of modulating cellular uptake of a cargo moiety C,
comprising: covalently attaching a cargo moiety C to a peptide B of
about 5 to about 20 basic amino acid residues to form a molecule
BC; linking said molecule BC to a peptide A of about 2 to about 20
acidic amino acid residues with cleavable linkers X1 and X2 of
about 3 to about 30 atoms to form a cyclic molecule of the
structure A-X1-BC-X2 where each of X1 and X2 link to both A and BC,
and cleaving said cleavable linkers X1 and X2 effective to separate
BC from said peptide A.
21. A molecule for transporting a cargo moiety across a cell
membrane of the structure A-X-B-C, wherein C is a portion
comprising a cargo moiety, B is a peptide portion of about 5 to
about 20 basic amino acid residues, which is suitable for cellular
uptake, is covalently linked to portion C, and is effective to
enhance transport of cargo portion C across a cell membrane, A is a
peptide portion of about 2 to about 20 acidic amino acid residues,
which when linked with portion B is effective to inhibit or prevent
cellular uptake of B-C, and X is a cleavable linker of about 2 to
about 100 atoms joining A with B-C, which can be cleaved under
conditions in or near a target cell.
22. The molecule for transporting a cargo moiety across a cell
membrane of claim 21, wherein said portion comprising a cargo
moiety C comprises a phage.
23. The molecule of claim 21, wherein said portion comprising a
cargo moiety C comprises a label.
24. The molecule of claim 23, wherein said label comprises a label
selected from the group consisting of fluorescent labels,
luminescent labels, radioactive labels, contrast agents, radiation
sensitizers, antibodies, and antigens.
25. The molecule of claim 21, wherein said portion comprising a
cargo moiety C comprises a radioactive technetium label.
26. The molecule of claim 21, wherein said portion comprising a
cargo moiety C comprises a contrast agent.
27. The molecule of claim 21, wherein said portion comprising a
cargo moiety C comprises a gadolinium contrast agent.
28. A method of guiding surgical resection of a cancer, the method
comprising the steps of administering to the subject a molecule of
claim 21, wherein the cargo is a fluorescent molecule.
29. The method of claim 28, wherein the cancer has metastasized to
lymph nodes which are resected during the surgery.
30. A method of diagnosing metastatic cancer in lymph nodes by in
vivo imaging, the method comprising the steps of administering to a
subject a molecule of claim 21, wherein the cargo is a gamma
emitting radionuclide, and imaging the lymph nodes using SPECT or
planar gamma camera imaging.
31. The method of claim 30, wherein the gamma emitting radionuclide
is selected from the group consisting of .sup.64Cu, .sup.99mTc,
.sup.111In, .sup.123I, .sup.124I or .sup.131I.
32. A method of diagnosing metastatic cancer in lymph nodes by in
vivo imaging, the method comprising the steps of administering to a
subject a molecule of claim 21, wherein the cargo is a contrast
agent, and imaging the lymph nodes using MRI.
33. The method of claim 32, wherein the contrast agent is
Gd.sup.3+.
34. A method of in vivo selection of protease cleavage sites in
linker X, the method comprising the steps of: (a) providing a
molecule of claim 21, wherein the cargo is a phage, the molecule is
attached to the phage via a coat protein attached to at portion B,
and X is a test cleavable linker from a random amino acid library;
(b) administering the phage to a non-human subject; (c) removing
tumor tissue comprising the phage; (d) isolating the phage from the
tumor tissue; (e) amplifying the phage by re-administering the
phage to a non-human subject; and (f) isolating and sequencing the
phage to identify protease cleavage sites.
35. The method of claim 34, wherein the phage is administered to
the non-human subject for a total of seven cycles.
36. The method of claim 34, wherein the molecule has a structure
A-X-B-C, wherein B is linked to C at the N-terminus of the pIII
coat protein of M13 phage, or C-B-X-A, wherein B is linked to C at
the C-terminus of the 10B capsid protein of T7 phage.
37. A method of in vitro selection of protease cleavage sites in
linker X, the method comprising the steps of: (a) provide a
molecule of claim 21, wherein the cargo is a phage, the molecule is
attached to the phage via a coat protein attached to at portion B,
and X is a test cleavable linker from a random amino acid library;
(b) incubating the phage with protease from a non-cancerous cell;
(c) isolating phage that has not entered the cell; (d) incubating
the phage with protease from a cancerous cell; (e) isolating phage
cleaved by protease from the cancerous cell; and (f) sequencing the
phage to identify protease cleavage sites.
38. The method of claim 37, wherein the phage is amplified for a
total of six cycles.
39. The method of claim 37, wherein the protease is purified.
40. The method of claim 37, wherein the protease is from a tissue
extract.
41. A method of imaging a tumor in vivo, the method comprising the
steps of administering to a subject a molecule of claim 21, wherein
the cargo is a contrast agent, and imaging the tumor using MRI.
42. The method of claim 41, wherein the contrast agent is
Gd.sup.3+.
43. A method of imaging a tumor in vivo, the method comprising the
steps of administering to a subject a molecule of claim 21, wherein
the cargo is a gamma emitting radionuclide, and imaging the tumor
using SPECT or planar gamma camera imaging.
44. The method of claim 43, wherein the gamma emitting radionuclide
is selected from the group consisting of .sup.64Cu, .sup.99mTc,
.sup.111In, .sup.123I, .sup.124I or .sup.131I.
45. A method of treating cancer in a subject, the method comprising
the steps of administering to the subject a molecule of claim 21,
wherein the cargo is a therapeutic radionuclide.
46. The method of claim 45, wherein the therapeutic radionuclide is
selected from the group consisting of .sup.90Y or .sup.131I,
.sup.32P, .sup.64Cu, .sup.67Cu, .sup.89Sr, .sup.111In, .sup.117mSn,
.sup.153Sm, .sup.177Lu, .sup.186Re, and .sup.188Re.
47. The molecule of claim 21, wherein peptide portion A is further
linked to a macromolecule having a size of about 50-70 kD.
48. The molecule of claim 21, wherein peptide portion A is further
linked to a macromolecule having a size of about 35-70 kD.
49. The molecule of claim 48, wherein the macromolecule is selected
from the group consisting of dextran, serum albumin, PSA, PEG, and
PAMAM dendrimer.
50. The molecule of claim 21, wherein the cargo is linked to a
nuclear localization signal.
51. The molecule of claim 21, wherein the cargo is linked to an
endosomal release signal.
52. The molecule of claim 21, wherein the cargo is selected from
the group consisting of .sup.64Cu, .sup.99mTc, .sup.111In,
.sup.123I, .sup.124I or .sup.131I.
53. The molecule of claim 21, wherein the cargo is selected from
the group consisting of .sup.90Y .sup.32P, .sup.64Cu, .sup.67Cu,
.sup.89Sr, .sup.111In, .sup.117mSn, .sup.153Sm, .sup.177Lu,
.sup.186Re, and .sup.188Re.
54. The molecule of claim 21, wherein the cargo is selected from
the group consisting of .sup.32P, .sup.99mTc, .sup.47Sc, .sup.64Cu,
.sup.67Cu, .sup.89Sr, .sup.86Y, .sup.87Y, .sup.90Y, .sup.105Rh,
.sup.111Ag, .sup.111In, .sup.117mSn, .sup.149Pm, .sup.153Sm,
.sup.166Ho, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.211At,
.sup.123I, .sup.124I, .sup.131I and .sup.212Bi.
55. The molecule of claim 21, wherein the cleavable linker is
cleaved by MMP-2, MMP-9, MMP11m MMP-13, MMP-14, uPA, or PSA.
56. A pharmaceutical composition comprising the molecule of claim
21.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/133,804, filed May 19, 2005 which is a
continuation-in-part of U.S. patent application Ser. No.
10/699,562, filed Oct. 31, 2003, from which priority is claimed
under 35 U.S.C. .sctn. 120, the disclosure of which application is
hereby expressly incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] This invention pertains to compositions and methods for
transporting material across cell membranes, and methods for making
such compositions.
[0004] Introduction
[0005] Cell membranes delimit the outer boundaries of cells, and
regulate transport into and out of the cell interior. Made
primarily of lipids and proteins, they provide a hydrophilic
surface enclosing a hydrophobic interior across which materials
must pass before entering a cell. Although many small, lipophilic
compounds are able to cross cell membranes passively, most
compounds, particles and materials must rely on active mechanisms
in order to gain entry into a living cell.
[0006] Transmembrane Transport
[0007] Regulation of transport into and out of a cell is vital for
its continued viability. For example, cell membranes contain ion
channels, pumps, and exchangers capable of facilitating the
transmembrane passage of many important substances. However,
transmembrane transport is selective: in addition to facilitating
the entry of desired substances into a cell, and facilitating the
exit of others, a major role of a cell membrane is to prevent
uncontrolled entry of substances into the cell interior. This
barrier function of the cell membrane makes difficult the delivery
of markers, drugs, nucleic acids, and other exogenous material into
cells.
[0008] Over the last decade, peptide sequences that can readily
enter a cell have been identified. For example, the Tat protein of
the human immunodeficiency virus 1 (HIV-1) is able to enter cells
from the extracellular environment (e.g., Fawell et al. P.N.A.S.
91:664-668 (1994)). A domain from Antennapedia homeobox protein is
also able to enter cells (Vives, E., et al., J. Biol. Chem. 272,
16010-16017 (1997)). Such uptake is reviewed in, for example,
Richard et al., J. Biol. Chem. 278(1):585-590 (2003).
[0009] Such molecules that are readily taken into cells may also be
used to carry other molecules into cells along with them. Molecules
that are capable of facilitating transport of substances into cells
have been termed "cell-penetrating peptides" (CPPs), protein
transduction domains, and "membrane translocation signals" (MTS)
(see, e.g., Tung et al., Advanced Drug Delivery Reviews 55:281-294
(2003)). The most important MTS are rich in amino acids such as
arginine with positively charged side chains. Molecules transported
into cell by such cationic peptides may be termed "cargo" and may
be reversibly or irreversibly linked to the cationic peptides. An
example of a reversible linkage is found in Zhang et al., P.N.A.S.
95:9184-9189 (1994)).
[0010] MTS molecules are discussed in, for example, Wender et al.,
P.N.A.S. 97:13003-13008 (2000); Hallbrink et al., Biochim. Biophys.
Acta 1515:101-109 (2001); Derossi et al., Trends in Cell Biology
8:84-87 (1998); Rothbard et al., J. Med. Chem. 45:3612-3618 (2002);
Rothbard et al., Nature Medicine 6(11):1253-1247 (2000); Wadia et
al., Curr. Opinion Biotech. 13:52-56 (2002); Futaki et al; Bioconj.
Chem. 12:1005-1011 (2001); Rothbard et al., U.S. Pat. No.
6,306,993; Frankel et al., U.S. Pat. No. 6,316,003; Rothbard et
al., U.S. Pat. No. 6,495,663; and Monahan et al., U.S. Pat. No.
6,630,351. All patents and publications, both supra and infra, are
hereby incorporated by reference in their entirety.
[0011] The uptake facilitated by MTS molecules is typically without
specificity, enhancing uptake into most or all cells. Thus,
although MTS molecules are capable of entering cells, and may be
capable of enhancing the transport of other molecules linked to MTS
molecules into cells, control and regulation of such transport
remains difficult. However, it would be desirable to have the
ability to target the delivery of cargo to a type of cell, or to a
tissue, or to a location or region within the body of an animal.
Accordingly, there remains a need in the art to target, to control
and to regulate the delivery of cargo molecules by MTS
molecules.
SUMMARY OF THE INVENTION
[0012] Molecules, compositions and methods for controlled delivery
of substances into cells by transport molecules are provided.
Molecules having features of the invention include peptide portions
linked by a cleavable linker portion which may be a peptide. The
inventors have found that the cellular uptake of MTS molecules with
multiple basic amino acids can be inhibited or prevented by the
addition of a portion having multiple negative charges at
physiological pH, such as a peptide portion having multiple acidic
amino acids. Thus, an embodiment of the invention provides
compounds including a peptide portion A of between about 2 to about
20 acidic amino acids linked by a cleavable linker X to a peptide
portion B of between about 5 to about 20 basic amino acids, so that
while the peptide portion A is linked to the peptide portion B,
uptake of the molecule into cells is inhibited or prevented. An
acidic portion A may include some amino acids that are not acidic
amino acids, or other moieties as well; similarly, a basic portion
B may include some amino acids that are not basic amino acids, or
other moieties as well. The inhibition or prevention of uptake of a
basic portion B by an acidic portion A is termed "veto" of uptake
of B. After cleavage of linker X so that peptide portion A may
separate from the peptide portion B, portion B is able to enter a
cell, the veto due to portion A having been removed. A cleavable
linker X is preferably cleavable under physiological
conditions.
[0013] In a further embodiment, a cargo portion C including a cargo
moiety may be attached to basic portion B for transport of a cargo
portion C along with B into a cell. Thus, an embodiment of the
invention provides compounds including a peptide portion A of
between about 2 to about 20 acidic amino acids in sequence linked
by a cleavable linker X to a peptide portion B of between about 5
to about 20 basic amino acids, the peptide portion B being
covalently attached to a cargo portion C to form a structure B-C,
effective that while the peptide portion A is linked to the portion
B, uptake of the MTS compound into cells is inhibited or prevented.
Acidic portion A is able to veto of uptake of B-C. Transport across
a cell membrane of cargo portion C linked to portion B is also thus
inhibited or prevented by acidic portion A. After cleavage of
linker X so that peptide portion A may separate from the peptide
portion B, cargo portion C linked to peptide portion B is able to
enter a cell as the uptake veto due to peptide portion A has been
removed. A cleavable linker X is preferably cleavable under
physiological conditions, allowing transport of cargo portion C
into living cells. Cargo portion C may also be cleavably attached
to basic portion B so that cargo portion C may separate from
portion B within a cell.
[0014] Thus, an embodiment of the invention provides molecules
including a peptide portion A having multiple acidic amino acids,
e.g., between about 2 to about 20, preferably between about 5 and
20 acidic amino acids, the peptide portion A being effective to
prevent the uptake of an MTS molecule having a peptide portion B
having multiple basic amino acids e.g., between about 5 to about
20, preferably between about 9 to about 16 basic amino acids.
Peptide portion A is also thus effective to prevent the enhancement
of transport of cargo C across a cell membrane by a peptide portion
B having multiple basic amino acids. Cleavage of a peptide portion
A from a molecule that has a peptide portion B is effective to
restore the ability of the remaining portion of the molecule
including the portion B to be taken up by a cell. Cleavage of a
peptide portion A from a molecule that has a cargo portion C
covalently attached to a peptide portion B to form a structure B-C
is effective to restore the ability of the structure B-C to be
taken up by a cell.
[0015] In one embodiment, a molecule for controllably transporting
a cargo moiety across a cell membrane includes a molecule or
material having the structure A-X-B-C, where C comprises a cargo
moiety, B comprises a peptide portion having multiple basic amino
acids (e.g., between about 5 to about 20, preferably between about
9 to about 16 basic amino acids), B and C being covalently linked,
A comprises a peptide portion having multiple acidic amino acids
(e.g., between about 2 to about 20, preferably between about 4 to
about 20 acidic amino acids), and X comprises a cleavable linker
joining A with B-C. When linked with B-C, peptide portion A is
effective to prevent the enhancement of transport of cargo C across
a cell membrane. When the cleavable linker X is cleaved, the
peptide portion A is freed from the rest of the molecule, including
being freed from portion B and cargo portion C. The cargo portion C
remains linked to portion B after cleavage of the cleavable linker
X. The portion B is effective to enhance transport of cargo portion
C across a cell membrane in the absence of portion A.
[0016] In embodiments of the invention, including molecules having
the schematic structure A-X-B and molecules having the schematic
structure A-X-B-C, acidic amino acids of portion A are glutamate,
aspartate, or phosphoserine. An acidic amino acid has a side chain
with a negative charge at pH 6.0, and may be glutamic acid,
aspartic acid, or other acidic amino acid An acidic portion A
having multiple acidic amino acids may have between about 2 to
about 20, or between about 5 to about 20, or preferably from about
5 to about 9 acidic amino acids. In preferred embodiments, portion
A comprises 5 to 9 glutamates or aspartates, and may comprise 5 to
9 consecutive glutamates or aspartates. In embodiments, acidic
amino acids of portion A are D amino acids. In preferred
embodiments, acidic amino acids of portion A are either
D-glutamate, D-aspartate, or both.
[0017] A basic amino acid has a side chain with a positive charge
at pH 6.0, and may be arginine, histidine, lysine, or other basic
amino acid. In embodiments of the invention, the basic amino acids
of portion B are either arginine, lysine or histidine. A basic
portion B having multiple basic amino acids may have between about
5 to about 20, or between about 9 to about 16 basic amino acids. In
preferred embodiments, portion B comprises about 9 to about 16
arginines, and may comprise about 9 to about 16 consecutive
arginines. In embodiments of the invention, the basic amino acids
of portion B are D amino acids. In preferred embodiments, basic
amino acids of portion B are either D-arginine, D-lysine,
D-histidine, or combinations thereof.
[0018] A cargo moiety may be any molecule, material, substance, or
construct that may be transported into a cell by linkage to a MTS.
A cargo portion C may include one or more cargo moieties. A cargo
moiety may be, for example, a fluorescent moiety, a
fluorescence-quenching moiety, a radioactive moiety, a radiopaque
moiety, a paramagnetic moiety, a nanoparticle, a vesicle, a
molecular beacon, a marker, a marker enzyme (e.g., horse-radish
peroxidase (HRP), beta-galactosidase, or other enzyme suitable for
marking a cell), a contrast agent (e.g., for diagnostic imaging), a
chemotherapeutic agent, a radiation-sensitizer (e.g., for radiation
therapy), a peptide or protein that affects the cell cycle, a
protein toxin, or other cargo suitable for transport into a cell.
In some embodiments where C is a fluorescent moiety, a
fluorescence-quenching moiety is attached to portion A effective to
quench the fluorescence of the fluorescent moiety C before cleavage
of the linker X, and removing the quenching of fluorescent moiety C
after cleavage of linker X.
[0019] A cleavable linker X serves to connect an acidic portion A
with a basic portion B. A cleavable linker X may include, for
example, between about 2 to about 100 atoms, or between about 6 to
about 30 atoms. Cleavable linker portion X may include amino acid
residues, and may be a peptide linkage of between about 1 to about
30, or between about 2 to about 10 amino acid residues. A cleavable
linker X suitable for the practice of the invention may be a
flexible linker. In preferred embodiments, a cleavable linker X
suitable for the practice of the invention is a flexible linker,
and may be about 6 to about 24 atoms in length. In embodiments of
the invention, X may include a peptide linkage. In some embodiments
of the invention, a cleavable linker X includes an aminocaproic
acid (also termed aminohexanoic acid) linkage.
[0020] A cleavable linker X may be configured for cleavage exterior
to a cell. In preferred embodiments of the invention, a cleavable
linker X may be configured to be cleaved in conditions associated
with cell or tissue damage or disease. Such conditions include, for
example, acidosis; the presence of intracellular enzymes (that are
normally confined within cells), including necrotic conditions
(e.g., cleaved by calpains or other proteases that spill out of
necrotic cells); hypoxic conditions such as a reducing environment;
thrombosis (e.g., a linker X may be cleavable by thrombin or by
another enzyme associated with the blood clotting cascade); immune
system activation (e.g., a linker X may be cleavable by action of
an activated complement protein); or other condition associated
with disease or injury.
[0021] For example, a cleavable linker X may be configured for
cleavage by an enzyme, such as a matrix metalloprotease. Other
enzymes which may cleave a cleavable linker include, for example,
urokinase plasminogen activator (uPA), lysosomal enzymes,
cathepsins, prostate-specific antigen, Herpes simplex virus
protease, cytomegalovirus protease, thrombin, caspase, and
interleukin 1.beta. converting enzyme. In embodiments of the
invention, cleavable linker X may include the amino acid sequence
PLGLAG (SEQ ID NO:1) or may include the amino acid sequence EDDDDKA
(SEQ ID NO:2). In other embodiments, a cleavable linker X may
include a S-S linkage, or may include a transition metal complex
that falls apart when the metal is reduced. A molecule embodying
features of the invention may have multiple linkers X linking a
plurality of portions A having acidic amino acids to a structure
B-C.
[0022] In embodiments of the invention, peptide portion A is
located at a terminus of a polypeptide chain comprising B-C, or
comprises the amino terminus of a polypeptide chain comprising B-C.
A may be linked near to or at the amino terminus of a polypeptide
chain comprising B-C, or A may be linked near to or at the carboxy
terminus of a polypeptide chain comprising B-C. The polypeptide
chain B-C may have ends that may be termed a B-side terminus and a
C-side terminus. A cleavable linker X may be disposed near or at
the B-side terminus, or may be disposed near or at the C-side
terminus. In further embodiments, a portion or portions may be
linear or may be cyclic. In embodiments, a cyclic molecule having
features of the invention may have a single linker X or may have
multiple linkers X.
[0023] In further embodiments of the invention, compositions and
solutions, including pharmaceutical compositions are provided which
include compounds of the invention having peptides capable of
controllable delivery of cargo into a cell and a suitable carrier.
Methods for producing such peptides capable of controllable
delivery of cargo into a cell, and pharmaceutical compositions
containing them are also provided. It will be understood that, in
embodiments of the invention, peptoids, carbamates, vinyl polymers,
and other molecules, with a cleavable linkage between an acidic and
a basic portion, may also be provided.
[0024] The molecules, compositions and methods embodying features
of the invention provide the advantages of controlling the uptake
of basic amino acid-containing molecules into cells, and of
controlling the delivery of cargo into cells. Such controlled
uptake and controlled delivery of cargo into cells may be useful,
for example, in treatment of patients having diseased cells or
tissues. For example, delivery of an imaging contrast agent or
antiproliferative agent as cargo may be directed to cancer cells,
and not to all cells in a patient, offering the advantage of
targeted delivery to the diseased cells, in order to enable
noninvasive imaging or increase the effectiveness and decrease
possible side effects of the treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0026] FIG. 1A is a schematic representation of a MTS molecule
having features of the invention comprising a basic portion B, a
linker portion X, and an acidic portion A.
[0027] FIG. 1B is a schematic representation of a cyclic MTS
molecule having features of the invention comprising a basic
portion B, two linker portions X, and an acidic portion A.
[0028] FIG. 2A is a schematic representation of a MTS molecule
having features of the invention comprising a cargo portion C, a
basic portion B, a linker portion X, and an acidic portion A.
[0029] FIG. 2B is a schematic representation of a MTS molecule
having features of the invention comprising a cargo portion C, a
basic portion B, a linker portion X, and an acidic portion A, the
linker portion X connecting to the cargo portion C.
[0030] FIG. 2C is a schematic representation of a MTS molecule
having features of the invention comprising a cargo C linked to
multiple copies of MTS molecules each comprising a basic portion B,
a linker portion X, and an acidic portion A.
[0031] FIG. 2D is a schematic representation of a MTS molecule
having features of the invention comprising a cargo portion C, a
basic portion B, multiple (two) linker regions X, and an acidic
portion A.
[0032] FIG. 2E is a schematic representation of a cyclic MTS
molecule having features of the invention comprising a cargo
portion C, a basic portion B, in which two linker regions X flank
an acidic portion A.
[0033] FIG. 2F is a schematic representation of a MTS molecule
having features of the invention comprising a fluorescent cargo
portion C, a basic portion B, a linker region X, and an acidic
portion A having a quencher Q attached.
[0034] FIG. 3 is a schematic representation of a MTS molecule
having features of the invention in which a cargo portion C is a
contrast agent or drug, a basic portion B is a sequence of eight to
ten D-arginine residues, a linker portion X is a cleavable linker
that may be cleaved by proteolytic enzymes or reducing environment
found near cancerous cells, and an acidic portion A is an
inhibitory domain comprising D-amino acids.
[0035] FIG. 4 is a schematic representation of a MTS molecule of
FIG. 3 having features of the invention in which the cleavable
linker is not cleaved near normal tissue, showing the inability of
a molecule of FIG. 3 to facilitate the entry of cargo into normal
tissue.
[0036] FIG. 5 is a schematic representation of a MTS molecule of
FIG. 3 having features of the invention in which the cleavable
linker is cleaved by proteolytic enzymes or by the reducing
environment found near cancer cells, showing the ability of a
molecule of FIG. 3 to facilitate cargo entry into diseased
tissue.
[0037] FIG. 6A illustrates a High Pressure Liquid Chromatography
(HPLC) chromatogram of a peptide having features of the invention
before cleavage of linker portion X that is a substrate for
enterokinase.
[0038] FIG. 6B illustrates a HPLC chromatogram of the peptide of
FIG. 6A after cleavage of linker portion X by enterokinase.
[0039] FIG. 7A illustrates a HPLC chromatogram of a peptide having
features of the invention before cleavage of linker portion X that
is a substrate for matrix metalloproteinase-2 (MMP-2).
[0040] FIG. 7B illustrates a HPLC chromatogram of the peptide of
FIG. 7A after cleavage of linker portion X by MMP-2.
[0041] FIG. 8 illustrates the mean fluorescence measured by
Fluorescence-Activated Cell Sorter (FACS) analysis of Jurkat cell
populations incubated for ten minutes with MTS molecules having
features of the invention, with fluorescent cargo moieties.
[0042] FIG. 9 illustrates the mean fluorescence measured by FACS
analysis of Jurkat cell populations incubated for ten minutes with
MTS molecules having features of the invention, with fluorescent
cargo moieties.
[0043] FIG. 10 illustrates the mean fluorescence measured by FACS
analysis of Jurkat cell populations incubated for ten minutes with
MTS molecules having features of the invention, with fluorescent
cargo moieties.
[0044] FIG. 11 illustrates the mean fluorescence measured by FACS
analysis of Jurkat cell populations incubated for ten minutes with
MTS molecules having features of the invention, with fluorescent
cargo moieties.
[0045] FIG. 12 illustrates the mean fluorescence measured in Jurkat
cells incubated for one hour with the MTS molecules of FIG. 11.
[0046] FIG. 13 illustrates the mean fluorescence measured in Jurkat
cells incubated for ten minutes with MTS molecules having a
disulfide linker connecting an acidic portion with a fluorescently
labeled basic portion, or with the fluorescently labeled basic
portion alone.
[0047] FIG. 14 illustrates some moieties suitable as part or all of
a cargo portion of an MTS molecules having features of the
invention.
[0048] FIG. 15 illustrates some moieties suitable for use as part
or all of an acidic portion A.
[0049] FIG. 16 illustrates some moieties suitable for use as part
or all of a linker X.
[0050] FIG. 17 illustrates some moieties suitable for use as part
or all of a basic portion B.
[0051] FIG. 18 illustrates some polymeric moieties suitable for use
as part or all of an acidic portion A.
[0052] FIG. 19 is a schematic diagram of activatable
cell-penetrating peptides (ACPPs). Cellular uptake induced by a
cationic peptide is blocked by a short stretch of acidic residues
attached by a cleavable linker. Once the linker is cleaved, the
acidic inhibitory domain drifts away, and the cationic
cell-penetrating peptide (CPP) is free to carry its cargo into
cells.
[0053] FIG. 20 illustrates association of ACPPs with live HT-1080
cells depends on cleavage by MMP-2, as demonstrated by FACS
analysis (A) and microscopy (B and C). (A) Trace 1 (blue) shows
untreated cells. Traces 2 (orange) and 3 (green) show cells
incubated for 10 min with 1 .mu.M uncleaved or precleaved peptide,
respectively. Cells incubated with 1 .mu.M r.sub.9k(Cy5) are shown
in red (Trace 4). (B) HT-1080 cells were incubated with 1 .mu.g/ml
Hoechst 33258 (Left) and 1.25 .mu.M uncleaved peptide (Center) and
imaged at Hoechst or Cy5 wavelengths (overlaid at Right). (C)
Results from a similar experiment with cleaved peptide. The
arrowheads indicate possible nucleoli.
[0054] FIG. 21 Nuclear Overhauser effects observed in
two-dimensional NMR of a simple ACPP,
succinyl-e.sub.8-XPLGLAG-r.sub.9-Xk, where X denotes
6-aminohexanoyl. Dashed red lines indicate observed nuclear
Overhauser effects, and the green line highlights the peptide
outline for clarity.
[0055] FIG. 22 illustrates visualization of HT-1080 tumor
xenografts with activatable CPPs. HT-1080 tumors were implanted
into the mammary fat pad of nude mice and allowed to grow until
they reached 5-7 mm in diameter. (A1) A live anesthetized animal
imaged 50 min after injection with 6 nmol of cleavable peptide. (A2
and A3) Tumor and muscle histology from a different animal killed
30 min after injection. (B1-B3) A similar experiment with the
scrambled peptide. (Scale bars, 30 .mu.m.)
[0056] FIG. 23 illustrates standardized uptake values (SUV) of
cleavable and non-cleavable peptides in various tissues in
mice.
[0057] FIG. 24. NMR spectra of the H.sup..beta./.gamma.
(.delta..sub.1)-H.sup.N(.delta..sub.2) region of the peptide.
Evidence for cross-strand interactions between D-arg and D-glu
residues. A. NOESY spectrum. Blue dashed line at 8.65 ppm
corresponds to C. B. TOCSY spectrum. Blue dashed lines at 8.65 and
8.59 correspond to D and E, respectively. C. 1D NOESY vector at the
H.sup.N of a D-arg. D. 1D TOCSY vector at the H.sup.N of a D-arg.
E. 1D TOCSY vector at the H.sup.N of a D-glu.
[0058] FIG. 25. NOESY spectrum of the
H.sup.N(.delta..sub.1)-H.sup.N(.delta..sub.2) region indicates
sequential H.sup.N-H.sup.N backbone interactions through the
residues of the linker region. Symmetry related cross-peaks are
labeled once at either side of the diagonal.
[0059] FIG. 26. TPEN inhibits staining of squamous cell carcinoma
specimens by a cleavable ACPP, (5 kDa
PEG)-eeeeeeeeeXPLGLAG-rrrrrrrrrXk(Cy5), where X denotes
6-aminohexanoyl (SEQ ID NO: 52). Tissue slices were stained with 1
.mu.M ACPP in the absence (A) or presence (B) of 1 .mu.M TPEN. The
slice shown in (A) contains regions of tumor (top right) as well as
normal tissue. The slice shown in (B) contains only tumor.
[0060] FIG. 27 illustrates cleavage kinetics for MMP-2 cleavage of
H.sub.2N-e.sub.6-XPLGLAG-r.sub.9-Xc(Cy5)-CONH.sub.2, where X is
aminohexanoic acid.
[0061] FIG. 28 illustrates the dependence of uptake on cleavage by
matrix metalloprotease-2 (MMP-2).
[0062] FIG. 29 illustrates the dependence of cargo uptake on
peptide cleavage.
[0063] FIG. 30 illustrates localization of MMP-2-positive tumors in
nude mice by imaging of fluorescence from cleaved peptides.
[0064] FIG. 31 illustrates the dependence of observed fluorescence
intensity on peptide cleavage in vivo.
[0065] FIG. 32 provides images of human HT1080 tumors xenografted
into nude mice illustrating improved contrast with cleavable
peptides.
[0066] FIG. 33 provides images of cleavable peptide-derived
fluorescence in spontaneous mammary tumors in mice, and provides
gel images showing presence of cleaved peptide in tumor.
[0067] FIG. 34 provides images of cleavable peptide-derived
fluorescence in metastasis and surrounding macrophages in lymph
nodes in mice.
[0068] FIG. 35 provides images of resected human squamous cell
carcinoma, and shows that gelatinase activity is increased in
tumor.
[0069] FIG. 36 provides images of resected human squamous cell
carcinoma.
[0070] FIG. 37 illustrates a scheme for generating phage particles
decorated with ACPPs suitable for directing phage to sites having
enzymatic activity for cleavage and delivery of phage to target
tissues and cells.
[0071] FIG. 38 illustrates a scheme for sequence-dependent phage
accumulation in tumors, and presents data illustrating increased
phage uptake in target tumors.
[0072] FIG. 39 illustrates a scheme for attachment of a radioactive
compound to an ACPP for delivery of the radioactive moiety to a
target cell.
[0073] FIG. 40 provides data demonstrating increased delivery of
radioactive cargo to target cells with enzymatic cleavage.
[0074] FIG. 41 provides data demonstrating enzymatic
cleavage-dependent increase in delivery of radioactive cargo to
target cells.
[0075] FIG. 42 illustrates a scheme for providing an MMP substrate
for enhancement of magnetic resonance imaging (MRI) images.
[0076] FIG. 43 illustrates increased uptake of MRI contrast agent
upon enzymatic cleavage of ACPP including contrast agent.
[0077] FIG. 44 illustrates a cyclic ACPP.
[0078] FIG. 45 illustrates a scheme for synthesizing a cyclic ACPP.
Polyethylene glycol (PEG) may be attached to the cyclic ACPP to
provide a PEGylated cyclic ACPP.
[0079] FIG. 46 illustrates a synthesized cyclic ACPP cleavable by
MMP, and a scheme for its modification by addition of PEG and of a
fluorescent label.
[0080] FIG. 47 provides data demonstrating self-quenching by cyclic
ACPPs prior to enzymatic cleavage.
[0081] FIG. 48 schematically illustrates cyclic ACPP peptides that
require cleavage at two sites for activation.
[0082] FIG. 49: Planar gamma imaging of HT1080 xenografts with
.sup.99mTc-labeled albumin-ACPP, (murine serum
albumin)-e.sub.9-XPLGLAG-r.sub.9-[DPK-.sup.99mTc(CO).sub.3]. Three
different nude mice, each at a different time point. Top row:
visible light images of mice, showing axillary location of
xenografted tumor. Middle row: Gamma images in grayscale. Bottom
row: Gamma images pseudocolored to highlight intensity differences.
The high-intensity central region labeled "liver" also includes
kidneys and spleen. Tumors are marginally visible.
[0083] FIG. 50: Biodistribution of .sup.99mTc-labeled albumin-ACPP.
Mice xenografted as in FIG. 49 were sacrificed at 6, 24, and 48 hr
after injection, organs were dissected, weighed, and radioactivity
counted within 2 hrs to obtain % injected dose (% ID)/g tissue as
shown in the bar graph (means.+-.s.d., n=4 per time point). The
inset table gives the corresponding mean standardized uptake values
(SUV) defined as animal weight.times.(% ID/g tissue).
[0084] FIG. 51: Magnetic resonance imaging of Gd.sup.3+-labeled
ACPP reveals metastatic lymph nodes in a transgenic MMTV-PyMT
mouse. The ACPP was Suc.sub.9-(70 KDa
dextran)-e.sub.9-XPLGLAG-r.sub.9-K(DOTA-Gd). T.sub.1-weighted
fat-saturated imaging was performed on a 7 Tesla small animal
scanner as described in the text. The six images were taken before
and at the indicated times after injection of 510 nmoles ACPP into
the tail vein. Yellow arrows point to primary tumors, blue to
liver, red to lymph nodes, and green to intestines. Note appearance
of bright lymph nodes starting at 2 hr. See text of section 1.b for
further explanation.
[0085] FIG. 52: ACPPs with far-red Cy5 cargoes and albumin or
dextran scaffolds reveal tumors by epifluorescence in MMTV-PyMT
transgenic mice. Images (excitation at 625-645 nm and emission at
665-695 nm) taken of intact mice through the belly, either
uninjected to measure autofluorescence background, or 36 hr after
injection of 4 different ACPPs whose quantity and composition are
indicated. Two of the ACPPs ("Albc, Dexc") are controls with
d-amino acids to hinder proteolysis compared to the test ACPPs
("Alb, Dex"). Red arrows point to tumors. Note bright fluorescence
from tumors in Alb and Dex-injected mice. Lower left graph shows
the time course of fluorescence from tumor regions vs. normal
tissue at center of thoracic horseshoe, indicating that the peak
optical contrast of about 4 is obtained 36 hr after injection.
Lower right bar graph shows standardized uptake values (SUVs)
obtained after final sacrifice at 48 hr; only tumors show marked
difference between test and control ACPPs. Although SUVs for kidney
and liver are comparable or greater than those for tumor, optical
imaging discriminates against kidney and liver because they are
more deeply buried.
[0086] FIG. 53: ACPP on 70 KDa dextran scaffold also accumulates in
lymph nodes near tumors. Top row shows epifluorescence views as in
FIG. 52 but with the body cavity opened to expose lymph nodes, some
of which have been individually identified as renal and lumbar. Not
all lymph nodes light up, as shown by the periaortic cluster in the
far right panel. The lower images are fluorescence views of
histological sections through the lymph nodes, comparing the test
ACPP with a cleavable PLGLAG linker with the control ACPP with all
d-amino acids, i.e., plglag. This difference is quantified in the
bar graph in the lower right.
[0087] FIG. 54: Sequence-dependent phage accumulation in
xenografted tumors. Mice with HT1080 xenografts were injected with
phage coated with ACPPs either containing the hydrolyzable PLGLAG
linker (maroon bars) or the less hydrolysable, scrambled sequence
LALGPG (blue bars). The bar graph indicates that the titer of phage
recovered from the tumor relative to blood was about 4 times higher
for PLGLAG than for LALGPG, whereas phage recovery from liver and
kidney was lower and not significantly different for the two
sequences.
[0088] FIG. 55: Fluorescence imaging of 3D cultures in Matrigel.
General flow chart for methodology.
[0089] FIG. 56: MDA-MB-231 cells in 3-D culture make enough MMPs to
take up ACPP over 24 hr. The left two columns show results with
e.sub.9-XPLGLAG-r.sub.9-k(Cy5) without or with 0.1 mM GM6001, a
general inhibitor of MMPs. GM6001 also reduces invasiveness of the
cells, explaining why the cell cluster is smaller. The right two
columns show e.sub.9-Xplglag-r.sub.9-k(Cy5) as a nonhydrolyzable
negative control and r.sub.9-k(Cy5) as a positive control. Upper
row shows Hoechst staining of nuclei, pseudocolored green, while
lower row shows Cy5 fluorescence. Note that these ACPPs have no
macromolecular carrier, because there is no need to hinder kidney
excretion and because the macromolecular carriers seem to hinder
diffusion through the Matrigel and into these organoids, which lack
active circulation.
[0090] FIG. 57: Mouse without tumors shows little or no contrast in
lymph nodes. In a mouse without tumors (bottom row of images), the
lymph nodes do not label when the same Gd-containing ACPP as was
used in FIG. 51 is administered to a mouse without tumor. The top
row of images shows the distribution of label in the lymph nodes in
a tumor-bearing mouse.
DETAILED DESCRIPTION OF THE INVENTION
[0091] In one embodiment, a generic structure for peptides having
features of the invention is A-X-B, where peptide portion B
includes between about 5 to about 20 basic amino acids, X is a
cleavable linker portion, preferably cleavable under physiological
conditions, and where peptide portion A includes between about 2 to
about 20 acidic amino acids. In some embodiments of molecules
having features of the invention, peptide portion B includes
between about 5 to about 20, or between about 9 to about 16 basic
amino acids, and may be a series of basic amino acids (e.g.,
arginines, histidines, lysines, or other basic amino acids). In
some embodiments of molecules having features of the invention,
peptide portion A includes between about 2 to about 20, or between
about 5 to about 20 acidic amino acids, and may be series of acidic
amino acids (e.g., glutamates and aspartates or other acidic amino
acids). A schematic representation of a MTS molecule having
features of the invention comprising a basic portion B, a linker
portion X, and an acidic portion A is presented in FIG. 1A. In
embodiments, MTS molecules having features of the invention may be
cyclic molecules, as schematically illustrated in FIG. 1B. Thus,
MTS molecules having features of the invention may be linear
molecules, cyclic molecules, or may be linear molecules including a
cyclic portion.
[0092] As discussed above, molecules including a multiple basic
amino acids, such as a series of basic amino acids, are often taken
up by cells. However, the present inventors have discovered that
molecules having structures including a basic portion B, a linker
portion X, and an acidic portion A are not taken up by cells. An
acidic portion A may include amino acids that are not acidic.
Acidic portion A may comprise other moieties, such as negatively
charged moieties. In embodiments of MTS molecules having features
of the invention, an acidic portion A may be a negatively charged
portion, preferably having about 2 to about 20 negative charges at
physiological pH, that does not include an amino acid. A basic
portion B may include amino acids that are not basic. Basic portion
B may comprise other moieties, such as positively charged moieties.
In embodiments of MTS molecules having features of the invention, a
basic portion B may be a positively charged portion, preferably
having between about 5 and about 20 positive charges at
physiological pH, that does not include an amino acid. Including an
acidic portion A is effective to inhibit or prevent the uptake of a
portion B into cells. Such a block of uptake that would otherwise
be effected by the basic amino acids of portion B may be termed a
"veto" of the uptake by the acidic portion A. The present inventors
have made the further surprising discovery that cleavage of linker
X, allowing the separation of portion A from portion B is effective
to allow the uptake of portion B into cells.
[0093] In a further embodiment, a generic structure for peptides
having features of the invention is A-X-B-C, where C is a cargo
moiety, X a linker, A an acidic portion, and B a basic portion. An
acidic portion A may include amino acids that are not acidic.
Acidic portion A may comprise other moieties, such as negatively
charged moieties. In embodiments of MTS molecules having features
of the invention, an acidic portion A may be a negatively charged
portion, preferably having about 2 to about 20 negative charges at
physiological pH, that does not include an amino acid. A basic
portion B may include amino acids that are not basic. Basic portion
B may comprise other moieties, such as positively charged moieties.
In embodiments of MTS molecules having features of the invention, a
basic portion B may be a positively charged portion, preferably
having between about 5 and about 20 positive charges at
physiological pH, that does not include an amino acid. In preferred
embodiments, the amount of negative charge in portion A is
approximately the same as the amount of positive charge in portion
B.
[0094] A cargo moiety C may be, for example, a contrast agent for
diagnostic imaging, or a chemotherapeutic drug or
radiation-sensitizer for therapy. B may be, for example, a peptide
portion having between about 5 to about 20 basic amino acids, such
as a series of basic amino acids (arginines are preferred, although
histidines are also suitable, as are lysines or other basic amino
acids). X is a cleavable linker that is preferably cleavable under
physiological conditions. A may be a peptide portion having between
about 2 to about 20 about 2 to about 20 acidic amino acids, such as
a series of acidic amino acids. In some embodiments of molecules
having features of the invention, glutamates and aspartates are
preferred acidic amino acids for peptide portion A. A schematic
representation of a MTS molecule having features of the invention
comprising a cargo portion C, a basic portion B, a linker portion
X, and an acidic portion A is presented in FIG. 2A.
[0095] The present inventors have made the surprising discovery
that including an acidic portion A is also effective to inhibit or
prevent the uptake into cells of molecules combining a portion B
and a portion C. The present inventors have made the further
discovery that cleavage of linker X, allowing the separation of
portion A from portion B is effective to allow the uptake of
portions B and C into cells. Thus, delivery of cargo C can be
controlled and enhanced by molecules having features of the
invention.
[0096] For example, when peptide portion A contains about 5 to
about 9 consecutive glutamates or aspartates, and X is a flexible
linker of about 2 to about 100, or about 6 to about 30 atoms in
length, the normal ability of a peptide portion B (e.g., a sequence
of nine consecutive arginine residues) to cause uptake into cells
is blocked. Cleavage of linker X allows the separation of portion A
from portion B and portion C, alleviating the veto by portion A.
Thus, when separated from A, the normal ability of portion B to
effect the uptake of cargo C into cells is regained. Such cellular
uptake typically occurs near the location of the cleavage event.
Thus, design of cleavable linker X such that it is cleaved at or
near a target cell is effective to direct uptake of cargo C into
target cells. Extracellular cleavage of X allows separation of A
from the rest of the molecule to allow uptake into cells.
[0097] A MTS molecule having features of the invention may be of
any length. In embodiments of MTS molecules having features of the
invention, a MTS molecule may be about 7 to about 40 amino acids in
length, not including the length of a linker X and a cargo portion
C. In other embodiments, particularly where multiple non-acidic (in
portion A) or non-basic (in portion B) amino acids are included in
one or both of portions A and B, portions A and B of a MTS molecule
may together be about 50, or about 60, or about 70 amino acids in
length. A cyclic portion of an MTS may include about 12 to about 60
amino acids, not including the length of a linker X and a cargo
portion C. For example, a linear MTS molecule having features of
the invention may have a basic portion B having between about 5 to
about 20 basic amino acids (preferably between about 9 to about 16
basic amino acids) and an acidic portion A having between about 2
to about 20 acidic amino acids (e.g., between about 5 to about 20,
preferably between about 5 to about 9 acidic amino acids). In some
preferred embodiments, a MTS molecule having features of the
invention may have a basic portion B having between about 9 to
about 16 basic amino acids and between about 5 to about 9 acidic
amino acids.
[0098] In healthy cells, the intact compound of structure A-X-B or
A-X-B-C would not be able to enter the cell because of the presence
of portion A. Thus, a strictly intracellular process for cleaving X
would be ineffective to cleave X in healthy cells since portion A,
preventing uptake into cells, would not be effectively cleaved by
intracellular enzymes in healthy cells since it would not be taken
up and would not gain access to such intracellular enzymes.
However, where a cell is injured or diseased, so that such
intracellular enzymes leak out of the cell, cleavage of A would
occur, allowing entry of portion B or B-C into the cell, effecting
targeted delivery of portion B or of cargo portion C to neighboring
cells.
[0099] Portions A and B may include either L-amino acids or D-amino
acids. In embodiments of the invention, D-amino acids are preferred
for the A and B portions in order to minimize immunogenicity and
nonspecific cleavage by background peptidases or proteases.
Cellular uptake of oligo-D-arginine sequences is known to be as
good or better than that of oligo-L-arginines. The generic
structures A-X-B and -A-X-B-C can be effective where A is at the
amino terminus and where A is at the carboxy terminus, i.e., either
orientation of the peptide bonds is permissible. However, in
embodiments where X is a peptide cleavable by a protease, it may be
preferable to join the C-terminus of X to the N-terminus of B, so
that the new amino terminus created by cleavage of X contributes an
additional positive charge that adds to the positive charges
already present in B.
[0100] Cargo portion C may be attached to B in any location or
orientation. A cargo portion C need not be located at an opposite
end of portion B than a linker X. Any location of attachment of C
to B is acceptable as long as that attachment remains after X is
cleaved. For example, a cargo portion C may be attached to or near
to an end of portion B with linker X attached to an opposite end of
portion B as illustrated in FIGS. 2A and 2B. A cargo portion C may
also be attached to or near to an end of portion B with linker X
attached to or near to the same end of portion B. In some
embodiments of the invention, a linker X may link to a cargo
portion C which is linked to a basic portion B as illustrated in
FIG. 2B. FIG. 2C is a schematic representation of a MTS molecule
having features of the invention comprising a cargo portion C
linked to multiple basic portions B, each of which basic portions B
are linked to a linker portion X, and via the linker to an acidic
portion A.
[0101] A linker X may be designed for cleavage in the presence of
particular conditions or in a particular environment. In preferred
embodiments, a linker X is cleavable under physiological
conditions. Cleavage of such a linker X may, for example, be
enhanced or may be effected by particular pathological signals or a
particular environment related to cells in which cargo delivery is
desired. The design of a linker X for cleavage by specific
conditions, such as by a specific enzyme, allows the targeting of
cellular uptake to a specific location where such conditions
obtain. Thus, one important way that MTS molecules having features
of the invention provide specific targeting of cellular uptake to
desired cells, tissues, or regions is by the design of the linker
portion X to be cleaved by conditions near such targeted cells,
tissues, or regions. After cleavage of a linker X, the portions B-C
of the molecule are then a simple conjugate of B and C, in some
instances retaining a relatively small, inert stub remaining from a
residual portion of linker X.
[0102] A linker portion X may be cleavable by conditions found in
the extracellular environment, such as acidic conditions which may
be found near cancerous cells and tissues or a reducing
environment, as may be found near hypoxic or ischemic cells and
tissues; by proteases or other enzymes found on the surface of
cells or released near cells having a condition to be treated, such
as diseased, apoptotic or necrotic cells and tissues; or by other
conditions or factors. An acid-labile linker may be, for example, a
cis-aconitic acid linker. Other examples of pH-sensitive linkages
include acetals, ketals, activated amides such as amides of 2,3
dimethylmaleamic acid, vinyl ether, other activated ethers and
esters such as enol or silyl ethers or esters, imines, iminiums,
enamines, carbamates, hydrazones, and other linkages. A linker X
may be an amino acid or a peptide. A peptide linker may be of any
suitable length, such as, for example, about 3 to about 30, or
preferably about 6 to about 24 atoms in sequence (e.g., a linear
peptide about 1 to 10 or preferably about 2 to 8 amino acids long).
A cleavable peptide linker may include an amino acid sequence
recognized and cleaved by a protease, so that proteolytic action of
the protease cleaves the linker X.
[0103] One important class of signals is the hydrolytic activity of
matrix metalloproteinases (MMPs), which are very important in the
invasive migration of metastatic tumor cells. MMPs are also
believed to play major roles in inflammation and stroke. MMPs are
reviewed in Visse et al., Circ. Res. 92:827-839 (2003). MMPs may be
used to cleave a linker X and so to allow separation of acidic
portion A from portions B and C, allowing cellular uptake of cargo
C so that cellular uptake of C is triggered by action of MMPs. Such
uptake is typically in the vicinity of the MMPs that trigger
cleavage of X. Thus, uptake of molecules having features of the
invention are able to direct cellular uptake of cargo C to specific
cells, tissues, or regions having active MMPs in the extracellular
environment.
[0104] For example, a linker X that includes the amino-acid
sequence PLGLAG (SEQ ID NO: 1) may be cleaved by the
metalloproteinase enzyme MMP-2 (a major MMP in cancer and
inflammation). Cleavage of such a linker X occurs between the
central G and L residues, causing cell uptake to increase by 10 to
20-fold (see Example 4). A great deal is known about the substrate
preferences of different MMPs, so that linkers X may be designed
that are able to bias X to be preferentially sensitive to
particular subclasses of MMPs, or to individual members of the
large MMP family of proteinases. For example, in some embodiments,
linkers X designed to be cleaved by membrane-anchored MMPs are
particularly preferred because their activity remains localized to
the outer surface of the expressing cell. In alternative
embodiments, linkers X designed to be cleaved by a soluble secreted
MMP are preferred where diffusion of cargo C away from the exact
location of cleavage may be desired, thereby increasing the spatial
distribution of the cargo. Other linkers X cleavable by other MMPs
are discussed in Example 9.
[0105] Hypoxia is an important pathological signal. For example,
hypoxia is thought to cause cancer cells to become more resistant
to radiation and chemotherapy, and also to initiate angiogenesis. A
linker X suitable for cleavage in or near tissues suffering from
hypoxia enables targeting of portion B and C to cancer cells and
cancerous tissues, infarct regions, and other hypoxic regions. For
example, a linker X that includes a disulfide bond is
preferentially cleaved in hypoxic regions and so targets cargo
delivery to cells in such a region. In a hypoxic environment in the
presence of, for example, leaky or necrotic cells, free thiols and
other reducing agents become available extracellularly, while the
O.sub.2 that normally keeps the extracellular environment oxidizing
is by definition depleted. This shift in the redox balance should
promote reduction and cleavage of a disulfide bond within a linker
X. In addition to disulfide linkages which take advantage of
thiol-disulfide equilibria, linkages including quinones that fall
apart when reduced to hydroquinones may be used in a linker X
designed to be cleaved in a hypoxic environment.
[0106] Necrosis often leads to release of enzymes or other cell
contents that may be used to trigger cleavage of a linker X. A
linker X designed for cleavage in regions of necrosis in the
absence of hypoxia, for example, may be one that is cleaved by
calpains or other proteases that may be released from necrotic
cells. Such cleavage of linkers X by calpains would release the
connected portions B-C from portion A, allowing cargo to be taken
up by diseased cells and by neighboring cells that had not yet
become fully leaky.
[0107] Acidosis is also commonly observed in sites of damaged or
hypoxic tissue, due to the Warburg shift from oxidative
phosphorylation to anaerobic glycolysis and lactic acid production.
Such local acidity could be sensed either by making an acid-labile
linker X (e.g., by including in X an acetal or vinyl ether
linkage). Alternatively, or in addition, acidosis may be used as a
trigger of cargo uptake by replacing some of the arginines within B
by histidines, which only become cationic below pH 7.
[0108] Molecules having features of the invention are suitable for
carrying different cargoes, including different types of cargoes
and different species of the same types of cargo, for uptake into
cells. For example, different types of cargo may include marker
cargoes (e.g., fluorescent or radioactive label moieties) and
therapeutic cargoes (e.g., chemotherapeutic molecules such as
methotrexate or doxorubicin), or other cargoes. Where destruction
of aberrant or diseased cells is therapeutically required, a
therapeutic cargo may include a "cytotoxic agent," i.e., a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. In some embodiments, a single molecule
having features of the invention may include more than one cargo
portion C so that a basic portion B may be linked to multiple
cargoes C. Such multiple cargoes C may include marker cargoes,
therapeutic cargoes, or other cargoes. Multiple cargo moieties may
allow, for example, delivery of both a radioactive marker and an
ultrasound or contrast agent, allowing imaging by different
modalities. Alternatively, for example, delivery of radioactive
cargo along with an anti-cancer agent, providing enhanced
anticancer activity, or delivery of a radioactive cargo with a
fluorescent cargo, allowing multiple means of localizing and
identifying cells which have taken up cargo.
[0109] Delivery of cargo such as a fluorescent molecule may be used
to visualize cells having a certain condition or cells in a region
exhibiting a particular condition. For example, thrombosis (clot
formation) may be visualized by designing a linker X to be cleaved
by any of the many proteases in the blood clot formation cascade
for delivery of a cargo including a fluorescent or other marker to
the region. Similarly, complement activation may be visualized by
designing a linker X to be cleaved by any one or more of the
proteases in the complement activation cascades for delivery of a
fluorescent or other marker to the region. Thus, fluorescent
molecules are one example of a marker that may be delivered to
target cells and regions upon release of a portion A upon cleavage
of a linker X.
[0110] A molecule having features of the invention may include one
or more linkers X so that an acidic portion A may be linked to
portions B and C by one or more linkages. Such linkages connecting
to portion A may be to portion B, to portion C, or to both portions
B and C. Where a molecule having features of the invention includes
multiple linkages X, separation of portion A from the other
portions of the molecule requires cleavage of all linkages X.
Cleavage of multiple linkers X may be simultaneous or sequential.
Multiple linkages X may include linkages X having different
specificities, so that separation of portion A from the other
portions of the molecule requires that more than one condition or
environment ("extracellular signals") be encountered by the
molecule. Cleavage of multiple linkers X thus serves as a detector
of combinations of such extracellular signals. FIG. 2D shows a MTS
molecule having features of the invention that includes two linker
portions Xa and Xb connecting basic portion B with acidic portion
A. FIG. 2E shows a cyclic MTS molecule having features of the
invention that includes two linker regions Xa and Xb connecting
basic portion B with acidic portion A. In the MTS molecules
schematically illustrated in FIGS. 2D and 2E, both linkers Xa and
Xb must be cleaved before acidic portion A is separated from basic
portion B allowing entry of portion B and cargo portion C (if any)
to enter a cell. It will be understood that a linker region may
link to either a basic portion B or a cargo portion C independently
of another linker that may be present, and that, where desired,
more than two linker regions X may be included.
[0111] Combinations of two or more linkers X may be used to further
modulate the targeting and delivery of molecules to desired cells,
tissue or regions. Boolean combinations of extracellular signals
can be detected to widen or narrow the specificity of the cleavage
of linkers X if desired. Where multiple linkers X are linked in
parallel, the specificity of cleavage is narrowed, since each
linker X must be cleaved before portion A may separate from the
remainder of the molecule. Where multiple linkers X are linked in
series, the specificity of cleavage is broadened, since cleavage on
any one linker X allows separation of portion A from the remainder
of the molecule. For example, in order to detect either a protease
OR hypoxia (i.e., to cleave X in the presence of either protease or
hypoxia), a linker X is designed to place the protease-sensitive
and reduction-sensitive sites in tandem, so that cleavage of either
would suffice to allow separation of the acidic portion A.
Alternatively, in order to detect the presence of both a protease
AND hypoxia (i.e., to cleave X in the presence of both protease and
hypoxia but not in the presence of only one alone), a linker X is
designed to place the protease sensitive site between at least one
pair of cysteines that are disulfide-bonded to each other. In that
case, both protease cleavage AND disulfide reduction are required
in order to allow separation of portion A.
[0112] The fact that capillaries are often leaky around tumors and
other trauma sites should enhance the ability of high molecular
weight molecules (e.g., molecular weight of about 40 kDa or more)
to reach the interstitial compartment. Since the cleavage of a
linker X is typically extracellular, some bystander labeling is
expected, i.e., cells that do not express the relevant protease but
that are immediately adjacent to expressing cells are likely to
pick up some of the cargo. For tumors, such bystander targeting is
considered beneficial because of the heterogeneity of cell
phenotypes and the wish to eliminate as high a percentage of
suspicious cells.
[0113] The fact that a single mechanism can mediate uptake of both
imaging and therapeutic cargoes will be particularly valuable,
because imaging with noninjurious tracer quantities can be used to
test whether a subsequent therapeutic dose is likely to concentrate
correctly in the target tissue.
[0114] D amino acids may be used in MTS molecules having features
of the invention. For example, some or all of the peptides of
portions A and B may be D-amino acids in some preferred embodiments
of the invention. In an embodiment of the invention suitable for
delivering a detectable marker to a target cell, a MTS having
features of the invention includes a contrast agent as cargo C
attached to a basic portion B comprising 8 to 10 D-arginines.
Acidic portion A may include D-amino acids as well. Similarly, a
drug may be delivered to a cell by such molecules having a basic
portion B including 8 to 10 D-arginines and an acidic portion A
including acidic D-amino acids. A schematic representation of such
MTS molecules is shown in FIG. 3.
[0115] It will be understood that a MTS molecule having features of
the invention may include non-standard amino acids, such as, for
example, hydroxylysine, desmosine, isodesmosine, or other
non-standard amino acids. A MTS molecule having features of the
invention may include modified amino acids, including
post-translationally modified amino acids such as, for example,
methylated amino acids (e.g., methyl histidine, methylated forms of
lysine, etc.), acetylated amino acids, amidated amino acids,
formylated amino acids, hydroxylated amino acids, phosphorylated
amino acids, or other modified amino acids. A MTS molecule having
features of the invention may also include peptide mimetic
moieties, including portions linked by non-peptide bonds and amino
acids linked by or to non-amino acid portions. For example, a MTS
molecule having features of the invention may include peptoids,
carbamates, vinyl polymers, or other molecules having non-peptide
linkages but having an acidic portion cleavably linked to a basic
portion having a cargo moiety.
[0116] The linker portion X may be designed so that it is cleaved,
for example, by proteolytic enzymes or reducing environment, as may
be found near cancerous cells. Such an environment, or such
enzymes, are typically not found near normal cells. FIG. 4
illustrates a MTS molecule as shown in FIG. 3, having a cleavable
linker X designed to be cleaved near cancerous cells. As
illustrated in FIG. 4, the cleavable linker is not cleaved near
normal tissue. FIG. 4 illustrates the ability of a MTS having a
portion A capable of vetoing cellular uptake of a portion B, and of
a portion B-C, blocking the entry of cargo into normal tissue.
[0117] However, as illustrated in FIG. 5, the linker portion X may
be cleaved, for example, by proteolytic enzymes or reducing
environment found near cancerous cells to deliver a marker or a
drug to cancerous cells. As shown in FIG. 5, a MTS molecule of FIG.
3 with a cleavable linker X that is cleaved by proteolytic enzymes
or by the reducing environment near cancer cells is able to
facilitate cargo entry into diseased tissue. Thus, the selective
cleavage of the linker X and the resulting separation of cargo C
and basic portion B from acidic portion A allows the targeted
uptake of cargo into cells having selected features (e.g.,
enzymes), or located near to, a particular environment. Thus,
molecules having features of the invention are able to selectively
deliver cargo to target cells without doing so to normal or
otherwise non-targeted cells.
[0118] In some embodiments, cargo C may be a fluorescent molecule
such as fluorescein. Fluorescent cargo moieties enable easy
measurement by fluorescence microscopy or flow cytometry in unfixed
cultured cells. However, oligoarginine sequences, such as make up
portion B, have been demonstrated to import a very wide varieties
of cargoes C, ranging from small polar molecules to nanoparticles
and vesicles (Tung & Weissleder (2003) Advanced Drug Delivery
Reviews 55: 281-294). Thus, in embodiments of the invention, a
cargo portion C may be any suitable cargo moiety capable of being
taken up by a cell while connected to a basic portion B.
[0119] For example, for in vivo imaging purposes, C may be labeled
with a positron-emitting isotope (e.g., .sup.18F) for positron
emission tomography (PET), gamma-ray isotope (e.g., .sup.99mTc) for
single photon emission computed tomography (SPECT), a paramagnetic
molecule or nanoparticle (e.g., Gd.sup.3+ chelate or coated
magnetite nanoparticle) for magnetic resonance imaging (MRI), a
near-infrared fluorophore for near-infra red (near-IR) imaging, a
luciferase (firefly, bacterial, or coelenterate) or other
luminescent molecule for bioluminescence imaging, or a
perfluorocarbon-filled vesicle for ultrasound. For therapeutic
purposes, for example, suitable classes of cargo include but are
not limited to: a) chemotherapeutic agents such as doxorubicin,
mitomycin, paclitaxel, nitrogen mustards, etoposide, camptothecin,
5-fluorouracil, etc.; b) radiation sensitizing agents such as
porphyrins for photodynamic therapy, or .sup.10B clusters or
.sup.157Gd for neutron capture therapy; or c) peptides or proteins
that modulate apoptosis, the cell cycle, or other crucial signaling
cascades. Existing chemotherapeutic drugs may be used, although
they may not be ideal, because they have already been selected for
some ability to enter cells on their own. In embodiments of the
molecules of the invention, cargoes that are unable to enter or
leave cells without the help of the polybasic portion B may be
preferred.
[0120] Cargo C may include a radioactive moiety, for example a
radioactive isotope such as .sup.211At, .sup.131I, .sup.125I,
.sup.90Y, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.212Bi, .sup.32P,
radioactive isotopes of Lu, and others.
[0121] Cargo portion C may include a fluorescent moiety, such as a
fluorescent protein, peptide, or fluorescent dye molecule. Common
classes of fluorescent dyes include, but are not limited to,
xanthenes such as rhodamines, rhodols and fluoresceins, and their
derivatives; bimanes; coumarins and their derivatives such as
umbelliferone and aminomethyl coumarins; aromatic amines such as
dansyl; squarate dyes; benzofurans; fluorescent cyanines;
carbazoles; dicyanomethylene pyranes, polymethine, oxabenzanthrane,
xanthene, pyrylium, carbostyl, perylene, acridone, quinacridone,
rubrene, anthracene, coronene, phenanthrecene, pyrene, butadiene,
stilbene, lanthanide metal chelate complexes, rare-earth metal
chelate complexes, and derivatives of such dyes. Fluorescent dyes
are discussed, for example, in U.S. Pat. No. 4,452,720, U.S. Pat.
No. 5,227,487, and U.S. Pat. No. 5,543,295.
[0122] A cargo portion C may include a fluorescein dye. Typical
fluorescein dyes include, but are not limited to,
5-carboxyfluorescein, fluorescein-5-isothiocyanate and
6-carboxyfluorescein; examples of other fluorescein dyes can be
found, for example, in U.S. Pat. No. 6,008,379, U.S. Pat. No.
5,750,409, U.S. Pat. No. 5,066,580, and U.S. Pat. No. 4,439,356. A
cargo portion C may include a rhodamine dye, such as, for example,
tetramethylrhodamine-6-isothiocyanate,
5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives,
tetramethyl and tetraethyl rhodamine, diphenyldimethyl and
diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101
sulfonyl chloride (sold under the tradename of TEXAS RED.RTM.), and
other rhodamine dyes. Other rhodamine dyes can be found, for
example, in U.S. Pat. No. 6,080,852, U.S. Pat. No. 6,025,505, U.S.
Pat. No. 5,936,087, U.S. Pat. No. 5,750,409. A cargo portion C may
include a cyanine dye, such as, for example, Cy3, Cy3B, Cy3.5, Cy5,
Cy5.5, Cy 7.
[0123] Some of the above compounds or their derivatives will
produce phosphorescence in addition to fluorescence, or will only
phosphoresce. Some phosphorescent compounds include porphyrins,
phthalocyanines, polyaromatic compounds such as pyrenes,
anthracenes and acenaphthenes, and so forth, and may be, or may be
included in, a cargo portion C. A cargo portion C may also be or
include a fluorescence quencher, such as, for example, a
(4-dimethylamino-phenylazo)benzoic acid (DABCYL) group.
[0124] A pair of compounds may be connected to form a molecular
beacon, having complementary regions with a fluorophore and a
fluorescent quencher associated together so that the fluorescence
of the fluorophore is quenched by the quencher. One or both of the
complementary regions may be part of the cargo portion C. Where
only one of the complementary regions (e.g., the fluorescent
moiety) is part of the cargo portion C, and where the quencher
moiety is part of the linker X or the acidic portion A, then
cleavage of the linker X will allow fluorescence of the fluorescent
portion and detection of the cleavage. Upon cellular uptake, the
fluorescent portion of a molecular beacon will allow detection of
the cell. For example, as illustrated in FIG. 2F, a quencher Q may
be attached to an acidic portion A to form a MTS molecule having
features of the invention Q-A-X-B-C where cargo C is fluorescent
and is quenched by Q. The quenching of C by Q is relieved upon
cleavage of X, allowing fluorescent marking of a cell taking up
portion B-C. The combination of fluorescence dequenching and
selective uptake should increase contrast between tissues able to
cleave X compared to those that cannot cleave X.
[0125] Cargo C may include a chemotherapeutic moiety, such as a
chemical compound useful in the treatment of cancer, or other
therapeutic moiety, such as an agent useful in the treatment of
ischemic tissue, or of necrotic tissue, or other therapeutic
agent.
[0126] MTS molecules having features of the invention may be
synthesized by standard synthetic techniques, such as, for example,
solid phase synthesis including solid phase peptide synthesis. An
example of peptide synthesis using Fmoc is given as Example 1
below. For example, conventional solid phase methods for
synthesizing peptides may start with N-alpha-protected amino acid
anhydrides that are prepared in crystallized form or prepared
freshly in solution, and are used for successive amino acid
addition at the N-terminus. At each residue addition, the growing
peptide (on a solid support) is acid treated to remove the
N-alpha-protective group, washed several times to remove residual
acid and to promote accessibility of the peptide terminus to the
reaction medium. The peptide is then reacted with an activated
N-protected amino acid symmetrical anhydride, and the solid support
is washed. At each residue-addition step, the amino acid addition
reaction may be repeated for a total of two or three separate
addition reactions, to increase the percent of growing peptide
molecules which are reacted. Typically, 1 to 2 reaction cycles are
used for the first twelve residue additions, and 2 to 3 reaction
cycles for the remaining residues.
[0127] After completing the growing peptide chains, the protected
peptide resin is treated with a strong acid such as liquid
hydrofluoric acid or trifluoroacetic acid to deblock and release
the peptides from the support. For preparing an amidated peptide,
the resin support used in the synthesis is selected to supply a
C-terminal amide, after peptide cleavage from the resin. After
removal of the strong acid, the peptide may be extracted into 1M
acetic acid solution and lyophilized. The peptide can be isolated
by an initial separation by gel filtration, to remove peptide
dimers and higher molecular weight polymers, and also to remove
undesired salts The partially purified peptide may be further
purified by preparative HPLC chromatography, and the purity and
identity of the peptide confirmed by amino acid composition
analysis, mass spectrometry and by analytical HPLC (e.g., in two
different solvent systems).
[0128] The invention also provides polynucleotides encoding MTS
molecules described herein. The term "polynucleotide" refers to a
polymeric form of nucleotides of at least 10 bases in length. The
nucleotides can be ribonucleotides, deoxynucleotides, or modified
forms of either type of nucleotide. The term includes single and
double stranded forms of DNA. The term therefore includes, for
example, a recombinant DNA which is incorporated into a vector,
e.g., an expression vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., a cDNA)
independent of other sequences.
[0129] These polynucleotides include DNA, cDNA, and RNA sequences
which encode MTS molecules having features of the invention, or
portions thereof. Peptide portions may be produced by recombinant
means, including synthesis by polynucleotides encoding the desired
amino acid sequence. Such polynucleotides may also include promoter
and other sequences, and may be incorporated into a vector for
transfection (which may be stable or transient) in a host cell.
[0130] The construction of expression vectors and the expression of
genes in transfected cells involves the use of molecular cloning
techniques that are well known in the art. See, for example,
Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) and
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
(Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., most recent
Supplement). Nucleic acids used to transfect cells with sequences
coding for expression of the polypeptide of interest generally will
be in the form of an expression vector including expression control
sequences operatively linked to a nucleotide sequence coding for
expression of the polypeptide. As used herein, "operatively linked"
refers to a juxtaposition wherein the components so described are
in a relationship permitting them to function in their intended
manner. A control sequence operatively linked to a coding sequence
is ligated such that expression of the coding sequence is achieved
under conditions compatible with the control sequences. "Control
sequence" refers to polynucleotide sequences which are necessary to
effect the expression of coding and non-coding sequences to which
they are ligated. Control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence. The
term "control sequences" is intended to include, at a minimum,
components whose presence can influence expression, and can also
include additional components whose presence is advantageous, for
example, leader sequences and fusion partner sequences. As used
herein, the term "nucleotide sequence coding for expression of" a
polypeptide refers to a sequence that, upon transcription and
translation of mRNA, produces the polypeptide. This can include
sequences containing, e.g., introns. As used herein, the term
"expression control sequences" refers to nucleic acid sequences
that regulate the expression of a nucleic acid sequence to which it
is operatively linked. Expression control sequences are operatively
linked to a nucleic acid sequence when the expression control
sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus,
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signals for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of the mRNA, and stop codons.
[0131] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the fluorescent
indicator coding sequence and appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. (See, for example,
the techniques described in Maniatis, et al., Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).
Transformation of a host cell with recombinant DNA may be carried
out by conventional techniques as are well known to those skilled
in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method by procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell or by electroporation.
[0132] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransfected with DNA
sequences encoding the fusion polypeptide of the invention, and a
second foreign DNA molecule encoding a selectable phenotype, such
as the herpes simplex thymidine kinase gene. Another method is to
use a eukaryotic viral vector, such as simian virus 40 (SV40) or
bovine papilloma virus, to transiently infect or transform
eukaryotic cells and express the protein. (Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
Techniques for the isolation and purification of polypeptides of
the invention expressed in prokaryotes or eukaryotes may be by any
conventional means such as, for example, preparative
chromatographic separations and immunological separations such as
those involving the use of monoclonal or polyclonal antibodies or
antigen.
[0133] It will be understood that the compounds of the present
invention can be formulated in pharmaceutically useful
compositions. Such pharmaceutical compositions may be prepared
according to known methods. For example, MTS compounds having
features of the invention, and having a cargo portion C that is,
for example, a therapeutic moiety, may be combined in admixture
with a pharmaceutically acceptable carrier vehicle. Suitable
vehicles and their formulation, inclusive of other human proteins,
e.g., human serum albumin are described, for example, in
Remington's Pharmaceutical Sciences by E. W. Martin, which is
hereby incorporated by reference. Such compositions will contain an
effective amount of the compounds hereof together with a suitable
amount of vehicle in order to prepare pharmaceutically acceptable
compositions suitable for effective administration. Dosages and
dosing regimens may be determined for the indications and compounds
by methods known in the art, including determining (e.g., in
experimental animals) the effective dose which causes half of those
treated to respond to the treatment (ED.sub.50) by providing a
range of doses to experimental animals or subjects and noting the
responses.
EXAMPLE 1
[0134] Peptide Synthesis
[0135] A number of peptides whose cell uptake could be modulated
were synthesized. In the following, the following symbols, where
used, are used with the indicated meanings: Fl=fluorescein;
aca=aminocaproic acid linker (--HN--(CH.sub.2).sub.5--CO--),
C=L-cysteine, E=L-glutamate, R=L-arginine, D=L-aspartate,
K=L-lysine, A=L-alanine, r=D-arginine, c=D-cysteine, e=D-glutamate,
P=L-proline, L=L-leucine, G=glycine, V=valine, I=isoleucine,
M=methionine, F=phenylalanine, Y=tyrosine, W=tryptophan,
H=histidine, Q=glutamine, N=arginine, S=serine, and T=threonine. In
sequences discussed below, lower case letters indicate the D isomer
of the amino acid.
[0136] Peptides were synthesized on a peptide synthesizer (Pioneer
Peptide Synthesis System by Applied Biosystems) using solid phase
synthesis method and commercial available Fmoc amino acids, resins,
and the other reagents. The peptides were cleaved with
TFA/thioanisole/triisopropylsilane or
TFA/thioanisole/triisopropylsilane/ethanedithiol. Peptides were
labeled with 5-(and-6)carboxyfluorescein succinimidyl ester on the
amino group on the peptide or with 5-iodoacetamidofluorescein on
the thiol group on the peptide. The crude peptide was purified on
HPLC and lyophilized overnight. Each peptide composition was
confirmed by mass spectrometry.
EXAMPLE 2
[0137] Peptide Cleavage by Enterokinase
[0138] 10 .mu.l 0.38 mM peptide dissolved in water stock solution
was added to 10 .mu.l 1 U/.mu.l Enterokinase (Invitrogen, EKmax)
and the cleavage progress was monitored by injecting 5 .mu.l of the
reaction mixture on HPLC monitored at 440 nm. The peptide was
designed to be a substrate for enterokinase, with cleavage by
enterokinase expected between the K and A residues. A High
Performance Liquid Chromatography (HPLC) chromatogram of the
peptide EDDDDKA-aca-R.sub.9-aca-C(Fl)-CONH.sub.2 (SEQ ID NO: 3)
(before cleavage of linker portion between K and A) is illustrated
in FIG. 6A. (The term "R.sub.9" indicates a sequence of nine
arginines.) The HPLC chromatograms showed that the peptide was
cleaved almost completely after 15 min reaction time. FIG. 6B
illustrates the HPLC chromatogram of the peptide of FIG. 6A after
cleavage by enterokinase. The new peak was collected and determined
on a mass spectrometer. The determined mass corresponded (as
expected) to cleavage between K and A in the sequence of
EDDDDKA-aca-R.sub.9-aca-C(Fl)-CONH.sub.2. (SEQ ID NO: 3)
EXAMPLE 3
[0139] Peptides Having Acidic Portions to Veto Uptake
[0140] Peptide molecules having features of the invention, having
fluorescent cargo moieties connected to basic portions (having
multiple arginine residues), these latter being linked by cleavable
linkers to an acidic portion (having multiple glutamate residues),
were synthesized and tested for ability to deliver cargo into
cells. Peptides showing ability of oligoglutamates to veto
oligoarginine-mediated cellular uptake include: TABLE-US-00001
Fl-aca-CRRRRRRRRR-aca-EEEEEEEEEC- (SEQ ID NO:5) CONH.sub.2 (linear
or cyclic, 5-47) Fl-aca-CEEEE-aca-RRRRRRRRRC-CONH.sub.2 (SEQ ID
NO:6) (linear or cyclic, 6-10) Peptides showing cleavage-dependent
uptake include: H.sub.2N-EEEEEDDDDKA-aca-RRRRRRRR-aca- (SEQ ID
NO:7) C(Fl)-CONH.sub.2 (6-14, Enterokinase substrate, (SEQ ID NO:8)
cleaved after DDDDK) H.sub.2N-EDDDDKA-
aca-RRRRRRRRR-aca-C(Fl)-CONH.sub.2 (6-16, Enterokinase substrate)
(SEQ ID NO:9) H.sub.2N-EEEEEDDDDKARRRRRRRRR-aca-C(Fl)- CONH.sub.2
(6-27, Enterokinase substrate) H.sub.2N- (SEQ ID NO:10)
EEDDDDKA-aca-rrrrrrrrr-aca-C(Fl)- CON-H.sub.2 (6-29, Enterokinase
substrate) H.sub.2N- (SEQ ID NO:11)
DDDDDDKARRRRRRRRR-aca-C(Fl)-CONH.sub.2 (7-2, Enterokinase
substrate) H.sub.2N- (SEQ ID NO:12)
EEDDDDKAR-aca-RR-aca-RR-aca-RR-aca- RR-aca-C(Fl)-CONH.sub.2 (7-4,
Enterokinase substrate) H.sub.2N- (SEQ ID NO:13)
eeeeee-aca-PLGLAG-rrrrrrrrr-aca- c(Fl)-CONH.sub.2 (7-6, MMP-2
substrate, cleaved between PLG and LAG)
EXAMPLE 4
[0141] Peptide Cleaved by Matrix Metalloproteinase-2 (MMP-2):
[0142] MMP-2 (5 .mu.g in 88 .mu.l) was activated from human
rheumatoid synovial fibroblast proenzyme (Invitrogen) in Tris-HCl
buffer as described by Stricklin et al (1983) Biochemistry 22: 61
and Marcy et al (1991) Biochemistry 30: 6476), then incubated with
32 .mu.l 0.5 mM peptide stock solution for one hour at room
temperature. FIG. 7A illustrates a HPLC chromatogram of the
substrate peptide before cleavage by MMP-2. Enzyme cleavage
progress was monitored by HPLC at 215 nm absorbance. FIG. 7B is a
HPLC chromatogram of the peptide after cleavage by MMP-2, showing
complete conversion to a new species.
EXAMPLE 5
[0143] FACS Analysis of Cell Uptake:
[0144] The human T cell line-wide type Jurkat cells were cultured
in RPMI 1640 media with 10% (v/v) deactivated fetal calf serum
(FBS) and reached density .about.1.times.10.sup.6 cells/ml. The
media was refreshed one day before being used. Before the
experiment, the Jurkat cells were washed with HBSS buffer three
times and resuspended in HBSS at (0.5-1).times.10.sup.6 cells/ml
density. In the cell uptake experiment, cells were stained with 1
.mu.M peptide or compound at room temperature for 10 min, then
washed twice with cold HBSS and submitted for FACS analysis. Cell
uptake was monitored by fluorescence at 530 nm run on FACS and
5,000 events were recorded from cells judged to be healthy by their
forward and side scatter. The data represent mean fluorescence of
the recorded cell population indicating uptake of the fluorescently
labeled compounds. In most experiments, Fl-GGR.sub.10-CONH.sub.2
(abbreviated as "R10" on the graphs; SEQ ID NO: 49) was included as
a positive control for uptake.
[0145] The mean fluorescence measured in Jurkat cells incubated for
ten minutes with the indicated peptides (each with fluorescent
cargo moieties) is shown in FIGS. 8, 9 and 10.
[0146] As shown in FIG. 9, compounds 6-14 (SEQ ID NO: 7) and 6-16
(SEQ ID NO: 8) showed greatly enhanced fluorescence, indicating
much greater uptake, of the cleaved form of the peptides than the
intact peptides. Similarly, as shown in FIG. 10, compounds 7-2 (SEQ
ID NO: 11) and 7-6 (SEQ ID NO: 13) also showed greatly enhanced
fluorescence after cleavage compared with the fluorescence of the
uncleaved compounds. Thus, these results demonstrate prevention of
cellular uptake of compounds having basic amino acids by linkage to
an acidic portion. Additionally, these results demonstrate enhanced
cellular uptake of fluorescent portions of these peptides (having
basic amino acids) following cleavage of the acidic portions.
[0147] Such cellular uptake increases as incubation time increases.
FIG. 11 illustrates the mean fluorescence measured in Jurkat cells
incubated for ten minutes with the indicated peptides having
fluorescent cargo moieties, basic and acidic portions, and
cleavable linker portions. As shown in FIG. 12, the mean
fluorescence measured in Jurkat cells incubated for one hour was
increased compared to the fluorescence measured as shown in FIG.
11.
[0148] The ability of MTS molecules having disulfide linkers X to
provide controlled delivery of a cargo portion was tested using
peptide 7-45 (SEQ ID NO: 14) having the structure ##STR1## in which
a disulfide bond between the two cysteines links the acidic portion
H.sub.2N-eeeeeec-CONH.sub.2 (SEQ ID NO: 15) with the basic portion
Fl-rrrrrrrrrc-CONH.sub.2 (SEQ ID NO: 16). The basic portion carries
the cargo portion, fluorescent moiety Fl (fluorescein). As
illustrated in FIG. 13, the mean fluorescence measured in Jurkat
cells incubated for ten minutes with the intact 7-45 peptide (SEQ
ID NO: 14) showed only a small amount of fluorescence above that of
the background measured from the Jurkat cells alone. However, when
the peptide was reduced with 25 mM tris(carboxyethyl)phosphine and
250 mM 2-mercaptoethanesulfonate for 15 min, which cleave the
disulfide linker X, then incubated with Jurkat cells for ten
minutes, the fluorescence taken up by the cells was comparable to
that of cells incubated for 10 minutes in the presence of R10.
Thus, a MTS molecule having features of the invention, with a
disulfide linker X, is able to provide controlled delivery of cargo
portion to cells.
EXAMPLE 6
[0149] MTS Molecules Having Varying Lengths
[0150] MTS molecules having features of the invention may have
different numbers of basic amino acids, different numbers of acidic
amino acids, and different linkers. Several examples of different
MTS molecules illustrating features of the invention are presented
in this Example, in which a fluorescent cargo moiety is exemplified
by fluorescein (Fl), a radioactive cargo moiety is exemplified by
.sup.125I, and a therapeutic cargo by doxorubicin (DOX).
[0151] EDA-aca-R.sub.5-aca-C(Fl)-CONH.sub.2 (SEQ ID NO: 17):
[0152] EDDDDKA-aca-R.sub.6-aca-C(DOX)-CONH.sub.2 (SEQ ID NO: 18)
TABLE-US-00002 EEEDDDEEEDA-aca-R.sub.9-aca-Y(.sup.125I)-CONH.sub.2
(SEQ ID NO:19) ededdAAeeeDDDDKA-aca-R.sub.11-aca-C(Fl)- (SEQ ID
NO:20) CONH.sub.2 eddedededDDDDKA-aca-R.sub.6-AGA-R.sub.6-aca-C
(SEQ ID NO:21) (DOX)-CONH.sub.2 Ggedgddeeeeeeddeed-aca-PLGLAG-aca-
(SEQ ID NO:22) R.sub.8-AAA-R.sub.12-aca-C(Fl)-CONH.sub.2
eeddeeddKA-aca-R.sub.7-aca-C(Fl)-CONH.sub.2 (SEQ ID NO:23)
eDDDDKA-aca-RGRGRRR-aca-C(Fl)-CONH.sub.2 (SEQ ID NO:24)
eddddeeeeeee-aca-PLGLAGKA-aca-R.sub.10- (SEQ ID NO:25)
aca-C(Fl)-CONH.sub.2 eeeeeeeeeeeeeeee-aca-DDDDKA-aca-R.sub.20- (SEQ
ID NO:26) aca-C(Fl)-CONH.sub.2
eeeeeeeeeddddd-aca-DDDDKA-aca-R.sub.17- (SEQ ID NO:27)
aca-Y(.sup.125I)-CONH.sub.2
dddddddddddddddd-aca-PLGLAG-aca-R.sub.14- (SEQ ID NO:28)
aca-C(DOX)-CONH.sub.2
EXAMPLE 7
[0153] Examples of Molecules Suitable for Use as Cargo Moieites
[0154] Examples of molecules suitable for attachment as cargo
moieties to a basic portion B of a MTS molecule having features of
the invention are illustrated in FIG. 14. The different exemplary
molecules shown in FIG. 14 are each labeled by an identifier letter
in parentheses. The molecules are shown having one bond that ends
in a dot; the bond ending in a dot may be used to attach the cargo
molecule to a basic portion B. A letter in brackets near the dotted
bond indicates a suitable atom to which the cargo molecule might
bind; for example, [N] indicates that the cargo molecule may bind
to a nitrogen, such as a nitrogen of a lysine epsilon amino group,
or a nitrogen of an alpha amino group of a peptide backbone of the
MTS molecule. An [S] indicates a linkage to a sulfur atom, such as
a cysteine sulfur atom.
[0155] More than one of these exemplary cargo molecules may be
attached to a basic portion B, and basic portions B carrying
multiple cargo molecules may have more than one type of cargo
molecule attached. The cargo molecules may form part of more
complex structures as well. For example, the dark circle in the
cargo moiety labeled (k) represents a particle including a
superparamagnetic iron oxide core, jacketed by crosslinked,
aminated dextran (such particles typically have a radius of about
22 nanometers). Although only one pendant group is shown, such
particles may have multiple pendant groups (typically about 4 to
about 20).
EXAMPLE 8
[0156] Examples of Acidic Moieties Suitable for Inclusion in an
Acidic Portion A
[0157] An acidic portion A may include acidic moieties such as
those illustrated in FIG. 15. Such moieties may be linked to a
linker X and an acidic portion A by peptide bonds, disulfide bonds,
or other bonds. A dashed line in the illustration indicates a
possible attachment point. In this and subsequent figures, a moiety
in brackets indicates a motif that may be repeated, with a letter
(e.g., "x") indicating the number of times that the motif may be
repeated (which may take on a number of possible values, typically
between about 1 and about 100, preferably between about 1 and about
20). It will be understood that such acidic moieties may be
attached to an acidic portion A in any suitable manner. In
embodiments, an acidic portion A of a MTS molecule having features
of the invention may be partly comprised of, or mainly comprised
of, or essentially completely comprised of acidic moieties such as
those illustrated in FIG. 15.
EXAMPLE 9
[0158] Examples of Linker Moieties
[0159] Linkers suitable for use in a MTS molecule having features
of the invention may be peptides or other molecules cleavable by
enzymes under physiological conditions. For example, linkers may be
cleavable by such enzymes as metalloproteases. Linkers cleavable by
MMP-2 have been discussed supra. In addition, for example, linkers
cleavable by other metalloproteases, such as MMP-9, MMP-11, and
MMP-14 are also suitable. For example, peptide linker cleavable by
MMP-9 may include the peptide sequence
[0160] PR(S/T)(L/I)(S/T) (SEQ ID NO: 29)
[0161] where the letters in parentheses indicate that either one of
the indicated amino acids may be at that position in the sequence.
A peptide linker cleavable by MMP-11 may include the peptide
sequence
[0162] GGAANLVRGG (SEQ ID NO: 30)
[0163] and peptide linker cleavable by MMP-14 (MT1-MMP) may include
the peptide sequence
[0164] SGRIGFLRTA (SEQ ID NO: 31).
[0165] A peptide linker cleavable by urokinase plasminogen
activator (uPA) may include the peptide sequence
[0166] SGRSA (SEQ ID NO: 32)
[0167] A peptide linker cleavable by lysosomal enzymes may include
one of more of the peptide sequences
[0168] GFLG (SEQ ID NO: 33),
[0169] ALAL (SEQ ID NO: 34), and FK.
[0170] A peptide linker may be cleavable by a cathepsin. For
example, a linker cleavable by cathepsin B may include a KK or a RR
sequence, or may include both, where the cleavage would typically
occur between the lysines or arginines. A peptide linker cleavable
by cathepsin D may include the peptide sequence
[0171] PIC(Et)F-F (SEQ ID NO: 35),
[0172] where C(Et) indicates S-ethylcysteine (a cysteine with an
ethyl group attached to the thiol) and the "-" indicates the
typical cleavage site in this and subsequent sequences. A peptide
linker cleavable by cathepsin K may include the peptide
sequence
[0173] GGPRGLPG (SEQ ID NO: 36).
[0174] A peptide linker cleavable by prostate-specific antigen may
include the peptide sequence
[0175] HSSKLQ- (SEQ ID NO: 37).
[0176] A peptide linker cleavable by Herpes simplex virus protease
may include the peptide sequence
[0177] LVLA-SSSFGY (SEQ ID NO: 38).
[0178] A peptide linker cleavable by HIV protease may include the
peptide sequence
[0179] GVSQNY-PIVG (SEQ ID NO: 39).
[0180] A peptide linker cleavable by Cytomegalovirus protease may
include the peptide sequence
[0181] GVVQA-SCRLA (SEQ ID NO: 40)
[0182] A peptide linker cleavable by Thrombin may include the
peptide sequence
[0183] f(Pip)R-S (SEQ ID NO: 41)
[0184] where "f" indicates D-phenylalanine and "Pip" indicates
piperidine-2-carboxylic acid (pipecolinic acid, a proline analog
having a six-membered ring).
[0185] A peptide linker cleavable by Caspase-3 may include the
peptide sequence
[0186] DEVD- (SEQ ID NO: 42).
[0187] A peptide linker cleavable by Interleukin 1.beta. converting
enzyme may include the peptide sequence
[0188] GWEHD-G (SEQ ID NO: 43).
[0189] In addition, linkers suitable for use in a MTS molecule
having features of the invention may be cleavable by agents other
than proteases under physiological conditions. Linkers may also be
non-peptide molecules. Some examples of enzymatically and
non-enzymatically cleavable moieties suitable as linkers are
illustrated in FIG. 16. Examples of different cleavable linkers are
shown along with an indication of conditions which lead to
cleavage. For example, cleavage of the linker labeled (a) may be
accomplished by beta-lactamase. Cleavage of the linker labeled (b)
may be accomplished by exposure to light, such as to a single
photon of violet light or to two photons of infrared light.
Cleavage of the linker labeled (c) may occur under reducing
conditions. Cleavage of the linkers labeled (d) and (e) may occur
in acidic conditions. Action of an esterase may cleave the linker
labeled (f), and a phosphatase may cleave the linker labeled
(g).
EXAMPLE 10
[0190] Examples of Basic Moieties Suitable for Inclusion in a Basic
Portion B
[0191] A basic portion B may include basic moieties such as those
illustrated in FIG. 17. Such moieties B may be linked to a linker
X, cargo C, or to another part of a basic portion B by peptide
bonds, disulfide bonds, or other bonds. A dot indicates a possible
attachment point, while a letter enclosed by brackets indicates a
possible atom to which such an attachment may be made (e.g., [S]
indicates that a bond, such as a diusulfide bond, may be made to a
sulfur atom; a [N] indicates a bond to a nitrogen may be made). It
will be understood that such basic moieties may be attached to a
basic portion B or other portions of a MTS molecule in any suitable
manner. For example, the "X" shown in compound (c) of FIG. 17
indicates attachment of a linker X to the side-chain of a D-lysine
residue. The amino acid portion of compound (c) of FIG. 17 is SEQ
ID NO: 44; the amino acid portion of compound (d) of FIG. 17 is SEQ
ID NO: 45; the amino acid portion of compound (e) of FIG. 17 is SEQ
ID NO: 46; and the amino acid portion of compound (f) of FIG. 17 is
SEQ ID NO: 47. In embodiments, a basic portion B of a MTS molecule
having features of the invention may be partly comprised of, or
mainly comprised of, or essentially completely comprised of basic
moieties such as those illustrated in FIG. 17.
[0192] It will be understood that some combinations of A and B may
be more suitable than others. For example, it is preferred that the
same backbone structure be present in both portions A and B in a
MTS molecule having features of the invention, so that, for
example, both A and B are peptides, or both A and B are peptoids,
or both A and B are carbamates. It is also preferred that the
absolute value of the net charge of one portion be similar, or the
same as, the absolute value of the net charge of the other portion
so that, for example, A has approximately the same number of
negative charges as B has positive charges.
EXAMPLE 11
[0193] Examples of Polymeric Acidic Portions
[0194] In another embodiment, an acidic portion A may include or be
part of a polymer. In preferred embodiments, the polymer has an
average molecular weight of about 50 kDa or above. Such high
molecular weights reduce immunogenicity and improve
pharmacodynamics by slowing excretion and lengthening the residence
time in the bloodstream. Furthermore, polymers of such size benefit
from "enhanced permeability and retention" (EPR) in tumors, whose
capillaries are much leakier than normal tissue and whose lymphatic
drainage is often impaired. These properties cause polymers to have
higher ratios of concentrations in tumor vs. normal tissue than
those of low-molecular-weight drugs. For recent discussions of the
benefits of polymeric carriers, see Kopecek et al (2001) J.
Controlled Release 74: 147-158; Luo & Prestwich (2002) Current
Cancer Drug Targets 2: 209-226; Maeda et al (2003) International
Immunopharmacology 3: 319-328; and Torchilin & Lukyanov (2003)
Drug Discovery Today 8: 259-266. This EPR effect leading to
enhancement of concentration in tumor tissue compared to normal
tissue should further reinforce the tumor selectivity resulting
from preferential cleavage of the linker X of MTS molecules having
features of the invention by enzymes or under conditions found near
tumors. Cleavage of X is effective to release basic portion B and
cargo C attached to B from a polymeric acidic portion A, allowing
the uptake of B and C into cells. In preferred embodiments, the
polymer carries a sufficient number of negative charges to veto
uptake of B and C while linker X is still intact. Examples of such
polymers are shown in FIG. 18. The amino acid portion of compound
(c) of FIG. 18 is SEQ ID NO: 48.
EXAMPLE 12
[0195] Examples of Tumor Imaging
[0196] The methods, compositions and systems disclosed herein may
be used for selectively delivering molecules to tumor cells.
Cellular association of polyarginine based, cell-penetrating
peptides (CPPs) is effectively blocked when they are fused to an
inhibitory domain made up of negatively charged residues. In this
example, such fusions are termed "activatable CPPs" (ACPPs) because
cleavage of the linker between the polycationic and polyanionic
domains, typically by a protease, releases the CPP portion and its
attached cargo to bind to and enter cells. Association with
cultured cells typically increases 10-fold or more upon linker
cleavage. In mice xenografted with human tumor cells secreting
matrix metalloproteinases 2 and 9, ACPPs bearing a
far-red-fluorescent cargo show in vivo contrast ratios of 2-3 and a
3.1-fold increase in standard uptake value for tumors relative to
contralateral normal tissue or control peptides with scrambled
linkers. Ex vivo slices of freshly resected human squamous cell
carcinomas give similar or better contrast ratios. Because CPPs are
known to import a wide variety of nonoptical contrast and
therapeutic agents, ACPPs offer a general strategy toward imaging
and treating disease processes associated with linker-cleaving
activities such as extracellular proteases. References cited in
this example are indicated by reference number, with the full
citation for each numbered reference provided at the end of the
example.
[0197] Molecular imaging and therapy in patients would greatly
benefit from generic, rational mechanisms to target contrast agents
and therapeutic drugs to diseased tissues, especially tumors (1).
Currently, the main strategies are based on antibodies against
surface markers or ligands for receptors preferentially expressed
in the target tissue (2). Although antibodies have occasionally
been successful in targeting tumors (3), their irreducible bulk
hinders penetration of solid tumors and excretion of unbound
reagent (4), and elaborate reengineering is required to minimize
immunogenicity (5, 6). A few small molecule ligands (2 kDa or less)
for endogenous receptors have been preliminarily explored, but
robust tumor specificity is rare or nonexistent (4). A fundamental
limitation of simple antibody or ligand binding is the lack of
amplification, where each target molecule (typically of low
abundance) can bind at most one probe. Some amplification can be
achieved by incorporating the probe into polymers or nanoparticles,
but the increase in bulk worsens access to diseased tissue and
removal from healthy organs. None of these approaches help get
drugs across the plasma membrane into the cytoplasm and nucleus of
diseased cells, the most desirable loci for modifying signal
transduction or triggering cell death. Certain polycationic
sequences [variously dubbed cell penetrating peptides (CPPs),
membrane-translocating sequences (MTS), or protein transduction
domains] can bring covalently attached payloads into mammalian
cells without requiring specific receptors. CPPs were first
discovered within a domain from Antennapedia homeobox protein and
the tat protein from HIV-1 (7, 8). A variety of multicationic
oligomers, including VP-22 and guanidinium-rich sequences, as
simple as 6-12 consecutive arginines are now known to be equally or
more effective (9-11). D-Amino acids are at least as good as
natural L-amino acids and possibly better because the unnatural
isomers resist proteolysis (10-12). Cargoes ranging in size from
metal chelates and fluorescent dyes (13, 14) to iron oxide
nanoparticles (15) and liposomes (16) can be imported, although the
detailed mechanisms and subcellular localizations remain poorly
understood and may differ, depending on cargo size, cell type, CPP
sequence, and other experimental variables (17, 18). Initial
attachment of the polycations to the cell surface is avid, rapid,
and probably mediated by electrostatic attraction for anionic
phospholipids and glycosaminoglycans. Much of the subsequent
internalization probably occurs by endocytosis, because delivery of
bioactive cargoes to the cytosol and nucleus can be enhanced by
inclusion of sequences known for acidification-dependent disruption
of endosomes (19, 20).
[0198] We now demonstrate a generic targeting mechanism based on
selective local unleashing of CPPs, as schematized in FIG. 19.
Cellular uptake of CPPs can be largely blocked by fusing them by
means of cleavable linkers to polyanionic sequences, which
neutralize the polycations by forming intramolecular hairpins of
.apprxeq.2-3 kDa. We call such constructs activatable CPPs (ACPPs),
because cleavage of the linkers dissociates the inhibitory
polyanions, releasing the polycationic peptides and their cargo to
attach to and enter cells. The mechanism (FIG. 19) is a flexible,
modular, amplifying strategy to concentrate imaging and therapeutic
agents on and within cells in the immediate vicinity of
extracellular cleavage activities, such as matrix
metalloproteinases (MMPs) in tumors. We chose MMP-2 and MMP-9 as
our primary initial targets because they are the best characterized
proteases overexpressed by tumors (21). Currently, at least 26
members of the MMP family have been identified. They play a crucial
role in extracellular matrix degradation, tissue invasion, and
metastasis (21-26).
[0199] Materials and Methods: Peptide Synthesis and Fluorophore
Labeling. Peptides were synthesized on an automatic peptide
synthesizer by using standard protocols for
fluorenylmethoxycarbonyl solid-phase synthesis. Further details and
further information regarding peptide synthesis, fluorophore
labeling, and poly(ethylene glycol) (PEG) attachment (PEGylation)
is presented in Example 13.
[0200] Peptide Cleaved by MMP-2 (PLGLAG). MMP-2 proenzyme (5 .mu.g
in 80 .mu.l of 50 mM Tris_HCl buffer) was activated with 2.5 mM
4-aminophenylmercuric acetate at 37.degree. C. for 2 h. Afterward,
we added 32 .mu.l of 0.5 mM peptide stock solution and incubated
the mixture for 1 hr at room temperature. Enzyme cleavage progress
was monitored by HPLC. The HPLC chromatograms showed that near
complete cleavage was accomplished after 30 min of incubation. The
new peak was collected, and its mass was determined by mass
spectroscopy. The mass spectrum indicated that the enzyme was cut
between glycine and leucine residues of the MMP-2 substrates as
predicted, giving products such as NH2-eeeeee-ahx-PLG (SEQ ID NO:
50) and LAG-rrrrrrrrr-ahx-c(Fl)-CONH2 (SEQ ID NO: 51), where "ahx"
indicates aminohexanoic acid (also termed "aminocaproic acid").
[0201] FACS Analysis and Microscopy. Jurkat cells were cultured in
RPMI medium 1640 plus 10% (vol_vol) FBS to a density of
0.5-1.times.10.sup.6 cells per ml. The media was refreshed 1 day
before the assay of ACPPs. Cells were washed with Hanks' balanced
salt solution (HBSS) buffer three times, resuspended in HBSS at
0.5-1.times.10.sup.6 cells per ml, stained with 1 .mu.M peptide in
HBSS at room temperature for 10 min, washed three times with cold
HBSS, and analyzed by flow cytometry at 530-nm emission for
fluorescein labeled peptides or at 675 nm for Cy5-labeled peptides.
We collected 10,000 events from cells judged to be healthy by their
forward and side scatter. Peptide association with HT-1080 cells
was similarly quantified by flow cytometry after release from
adherence with trypsin. For microscopic imaging, HT-1080 cells
grown to 70% confluency were washed with HBSS three times, stained
with 1.25 .mu.M peptide and 1 .mu.g/ml Hoechst 33258 (a nuclear
stain), rinsed twice, trypsinized, replated on polylysinecoated
dishes, and imaged for Cy5 content (excitation, 625-645 nm;
emission, 665-695 nm) and Hoechst 33258 (excitation, 375-385 nm;
emission, 420-460 nm).
[0202] Xenografts in Mice. Nude mice (age, 4-6 weeks) were injected
s.c. with .apprxeq.10.sup.6 HT-1080 cells. Once the tumors had
reached .apprxeq.5-7 mm in size (typically 1-2 weeks later),
animals were anesthetized with 100 mg/kg ketamine and 5 mg/kg
midazolam), weighed, and injected with .apprxeq.100 .mu.l of 60
.mu.M peptide through the tail vein. Animals were then imaged at
various times by using a Nikon f/1.2 camera lens in front of a
cooled charge-coupled device camera (SenSys, Photometrics, Tucson,
Ariz.). For longer lasting imaging studies, animals were allowed to
wake up after 2 h of anesthesia and were reanesthetized at
.apprxeq.4 and 6 hr for further data collection. Plasma half-lives
were determined by the decrease in fluorescence intensity of
.apprxeq.5-.mu.l blood samples withdrawn periodically into
heparinized capillaries. After imaging was ended, animals were
killed with halothane, and organs of interest were harvested and
weighed. For frozen sectioning, tissues were added to OCT
cryopreservative and frozen on dry ice and hexane. Samples were
stored at -80.degree. C. and cut into 5-.mu.m sections at
-20.degree. C. by using a cryotome. Cy5 fluorescence was imaged as
described above. To measure standardized uptake values (SUVs), 30
mg of each tissue was added to 100 .mu.l of a buffered 1% SDS
mixture (pH 7.6) and protease inhibitor mixture (Roche
Diagnostics). The tissue was then homogenized, heated to 70.degree.
C. for 15 min, microwaved for 15 sec, centrifuged at 20,500.times.g
for 15 min, then imaged on the same system used for whole mice. Two
sets of standards (liver and kidney) were used to calibrate
fluorescence intensity in terms of peptide concentration. From this
calibration, the quantity of peptide in 30 mg of tissue for each
organ was calculated. SUVs were calculated as the molality of
peptide in the tissue divided by the total injected dose as mol/kg
of body weight.
[0203] Squamous Cell Carcinoma Specimens. Human squamous cell
carcinoma specimens from planned resections of neoplasms were
collected postoperatively according to a protocol with
institutional review board approval. The specimens were in ice-cold
normal saline for 30 min during transport back to the laboratory,
where they were cut by hand to .apprxeq.1-mm-thick slices, added to
1 ml of 1 .mu.M peptide for 15 min at room temperature, rinsed five
times for 2 min in 1 ml of HBSS, cryosectioned, and imaged as
described above.
[0204] Results
[0205] Until Cleaved Off, Polyanionic Sequences Inhibit Association
of CPPs with Cells. Given that the initial binding of CPPs to cells
is believed to be electrostatic, we asked whether association with
cells could be prevented by appending polyanionic sequences to give
the polycations intramolecular diversions. Fluorescently labeled
peptides were synthesized with nine arginine residues fused by
means of cleavable linkers to six to nine consecutive acidic
residues, usually glutamate. We incubated these peptides, either
intact or with linkers precleaved, with Jurkat lymphocytes or
HT-1080 fibrosarcoma cells and assessed cell fluorescence by flow
cytometry and fluorescence microscopy of the live unfixed cells
after washing away unbound peptides. FIG. 20 shows results with
HT-1080 cells and an ACPP cleavable by MMP-2. The intact peptide
showed 18-fold less uptake than the equimolar mixture of the two
fragments resulting from linker cleavage, which in turn was similar
to a control CPP with only the polycation. The flow cytometric
histograms showed that fluorescence on or in healthy cells was
unimodal and reasonably homogeneous (FIG. 20A). Single cell
microscopy (FIG. 20B) confirmed that cargo uptake was far greater
after linker cleavage and indicated that a significant fraction
reached the nucleus, as judged by accumulation of fluorescence in
the nucleoli, similar to results previously reported for
polycation-mediated transduction (17). Analogous cleavage-dependent
association with cells was observed with a variety of ACPPs
containing different numbers of arginine residues, different
polyanionic sequences, and linkers cleavable by a variety of
proteases, including enterokinase, MMP-2, MMP-9, and urokinase
plasminogen activator, or even by simple reduction of a disulfide
bond (Table 1). In the best case, cell labeling increased
>100-fold when the polyanion was cut off from the polycation.
Both the arginine residues and the acidic residues could be D-amino
acids, as desirable to restrict in vivo proteolysis to the central
linker between the two domains. Greater contrast was obtained when
the polycationic, not the polyanionic, region was closer to the C
terminus. We hypothesize that this preference is because the new
amino terminus created by proteolytic cleavage would reinforce the
polycationic charge, whereas, if the polycation is at the N
terminus, proteolysis would append a negatively charged carboxylate
to the polycation. Cleavage-dependent contrast was equally
observable with fluorescein or the far-red fluorophore Cy5 as cargo
and with or without a PEG tail (Table 1). Such PEGylation increases
solubility and slows in vivo excretion but is not necessary to
block CPP activity. TABLE-US-00003 TABLE 1 Effect of different
linkers and acidic inhibitory domains on ACPP association with
Jurkat and HT-1030 cells assayed by flow cytometry Uptake Increase
Uptake after caused before cleavage by Sequence cleavage (reagent)
cleavage EEEEEDDDDK*AXRRRRRRRRR 0.18 2.4 (EK) 13 XC(Fl) SEQ ID
NO:52 EEEEEDDDDK*ARRRRRRRRRX 0.07 1.2 (EK) 17 C(Fl) SEQ ID NO:53
EDDDDK*AXRRRRRRRRRXC 0.30 2.3 (EK) 8 (Fl) SEQ ID NO:54
EEDDDDK*ARXRRXRRXRRXRR 0.015 0.11 (EK) 7 XC(Fl) SEQ ID NO:55
DDDDDDK*ARRRRRRRRRXC 0.05 0.77 (EK) 16 (Fl) SEQ ID NO:56
EEDDDDK*AXrrrrrrrrrXC 0.07 1.2 (EK) 17 (Fl) SEQ ID NO:57
eeeeeeXPLG*LAGrrrrrrrr 0.086, 1.3, 1.3 16, 39 rXc(Fl) 0.034 (MMP-2)
(SEQ ID NO:58) 10 min eeeeeeXPLG*LAGrrrrrrrr 0.11 2.1 (MMP-2) 19
rXc(Fl) (SEQ ID NO:58) 60 min UeeeeeeeeXPLG*LAGrrrrr 0.006 0.74 123
rrrrXk(Fl) (MMP-2) SEQ ID NO:59 eeeeeeXPLG*LAGrrrrrrrr nc nc
(MMP-2) 36 rXc(Cy5) SEQ ID NO:60 UeeeeeeXPLG*LAGrrrrrrr nc nc
(MMP-2) 20 rrXc(Cy5) SEQ ID NO:61 UeeeeeeeeXPLG*LAGrrrrr nc nc
(MMP-2) 17 rrrrXk(Cy5.5) SEQ ID NO:62 [11-kDa PEG]Xeeeeeeeee 0.012,
0.82, 68, 21 XPLG*LAGrrrrrrrrrXk 0.10 2.1 (Cy5) (MMP-2) SEQ ID
NO:63 0.10 1.87 18 (MMP-9) [11-kDa PEG]Xeeeeeeeee 0.019 0.021 1.13
XLALGPGrrrrrrrrrXk (MMP-2) (Cy5).dagger. -- 0.020 1.07 SEQ ID NO:64
(MMP-9) Fl-XrrrrrrrrrXPLG*LAGe 0.004 0.06 16 eeeeeee-.beta.Ala
(MMP-2) SEQ ID NO:65 Fl-XrrrrrrrrrXSGRS*Aee 0.012 0.05 (uPA) 4
eeeeee-.beta.Ala SEQ ID NO:66 eeeeeeXSGRS*AXrrrrrrrr nc nc (uPA) 11
rXc(Cy5) SEQ ID NO:66 Fl-rrrrrrrrrc-*-ceeeeee.dagger-dbl. 0.092
0.72 8 SEQ ID NO:67 (reduction) For sequences, lowercase characters
indicate D-amino acid. All peptides were amidated at C terminus.
Values represent the results of triplicate experiments performed on
the same day. Some entries have two values because the triplicate
experiments were repeated on another day. *Cleavage site; U,
succinoyl; X, 6-aminohexanoyl; Fl, fluorescein. Uptake before and
after cleavage was measured by FACS, normalized to Fl-GGRRRRRRRRRR
(SEQ ID NO:68) or rrrrrrrrrk(Cy5) (SEQ ID NO:69), except for some
measurements not calibrated (nc) with respect to either reference
peptide. EK, enterokinase; uPA, urokinase plasminogen activator.
.dagger.This scrambled control should be uncleavable, so the
rightmost column refers to increase due to enzyme exposure rather
than cleavage. .dagger-dbl.Disulfide-linked. In "ceeeeee", the N
terminus is D-glu and the amidated C terminus is D-cys.
[0206] ACPPs Adopt a Hairpin Conformation Before Cleavage.
Polyanion inhibition of polycation uptake would be most easily
understood if the oppositely charged segments zippered together as
shown in FIG. 19. Direct evidence for such a hairpin structure was
obtained by homonuclear two-dimensional NMR analysis (see
supporting information for methods). FIG. 21 shows nuclear
Overhauser effects observed in two-dimensional NMR of a simple
ACPP, succinyl-e.sub.8-XPLGLAG-r.sub.9-Xk, where X denotes
6-aminohexanoyl. (The cleavable peptide was [11 kDa
PEG]-X-e.sub.9-XPLG*LAG-r.sub.9 and the scrambled peptide was [11
kDaPEG]-X-e.sub.9-XLALGPG-r.sub.9). The observed nuclear Overhauser
effect correlations shown in FIG. 21 reflect proton-proton
proximities. Dashed red lines indicate observed nuclear Overhauser
effects, and the green line highlights the peptide outline for
clarity. The observed short-range couplings within the PLGLAG (SEQ
ID NO: 1) linker indicate a turn conformation (see Example 13 for
more detail). In addition, the numerous nuclear Overhauser effects
between the strings of D-arginine and D-glutamate clearly indicate
pairings that would stabilize the hairpin turn. Taken together, the
data indicate that the presence of a hairpin structure, although
they are not sufficient to define a complete atomic-level structure
owing to chemical shift overlap.
[0207] MMP-2 Cleavable ACPPs Concentrate in Human Tumors
Xenografted into Mice. We next tested whether ACPPs could light up
protease expressing human tumor xenografts in whole mice. We chose
HT-1080 tumors in the axilla of nude mice because these tumors
express both MMP-2 and MMP-9 and have been used to test other MMP-2
cleavable contrast agents (22, 26). Adding a PEG tail to the
peptide proved helpful to prevent excessively rapid excretion; PEGs
of 5, 11, and 21 kDa gave plasma half-lives of 5, 15, and 38 min,
respectively, consistent with trends reported in ref. 27.
Anesthetized mice were injected through the tail vein with either
an MMP-2 cleavable ACPP, an isomeric scrambled version verified not
to be a substrate for MMP-2 or MMP-9, or an all-D-amino acid
version. All peptides had Cy5 attached to permit in vivo imaging of
the far-red fluorescence through the skin. FIG. 22A1 shows that the
tumor is the brightest fluorescence visible in the live animal
injected with the MMP-2-cleavable ACPP, whereas FIG. 22B1 shows
much less tumor contrast from a different animal injected with the
scrambled analog. Similar cleavage-dependent contrast was seen in
frozen sections at higher magnifications (FIGS. 22 A2, A3, B2, and
B3). To quantitate the results, we measured the contrast index
defined as (fluorescence intensity of
tumor-autofluorescence)/(fluorescence of normal contralateral
region-autofluorescence). This index was 2.1.+-.0.17 (mean.+-.SE,
n=6) for the cleavable ACPP, which was modestly but significantly
higher (P<0.02, two-tailed t test) than the values obtained for
both the scrambled isomer (1.3.+-.0.16, n=2) and the all-D-amino
acid control (1.5.+-.0.11, n=4). The latter values may differ from
1.0 because of the phenomenon of enhanced permeability and
retention, whereby macromolecules passively accumulate in tumors
because their vasculature is leakier than that of healthy tissue
(28). Nevertheless, the amount of cleavable ACPP that accumulates
in the tumor is significantly more than can be accounted for by the
enhanced-permeability-and-retention effect, arguing for local
unmasking of the CPP by enzymes secreted by the tumor.
[0208] Although FIG. 22A1 shows that tumors become visible in
intact live animals, such fluorescence images are highly biased in
favor of superficial tissues, skin>s.c. tumors>deep organs.
To measure the true distribution of the peptides unbiased by
anatomical depth, postmortem tissue samples from different organs
were homogenized in detergent to release the labeled probe,
clarified by centrifugation, and quantified by Cy5 fluorescence
relative to tissue standards spiked with known amounts of dye.
Standardized uptake values (SUVs), defined as (moles of recovered
peptide/weight of tissue sample)/(moles injected into animal/total
body weight), are shown in FIG. 23 as SUVs (mean.+-.SD) 1 hr after
injection of peptide into mice, comparing a cleavable ACPP with its
all-D-amino acid control. The data shown in FIG. 23 are
fluorescence measurements from solubilized tissue. The cleavable
peptide was [11-kDa PEG]-X-e.sub.9-XPLG*LAG-r.sub.9-Xk(Cy5), and
the uncleavable peptide was [11-kDa
PEG]-X-e.sub.9-Xplglag-r.sub.9-Xk(Cy5). Although the kidney and
liver have the highest absolute SUVs, as typical for peptides, the
tumors gave a higher ratio of SUVs between the cleavable and
control peptide: 3.1. Also, of the tissues with appreciable uptake,
only in the tumors did the difference between the two peptides
attain statistical significance (P<0.05, two-tailed t test). The
standard deviation for the cleavable peptide was <0.05 for
muscle, brain, and spleen and <0.05 for muscle, spleen, heart
and pancreas for the uncleavable peptide.
[0209] ACPPs Light Up Human Squamous Cell Carcinomas. Although
human tumor cell lines xenografted into immunodeficient mice are
popular cancer models, they fail to mimic many aspects of real
human tumors. To get a preliminary indication whether ACPPs would
work on clinically relevant neoplasms, we applied ACPPs to coarse
sections cut from tissue freshly resected from patients undergoing
surgery for squamous cell carcinoma of the aerodigestive tract.
These surgical samples contained both neoplastic and normal tissue,
distinguishable by cell morphology and histological staining. The
ACPP, whose covalently attached PEG was reduced to 5 kDa to
facilitate diffusion, consistently stained tumor tissue more
brightly than normal tissue, whereas the scrambled peptide or the
ACPP coadministered with
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (a Zn.sup.2+
chelator and broad-spectrum MMP inhibitor), showed no such
consistent pattern. In FIG. 23, A Upper-D Upper are Cy5
fluorescence images displayed at a uniform gain, whereas A Lower-D
Lower are transmitted light views of the same fields. The squamous
cell carcinoma tumor tissue exposed to cleavable peptide (FIG. 23A)
was much more fluorescent than normal tissue exposed to cleavable
peptide (FIG. 23B) or either tissue exposed to scrambled peptide
(FIGS. 23 C and D). Contrast, defined as (tumor tissue
fluorescence-autofluorescence)/(normal tissue
fluorescence-autofluorescence), was almost eight in this example.
Contrast tended to be greatest where the tumor tissue had a high
histologic grade of malignancy. An example in FIG. 23A is that the
keratin pearl, characteristic of differentiated squamous epithelium
(29), was less fluorescent than the surrounding tumor. The contrast
averaged 2.7.+-.0.2 (mean.+-.SD) from two patients with relatively
differentiated oral cavity_oropharynx tumors (low to moderate
histologic grade of malignancy), whereas two high-grade laryngeal
tumors gave more contrast, 6.5.+-.3.4. Also, lymphocytic
granulation tissue was nearly as bright as the tumors themselves,
possibly because of the release of MMPs from lymphocytes. Normal
tissue immediately adjacent to tumor tissue was noticeably brighter
than more remote normal tissue, possibly because of the presence of
immune cells or to diffusion of the soluble proteases.
[0210] Discussion
[0211] We believe the selective activation of CPPs as disclosed
herein offers many advantages, including the following advantages:
(i) It should be adaptable to a wide variety of imaging and
therapeutic modalities, including radioactivity, because the
payload or cargo need not have any particular spectroscopic
properties. CPP-mediated uptake has already been demonstrated with
gamma-ray emitters and MRI contrast agents as well as potential
therapeutic agents (30). Close integration between imaging and
therapy would thus be facilitated; for example, providing ACPPs
having nonoptical cargoes is a useful application of the present
methods and compositions. (ii) Catalytic amplification is inherent
in the methods disclosed herein; i.e., each protease molecule can
cleave multiple substrate molecules, whereas with antibodies, for
example, each epitope can only bind one antibody at a time. (iii)
ACPPs help deliver the cargo not just to the surface of the target
cell but inside and to the nucleus, which is important for
therapeutic payloads and other payloads and applications. (iv)
Molecular masses can be varied over a wide range from quite small
(.apprxeq.18 aa or .apprxeq.2 kDa, where "aa" indicates amino
acid(s) and "kDa" indicates kiloDaltons) up to nanoparticles of
several nanometers in diameter (15, 16, 31). Depending on whether
polymers are appended to the polyanionic versus polycationic
portion, one can choose whether they are discarded or retained
after linker cleavage. Excessive molecular mass typically has the
disadvantage of decreasing penetration into solid tumors,
particularly when they have high interstitial fluid pressure (29).
(v) The highly modular substrates are synthesized by standard
methods of peptide synthesis and bioconjugation, without requiring
fermentation or high-level expression systems, yet they contain
enormous scope for rational or combinatorial variation. (vi) The
high content of D-amino acids would be expected to reduce
immunogenicity. Other guanidinium decorated nonpeptidic backbones,
such as carbamates and peptoids, are known to be competent for cell
uptake (32) and may be modulatable in analogy to the peptides
discussed above. (vii) Extracellular proteases are mechanistically
important in cancer (33), particularly in angiogenesis and
metastasis, unlike many tumor antigens of unknown function. In
principle, tumor cells that try to become resistant by
down-regulating their proteases are likely to become less
aggressive and metastatic. In addition, it is believed that
multiple subtypes of cancers may share similar properties of
up-regulating a relatively limited repertoire of proteases, giving
each successful substrate a wider range of clinical indications.
(viii) Proteases that are or can become extracellular are crucial
to many other disease processes, including thrombosis, congestive
heart failure, inflammation, neurodegeneration, and infectious
pathogens (34-37). Uses of the methods, compositions and systems
disclosed herein are not limited to proteases: any conditions that
sever the vetoing polyanion from the polycation (e.g., agents that
reduce disulfide bonds) may be used and exploited as mechanisms for
localization.
[0212] The present examples in vivo have included substrates for
soluble proteases, such as MMP-2 and MMP-9, mainly because these
MMPs have well established roles in metastasis and angiogenesis,
clear substrate preferences, and commercial sources for in vitro
testing. However, soluble proteases may have the potential
disadvantage of gradually leaking from the tumor into the general
circulation, where they would contribute to background signal and
reduced contrast. MMPs have been detected in the plasma and urine
of cancer patients at levels that show positive correlations with
the severity of metastatic disease (23), although the relative
enzyme activities in tumors versus blood do not seem to be known.
To circumvent diffusion of soluble MMPs, substrates for
membrane-bound MMPs, such as MT1-MMP (24, 25) may be used. Other
membrane-bound proteases including members of the ADAM (a
disintegrin and metalloprotease) family (38) are also suitable
alternatives to soluble MMPs and substrates for these and other
proteases may be used in the practice of the methods disclosed
herein.
[0213] Although the examples disclosed herein have not included
incorporation of additional contrast mechanisms, such as
fluorescence dequenching (22, 26) or enhanced permeability and
retention of adequately large polymers (28), within tumors with
leaky vasculature, such additional contrast mechanisms may be used
in or with these methods. For example, if maximum contrast and
sensitivity are desired, attachment of ACPPs to nanoparticles or
large polymers may be done in order to harness the enhanced
permeability- and-retention effect of such cargoes. In addition,
for example, in the case of fluorescence, crowding fluorophores
together on a polymer or nanoparticle (22, 26, 39, 40) or including
a quencher on the end of the polyanion may be used to improve
contrast by suppressing fluorescence of the uncleaved
substrate.
[0214] Far-red fluorescence was used as an imaging modality in the
examples, and offers at least the following advantages: the cyanine
dyes are stable and easy to conjugate, the imaging equipment is
relatively simple to use and cheap, and its spatial resolution
spans the full range from subcellular to whole animal. In mice,
fluorescence imaging can reach a significant fraction of the intact
animal, especially when aided by tomographic techniques (40). In
larger animals and in patients, the few-millimeters-deep
penetration may restrict the utility of fluorescence to (i) the
most superficial dermatological tumors, (ii) the retina, (iii)
tumors near the surface of a body cavity accessible by endoscopy
(41), and (iv) the margins of a surgical resection. An exemplary
use of the present methods is the real-time molecular imaging of
the margins of a resection while the patient is still on the
operating table. Such a use would be of great value to the surgeon
to decide whether any invasive carcinoma tissue remained lurking at
or just beyond the tissue just removed. Instrumentation for
infrared image-guided surgery has been described (42) and may be
useful for such methods. Contrast agents comprising compositions as
described herein may be provided to target tissue by topical
application, by intravenous infusion, or other means.
[0215] The ability of a polyanionic peptide domain to inhibit
binding and entry of a closely apposed polycationic CPP is
functionally reminiscent of intramolecular fluorescence resonance
energy transfer, in which an acceptor chromophore quenches the
fluorescence of a nearby donor fluorophore. In each case, if the
linker is cleaved and the inhibitory moiety diffuses away, the
active partner (the CPP or the donor fluorophore) is unmasked. The
unmasking of CPPs has a completely different underlying mechanism
and a much slower time scale than intramolecular fluorescence
resonance energy transfer, but offers a much broader range of
useful imaging modalities and cargoes than does fluorescence
resonance energy transfer.
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EXAMPLE 13
[0258] Further Exemplary Material Regarding Tumor Imaging via
Proteolytic Activation of Cell Penetrating Peptides
[0259] Reagents: Fmoc protected amino acids and synthesis resins
were purchased from EMD Chemicals Inc. Dimethylformamide (DMF),
piperidine, and
2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) were from Applied Biosystems.
Trifluoroacetic acid (TFA), thioanisole, triisopropylsilane,
ethanedithiol, and diisopropylethylamine (DIEA) were from
Sigma-Aldrich. 5(6)-Carboxyfluorescein succinimidyl ester and
5-iodoacetamidofluorescein were from Molecular Probes. Cy5
monoreactive NHS ester and Cy5 monomaleimide were from Amersham
Biosciences. MMP-2 proenzyme and MMP-9 were from EMD. Enterokinase
and urokinase plasminogen activator (uPA) were from Invitrogen and
Alexis, respectively. Methoxy PEG-maleimide (5 KDa, 21 KDa) was
purchased from Nektar and methoxy PEGmaleimide (11 KDa) was
supplied by SunBio PEG-SHOP, Korea. All reagents were used as
obtained without further purification.
[0260] Peptide Synthesis and Fluorophore Labeling: Peptides were
synthesized on an automatic peptide synthesizer (Pioneer Peptide
Synthesis System by Applied Biosystems) using standard protocols
for Fmoc solid phase synthesis. After the peptide was synthesized,
the resin was washed with dimethylformamide, dichloromethane, and
methanol 3 times each and vacuum dried for 3 hr. The peptides were
cleaved off the resin overnight with either
CF3COOH/thioanisole/triisopropylsilane (96/2/2, v/v) for peptides
without a sulfhydryl group, or
CF3COOH/thioanisole/triisopropylsilane/ethanedithiol (94/2/2/2,
v/v) for peptides with a sulfhydryl group. The cleavage solution
was evaporated nearly to dryness, and the crude peptide was
triturated with ether and vacuum dried for 3 hr. Fluorophores were
attached to peptides either before or after cleavage from the
resin; 5(6)-carboxyfluorescein Nhydroxysuccinimidyl ester and Cy5
monoreactive N-hydroxysuccinimidyl ester labeled amino groups,
whereas 5-iodoacetamidofluorescein and Cy5 monomaleimide reacted
with sulfhydryl groups. Finally, fluorophore labeled peptides were
purified on HPLC (C18 reverse phase column, eluted with 10-40%
acetonitrile in water with 0.1% CF3COOH) and lyophilized overnight.
The molecular weight of all peptides was confirmed by mass
spectroscopy, and the concentration of each peptide stock solution
was verified by UVvis absorbance.
[0261] Peptide disulfide bond formation and reduction: Peptides
with cysteine residues were cleaved off the resin via standard
procedures. To form a cyclic disulfide, vacuum dried crude peptide
was diluted to 1 mg/ml in 5 mM NH4HCO3 and vigorously stirred in
air for 3 hr. The crude cyclic peptide was purified on HPLC (C18
reverse phase column, eluted with 10-40% acetonitrile in water with
0.1% TFA) and lyophilized overnight. As before, the molecular
weight of each peptide was confirmed by mass spectroscopy, and the
concentration of the stock solution was verified by UV-vis
absorbance. To obtain the linear peptide, the disulfide bond was
reduced by mixing equal volumes of 100 .mu.M cyclic peptide, 10 mM
TCEP [tris(2-carboxyethyl)phosphine], and 100 mM MES
[2-mercaptoethanesulfonic acid, sodium salt] in PBS and incubating
at room temperature for 30 min. Reduction was confirmed by HPLC and
mass spectroscopy. The final concentrations of TCEP and MES in the
media during cell uptake assays were 0.5 and 5 mM respectively.
[0262] PEGylated peptide synthesis and Cy5 labeling: Peptides with
free thiol groups at the N-terminus were synthesized using a
standard Fmoc peptide synthesis protocol, except that the final
amino acid coupled to the resin was tritylmercaptoacetic acid. The
peptide was cleaved off the resin through the standard procedure
described earlier and reacted with 0.5-0.8 equivalent methoxy
PEGmaleimide in DMF and 100-fold excess 4-methymorpholine as base
at room temperature for over 12 hours. Solvent and excess base were
evaporated off under vacuum. The pegylated peptide was labeled with
Cy5 by reacting with 2-3 equivalent Cy5 mono NHS ester in 50 mM
sodium bicarbonate solution at room temperature overnight. The
crude product was purified on HPLC and then lyophilized.
[0263] ACPP Cleavage by Enterokinase: 10 .mu.l of a 0.38 mM peptide
stock solution dissolved in water was mixed with 10 .mu.l 1 U/.mu.l
enterokinase (Invitrogen) and incubated at 37.degree. C. Enzymatic
cleavage was monitored by injecting 5 .mu.l of the reaction mixture
on HPLC and observing either UVvis absorbance at 440 nm for
fluorescein labeled or 650 nm for Cy5 labeled peptide. The HPLC
chromatograms showed that cleavage by enterokinase was nearly
complete after 15 min incubation time. The new peaks were collected
and their identities were determined by mass spectroscopy. The mass
spectra indicated that the enzyme cut between lysine and alanine
residues of the enterokinase substrates as expected.
[0264] ACPP cleaved by urokinase plasminogen activator (uPA): 100
.mu.M peptide in 400 .mu.l PBS (Phosphate Buffered Saline, pH7.4)
was incubated at 37.degree. C. with 6 .mu.g uPA for over 3 hours.
The cleavage progress was monitored on HPLC. Mass spectroscopy on
HPLC fractions indicated that the cleavage was close to completion
after 3 hr and that the enzyme cleavage site was between arginine
and serine residues in the peptide as expected (1) (MS: 2688.6
found, 2688.2 calculated).
[0265] Conformational analysis by two-dimensional NMR: We have
studied the peptide using homonuclear two-dimensional NMR spectra
in order to assess structural proclivities of the native ensemble.
The NMR samples were prepared in 90% H2O, 10% D2O buffer containing
50 mM potassium phosphate, pH 6.5. Peptide concentration was 2.69
mM and spectra were recorded at 5.degree. C. NMR spectra were
collected using a Bruker DMX 500 MHz spectrometer and a Varian
UnityPlus 800 MHz spectrometer. DQF-COSY, TOCSY, and NOESY spectra
were collected using standard pulse sequences (see (2) and
references therein). All spectra were collected using the 3-9-19
pulse sequence with gradients for water suppression (3). The NOE
mixing time was 500 ms and the TOCSY mixing time was 60 ms.
[0266] Spectral processing was performed using Felix (Molecular
Simulations Inc., San Diego, Calif.). Apodizations in the t2 and t1
dimensions were with cosine squared window functions and the
solvent was deconvoluted from the spectra using the time domain
convolution method (4) with a sine bell function.
[0267] As expected, there is chemical shift degeneracy and spectral
overlap introduced by the strings of D-glu and D-arg residues
(DQF-COSY data not shown). We can classify the resonances of the
D-glu and D-arg residues by type and make sequence specific
assignments of the resonances of linker region, i.e., XPLGLAG (SEQ
ID NO: 70). In FIGS. 24 and 25, the consistency of the observed
NOEs are assessed relative to the sequential and medium-range NOEs
expected for a .beta.-turn and the long-range NOES for cross-strand
interactions.
[0268] FIG. 24A shows the H.sup..beta./H.sup..gamma./sidechain
(.delta..sub.1)-H.sup.N(.beta..sub.2) region of the NOESY spectrum,
and FIG. 24B shows the identical region for the TOCSY spectrum. The
H.sup..beta. and H.sup..gamma. shifts of the D-glu and D-arg
resonances are labeled in the TOCSY. Significant chemical shift
overlap is present among H.sup..beta. and H.sup..gamma. resonances,
but resonances by amino acid types are well resolved. In both FIGS.
24A and 24B, there is a cluster of H.sup..beta. and H.sup..gamma.
shifts at 1.7/1.78 and 1.55/1.66 respectively for D-arg and
H.sup..beta. and H.sup..gamma. shifts at 1.95/2.03 and 2.25/2.3
respectively for D-glu. In the NOESY spectrum (FIG. 24A), we see
evidence for crossstrand interactions between the string of D-arg
and the string of D-glu residues. NOE cross-peaks at 1.92, 2.03,
2.25, and 2.29 (.delta..sub.1) and 8.65 (.delta..sub.2) are
consistent with through space interactions between the H.sup..beta.
and H.sup..gamma. sidechain of one or more D-glu and the H.sup.N
backbone of one or more D-arg.
[0269] For clarity, 1D vectors drawn from selected D-arg and D-glu
backbone H.sup.N resonances (.delta..sub.2) (see dashed blue lines
in FIGS. 24A and 24B) are depicted in FIG. 24C-24E. FIG. 24C is
drawn from the NOESY spectrum at 8.65 ppm (.delta..sub.2), the
H.sup.N resonance of a D-arg, with H.sup..beta. and H.sup..gamma.
signals of both D-glu and D-arg present. FIG. 24D is drawn from the
TOCSY spectrum at the same .delta..sub.2 shift and H.sup.N
resonance, with only the H.sup..beta. and H.sup..gamma. signals of
D-arg. FIG. 24D is also drawn from the TOCSY spectrum but 8.59 ppm
(.delta..sub.2), the H.sup.N resonance of a D-glu, with only the
H.sup..beta. and H.sup..gamma. signals of D-glu.
[0270] FIG. 25 shows the H.sup.N
(.delta..sub.1)-H.sup.N(.delta..sub.2) region of the NOESY
spectrum. There are five cross-peaks labeled with sequence-specific
identification, indicating sequential H.sup.N-H.sup.N backbone
interactions among the residues of the linker region and the
neighboring D-glu and D-arg on either side. This type of
short-range NOE is consistent with turn or helical secondary
structure (5).
[0271] Potential interactions with furin: Up to now, we have relied
almost exclusively on 9 arginines, usually D-, in a row, partly for
simplicity and partly because they are amongst the most effective
uptake sequences (6, 7). However, very recently nona-D-arginine
amide has been reported to be a potent inhibitor of furin, a
well-known processing protease (8). Given the highly electrostatic
nature of the binding, it is quite likely that the intact substrate
with polyanionic domain still attached will be a much poorer
inhibitor of furin. If this prediction is verified experimentally,
then the furin inhibition may be unimportant or beneficial, because
it will be mainly in target tissue such as tumors that furin is
acutely inhibited.
[0272] Imaging of SCCA samples with ACPP's following administration
of an MMP inhibitor: Since MMP's are dependent on zinc for
activation, we used the lipid soluble, high affinity Zn.sup.2+
chelator TPEN (N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine)
(9) as a broad spectrum MMP inhibitor to preliminarily assess
whether cleavage and retention of our peptide in SCCA tumors was
MMP dependent. Fresh SCCA slices were incubated in HBSS (FIG. 26A)
or 1 .mu.M TPEN in HBSS (FIG. 26B) at room temperature for 15
minutes. Slices were then stained with 1 .mu.M cleavable peptide
alone (FIG. 26A) or 1 .mu.M cleavable peptide plus 1 .mu.M TPEN
(FIG. 26B) before being washed five times in fresh HBSS and
cryosectioned. The images shown in FIGS. 26A and 26B were taken
using a 10.times. objective, and hematoxylin/eosin staining was
used to verify tissue type.
REFERENCE LIST FOR EXAMPLE 13
[0273] 1. Ke, S. H., Coombs, G. S., Tachias, K., Corey, D. R. &
Madison, E. L. (1997) J. Biol. Chem. 272, 20456-20462. [0274] 2.
Ernst, R. R., Bodenhausen, G. & Wokaun, A. (1990) Principles of
Nuclear Magnetic Resonance in One and Two Dimensions (Oxford
University Press, Oxford). [0275] 3. Piotto, M., Saudek, V. &
Sklenar, V. (1992) J. Biomol. NMR 2, 661-665. [0276] 4. Marion, D.,
Ikura, M. & Bax, A. (2004) J. Magn. Reson. 84, 425-430. [0277]
5. Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids (John
Wiley & Sons, New York). [0278] 6. Mitchell, D. J., Kim, D. T.,
Steinman, L., Fathman, C. G. & Rothbard, J. B. (2000) J.
Peptide Res. 56, 318-325. [0279] 7. Gammon, S. T., Villalobos, V.
M., Prior, J. L., Sharma, V. & Piwnica-Worms, D. (2003)
Bioconjugate Chem. 14, 368-376. [0280] 8. Kacprzak, M. M., Peinado,
J. R., Than, M. E., Appel, J., Henrich, S., Lipkind, G., Houghten,
R. A., Bode, W. & Lindberg, I. (2004) J. Biol. Chem. 279,
36788-36794. [0281] 9. Arslan, P., Di Virgilio, F., Beltrame, M.,
Tsien, R. Y. & Pozzan, T. (1985) J. Biol. Chem. 260,
2719-2727.
EXAMPLE 14
[0282] Cleavage kinetics for MMP-2 cleavage of
H.sub.2N-e.sub.6-XPLGLAG-r.sub.9-Xc(Cy5)-CONH.sub.2 (where
X.ident.aminohexanoic acid) are illustrated in FIG. 27. As shown in
this figure, the K.sub.m for this cleavage is 534 .mu.M; the
k.sub.cat is 15.0 s.sup.-1 and the ratio k.sub.cat/K.sub.m is
28,037 M.sup.-1s.sup.-1.
EXAMPLE 15
[0283] Cleavage of a MMP-2 substrate ACPP is illustrated in FIG.
27. The kinetics of cleavage of the ACPP peptide
H.sub.2N-e.sub.6-XPLGLAG-r.sub.9-Xc(Cy5)-CONH.sub.2 (SEQ ID NO: 71)
(where (X.ident.aminohexanoic acid, also termed aminocaproic acid)
have a K.sub.m of 534 .mu.M, a k.sub.cat of 15.0 s.sup.-1 and a
k.sub.cat/K.sub.m of 28,037 M.sup.-1s.sup.-1. As shown in FIG. 28,
uptake into live, unfixed cells is dependent on enzymatic cleavage
of the ACPP peptide by MMP-2. The cleavage site is between the
first G and the second L in the sequence PLGLAG, as indicated in
the figure. The ACPP peptides may be labeled with Cy5 dye (SEQ ID
NO: 71), with fluorescein (Fluor) (e.g., H.sub.2N-eeeeee-(ahx)-PLG
LAG-rrrrrrrrr-(ahx)-c(Fluor)-CONH.sub.2 (SEQ ID NO: 72), or with
other labels. FIG. 29 shows fluorescence images from HT-1080 cells
treated with the Cy5-labeled ACPP peptide
XeeeeeeeeeXPLGLAGrrrrrrrrXk (SEQ ID NO: 52) that has been PEGylated
with an 11 kDa PEG moiety. The images shown in FIG. 29 demonstrate
increased uptake of the cleaved peptide compared to the uncleaved
peptide, and demonstrate localization of the cleaved peptide to the
nucleus of these cells as well as to cytoplasmic compartments.
EXAMPLE 16
[0284] Images taken from nude mice bearing MMP-2-positive tumors
demonstrate that cleavage leads to uptake and localization of the
ACPP peptide fragments in tumor tissue (FIG. 30). Control
"scramble" peptides lacking a MMP-2 cleavage site
(eeeeeeeeeXLALGPG-rrrrrrrrrXk(Cy5) (SEQ ID NO: 73) are not
concentrated in tumor tissue, while fluorescence from cleavable
peptides including a MMP-2 cleavage site is much higher in tumor
tissue than in other tissues and higher than in tumor tissue of
mice receiving the control peptides. Images were taken 17 min after
injection of Cy-5-labeled peptides into tail vein. Note that
bladder and salivary gland also fluoresce, and that the gut also
shows some autofluorescence. As shown in FIG. 31, the greatest Cy5
fluorescence intensity was found over tumor tissue treated with
cleavable peptides. Images shown in FIG. 32 show contrast
enhancement by the cleaved peptides compared to the uncleaved
peptides. Human HT-1080 tumors xenografted into mice are more
readily discernable in live and in histological images with cleaved
peptides than with uncleaved peptides. Similar images are shown in
FIG. 33 showing that cleaved peptides improve imaging of
spontaneous mammary tumors in MMTV-polyoma middle T, iNOS -/- mice.
An image taken 55 min after tail vein injection into a mouse having
a tumor shows significant intensity over a tumor. Histology of a
similar tumor suggests accumulation in stromal annulus rather than
tumor core. Gel images confirm that such tumors contain the cleaved
peptide. The figure also indicates that uncleaved and uncleavable
(all-D amino acid version) are not significantly taken up into
tumors. As shown in FIG. 34, a PEGylated cleavable peptide with RGD
labels tumor metastasis and labels surrounding macrophages in lymph
nodes in MMTV-polyoma middle T, iNOS -/- mice.
EXAMPLE 17
[0285] FIG. 35 shows human squamous cell carcinoma tissue resected
from a patient, including adjacent normal tongue tissue as a
control. Images from tumor tissue treated with cleavable peptide
are much clearer than are images from similar tumor tissue treated
with "scrambled" uncleavable peptides and are much clearer than
images from normal tongue tissue treated with cleavable peptide.
The tumor tissue contains much more gelatinase than normal tongue
tissue. Similar images in FIG. 36 further demonstrate the utility
of cleavable peptides for imaging of tumor tissue. Fresh tumor
tissue was sliced in 1-mm slices and incubated in cleavable or
uncleavable peptide for 15 min, washed, and frozen. Sections were
taken for fluorescence microscopy using a low-power objective, and
tissue type was verified by staining tissue. The arrow in the
picture on the left indicates a differentiated keratin pearl. As a
control, histologically normal tissue from the same patient was
treated similarly with MMP-2 cleavable peptide or scrambled
peptide. The arrow in the third figure form the left indicates
tumor cells.
EXAMPLE 18
[0286] Phage may be transported into cells by ACPP peptides. As
shown in FIG. 37, which presents a scheme for coating Filamentous
M13 phage with ACPPs, and FIG. 38, which provides further methods
and shows increased uptake of coated phage in tumor tissue, phage
particles may be coated with cleavable peptides for directed
delivery into cells upon cleavage of the inactivating portion or
the cleavable peptides. About 30-50 copies of the ACPP may be
incorporated per phage, where as indicated in the example shown,
the ACPP may be attached to pIII coat protein or other attachment
moieties. Cleavable peptides may include, for example, PLGLAG,
while the uncleavable peptides may include the scrambled peptide
LALGPG. The masked M13 phage is one that includes the ACPP
particles before cleavage. Upon enzymatic activation, the phage
becomes activated, having a positively charged exterior, and
becomes a tumor cell binding phage. M13 phage are indicated in FIG.
37 for coating and activation, and such activatable phage are shown
to be taken up by tumors in FIG. 38. Sequence-dependent phage
accumulation in xenografted tumors was demonstrated as shown in
FIG. 38. Phage was injected into the tail vein of mice bearing
xenografted tumors; after 3 hours, the organs and tumor xenograft
were removed from the mice and ground up. The ground up tissue was
added to bacteria. Bacteria were then plated for calculation of the
resulting phage titer. Phage carrying cleavable peptides were much
more readily taken up by tumors than were phage carrying
uncleavable peptides. In addition, the background labeling of liver
and kidney seems less severe with phage than with dye cargoes. It
is not essential that the charged amino acids be D-amino acids.
Thus, a selective uptake mechanism has been demonstrated and can
work on cargo as large as phage, demonstrating that large phage
libraries may be built to find optimal tumor-specific sequences.
Other phage may be used as well, including T7 phage, .lamda. phage,
P4 phage, T4 phage, MS2 phage, and others. For example, T7 phage
type 10-3b, T7 phage type 415-1b as well as Pcomb type M13 phage
may be used to provide coated, activatable phage for delivery of
phage and other cargo to target cells and tissues.
EXAMPLE 19
[0287] ACPPs may be used to deliver radioactive cargo to target
cells and tissues. An ACPP may be linked with a radioactive moiety
(either directly or indirectly, covalently or non-covalently. FIG.
39 provides a scheme for production of an MMP substrate with a
.sup.99mTc Chelator as a payload. As illustrated in FIG. 39, a
PEGylated MMP substrate peptide mPEG(11
kd)-S--CH.sub.2--CONH-ahx-e.sub.9-ahx-PLGLAG-r.sub.9-ahx-k-CONH.s-
ub.2 (SEQ ID NO: 74) may be linked with a radioactive technetium
atom for use in a cell uptake assay. Such ACPPs were produced and
tested for cargo-delivery efficacy with Jurkat cells. In the
experiments summarized in FIG. 40, 2 .mu.M peptide was incubated
either intact or following cleavage by the matrix metalloprotease
MMP-9. Spiked with 0.25 mCi Tc label, 25 nM intact or cleaved
substrate. The target Jurkat cells were incubated with the peptides
for 30 min at 37.degree. C., and then washed 3 times with HBSS
(HEPES-buffered saline solution) at room temperature (RT). 20 hours
later, cells were counted by Gamma counter. As shown in FIG. 40,
although little radioactivity was associated with the Jurkat cells
before cleavage, after cleavage of the ACPP to activate cargo
delivery, there was a 38-fold increase in radioactivity associated
with the target cells.
[0288] Further experiments demonstrating delivery of radioactive
cargo to target cells with a technetium chelating moiety is shown
in FIG. 41. Spiked (4%) with 60 .mu.Ci .sup.99mTc-labeled peptide,
the target Jurkat cells were loaded with 2 .mu.M intact or
MMP-9-cleaved peptide at 37.degree. C. for 30 min. Then, the cells
were washed with RT HBSS 3 times, and counted (Gamma counter) 20
hours later. In the experiment illustrated in FIG. 41, cleavage of
the ACPP resulted in a 57-fold increase in radioactivity associated
with the target cells. The technetium chelating moieties used in
these studies are shown in the Figures. These experiments
demonstrate in particular that .sup.99mTc can be selectively
accumulated in target cells.
EXAMPLE 20
[0289] ACPPs may be used to deliver contrast materials to target
tissues and cells, such as materials that enhance contrast for
imaging. An ACPP may be linked with a contrast-enhancing agent
directly or indirectly, covalently or non-covalently. Such imaging
may be by magnetic resonance (MRI), x-ray (e.g., computer assisted
tomography (CAT)), positron emission tomography (PET), single
photon emission computed tomography (SPECT), neutron computed
tomography (NCT), ultrasound, near infra-red (NIR) imaging, or
other imaging means or methods, as well as radiation sensitizers
(.sup.10B, .sup.157Gd) and chemotherapeutic agents (e.g.,
doxorubicin) for therapy, research, identification, of target
tissues and cells or other purposes. For example, these methods are
cascadable with other contrast mechanisms such as EPR (enhanced
permeability and retention), retention by intracellular enzymes,
fluorescence dequenching, rotational immobilization. .sup.157Gd is
particularly attractive for combined MRI and NCT. FIG. 42 provides
a scheme for production of an MMP substrate with an attached
contrast enhancing agent (in this example, gadolinium).
[0290] Gadolinium chloride (GdCl.sub.3) is used to add gadolinium
(Gd) to provide a contrast-enhancing, PEGylated ACPP
mPEG-S--CH.sub.2CONH-e.sub.9-ahx-PLGLAG-r.sub.9-K[DOTA(Gd)]-CONH.sub.2
(SEQ ID NO: 75). It will be understood that in other embodiments
the ACPP need not include a PEG moiety, and may include other
moieties as well. As shown in FIG. 43, cleavage of the ACPP
activates the uptake of MRI imaging reagent to nearly mM
concentrations. Jurkat cells were incubated with 8 .mu.M of the
intact or of the cleaved substrate at 37.degree. C. for 30 min;
then the cells were washed four times with RT HBSS. Gd
concentration was determined by ICP-MS (inductively coupled plasma
mass spectroscopy). Cleavage led to a 19-fold increase in uptake by
target cells.
EXAMPLE 21
[0291] yclic Substrate for MMPs is illustrated in FIG. 44. A cyclic
ACPP molecule can be linked to a cargo, PEGylated, or otherwise
modified as indicated in the figure. Cyclic ACPP molecules offer
the advantages of requiring cleavage at two sites to activate
cell-uptake (e.g., enzymatic cleavage by MMP as illustrated in FIG.
44). A requirement for cleavage at two sites is an advantage, since
then cleavage will be more sensitive to enzymatic concentration
than molecules with a single cleavage site. Such a requirement is
useful to improve contrast. FIGS. 45 and 46 illustrate exemplary
synthetic schemes for producing cyclic ACPPs. Note that these
molecules may be PEGylated.
[0292] As indicated in FIG. 47, a further advantage of cyclic ACPPs
is that they may be self-quenching. As shown in FIG. 47,
fluorescence from the cyclic ACPP shown in the example increases
upon enzymatic cleavage. In vivo experiments demonstrate uptake,
accumulation, cleavage and excretion of such peptides by liver and
kidney. Thus, such molecules may be administered in vivo, and are
cleaved in vivo.
[0293] FIG. 48 further illustrates ACPPs that require enzymatic
action at multiple sites, including cyclic ACPPs and branched
ACPPs. Where both of the cleavage sites are substrates for a single
enzyme, the uptake should be proportional to the square of the
protease concentration. However, with multiple enzymatic sites, an
ACPP molecule may include cleavage sites that are substrates for
more than one enzyme. Where an ACPP has two cleavage sites, each
being a substrate of different enzymes, the uptake should be
proportional to the product of the concentration of the first
enzyme times the concentration of the second enzyme. ACPPs may
include more than two cleavage sites, with uptake kinetics roughly
following the number of cleavage sites (e.g., with three identical
cleavage sites, the uptake should be approximately proportional to
the cube of the enzyme concentration).
[0294] Thus, ACPPs may be linear, cyclic, branched or have other or
mixed geometries. They may be used to deliver fluorescent,
radioactive, or other labels, may deliver contrast agents,
therapeutic agents, or multiple agents. Linkers can be cleaved by
proteases, by reduction of disulfide bond, or by acidic or other
conditions. Suitable enzymes and exemplary targets related to these
enzymes include matrix metalloproteinases (for, e.g., cancer,
stroke, and other conditions); Urokinase plasminogen activator
(uPA) (for, e.g., cancer and other conditions); Prostate-specific
antigen (for, e.g., cancer and other conditions); Thrombin and
clotting cascade (for, e.g., thrombosis and other blood-related
conditions); Reduction by leaked thiols under hypoxic conditions
(for, e.g., cancer, infarcts and other conditions that lead to, or
are caused by, hypoxic conditions); phosphatases (for, e.g.,
osteoporosis or other conditions); calpains (for, e.g., necrotic
cells or other conditions); light (for, e.g., refining the
specificity of photodynamic therapy and other uses).
[0295] Delivery of cargo by ACPPs provides advantages of other
transport or therapeutic systems. Targeting is accomplished without
the need for antibodies or antigens. ACPP targeting offers
enzymatic amplification and transport of cargo into the nucleus of
target cells. The ACPP peptides are relatively easy to synthesize
and to vary combinatorially, so their ease of production offers
advantages. Protease activities are mechanistically important and
interesting, and the exploitation of such activities offers
advantages including that the cleavage routes are well-studied and
will continue to be studied and characterized. Membrane-bound
proteases may be used to cleave ACPPs and to give higher contrast
than soluble secreted proteases. The cleavable ACPPs are active in
vivo. Some labeling of local bystander cells is expected and
desirable. The cleavable ACPPs disclosed herein provide an
extracellular analog of fluorescence resonance emission transfer
(FRET) at least in that the anionic portions of ACPPs may serve to
neutralize the cationic portions before cleavage. The ACPPs are
applicable to all imaging modalities, including fluorescence and
other imaging modalities.
EXAMPLE 22
[0296] Imaging Using ACPP Labeled with Technetium-99m
[0297] The use of ACPP labeled with technetium-99m (.sup.99mTc) as
described in Example 19 can be extended for use as an imaging agent
for mouse models of cancer. One obvious advantage of gamma
scintigraphy as compared to fluorescent imaging is that gamma
imaging reaches much deeper into tissue. As described above,
various carrier-conjugated ACPPs that incorporate
N-epsilon-bis(2-pyridylmethyl)lysine (DPK), a high-affinity
chelator for technetium tricarbonyl, Tc(CO).sub.3, have been
prepared. DPK appears superior to previous Tc chelators because it
can be site-specifically incorporated into the peptide chain during
solid-phase synthesis, and the resulting chelate with Tc(CO).sub.3
is extremely stable and has no exposed negative charges. The
simplest of these compounds, (11
KDa-mPEG)-e.sub.9-XPLGLAG-r.sub.9-[DPK-.sup.99mTc(CO).sub.3] was
first tested on isolated cultured cells. (Note: X denotes a
6-aminohexanoyl spacer, and lower case letters denote D-amino
acids.) Before cleavage, the uptake of this CPP into Jurkat
lymphocytes was only 1.9% of the positive control CPP,
r9-[DPK-.sup.99mTc(CO).sub.3]. After treatment of the peptide with
MMP-9 to cleave between PLG and LAG in the sequence, uptake of
radioactivity increased 57-fold to 107% of the positive
control.
[0298] Based on these results observed in tissue culture cells,
this peptide and analogous ACPPs were injected intravenously into
nude mice bearing subcutaneous xenografts of HT1080 human
fibrosarcoma cells. Serial scans were performed using the Biospace
small animal planar scintigraphic imager. The % ID/g (% injected
dose retained per gram of tissue) was measured by scintillation
counting of tissues harvested post-mortem. The inventors have
tested the above ACPPs and three other .sup.99mTc-labeled ACPPs:
(70
KDa-dextran)-e.sub.9-XPLGLAG-r.sub.9-[DPK-.sup.99mTc(CO).sub.3],
(murine serum
albumin)-e.sub.9-XPLGLAG-r.sub.9-[DPK-.sup.99mTc(CO).sub.3], and
(PAMAM generation 5
dendrimer)-e.sub.9-XPLGLAX-r.sub.9-[DPK-.sup.99mTc(CO).sub.3]. Thus
far, radioactivity has been observed to accumulate to a much
greater extent in the liver, kidney, and spleen than at the tumor
site. The tumor margins, however, appear relatively well-defined at
earlier time points (see FIG. 48). The albumin-conjugated ACPP
showed the most potential, in that tumor uptake was 1-3% ID/g
compared to liver uptake of 12-18% ID/g (see FIG. 50).
[0299] Because the ratio of tumor to liver uptake was at variance
with the results obtained with fluorescent and magnetic labels and
because of the limited literature on the use of DPK, the inventors
tested whether the DPK chelator could be responsible for the
affinity for normal tissues. Thus, the inventors synthesized and
tested (70 KDa dextran)-e.sub.9-XPLGLAX-r.sub.9-(DOTA-.sup.111In).
However, this compound showed a biodistribution pattern favoring
the liver, spleen, and kidneys, similar to (70 KDa
dextran)-e.sub.9-XPLGLAG-r.sub.9-[DPK-.sup.99mTc(CO).sub.3],
suggesting that DPK-Tc(CO).sub.3 was not the cause of the
distribution observed.
EXAMPLE 23
[0300] Development of MRI Contrast Agents Containing Gd.sup.3+
Cargoes.
[0301] Three ACPPs carrying Gd.sup.3+ were synthesized:
(11-KDa-mPEG)-e.sub.9-XPLGLAG-r9-K(DOTA-Gd), Suc.sub.9-(70 KDa
dextran)-e.sub.9-XPLGLAG-r.sub.9-K(DOTA-Gd), and Suc.sub.9-(70 KDa
dextran)-e.sub.9-XPLGLAX-r.sub.9-K(DOTA-Gd). The first of these
ACCPs was tested at 8 .mu.M on isolated Jurkat lymphocytes.
Gd.sup.3+ uptake was measured by inductively coupled plasma mass
spectroscopy. The intact peptide accumulated to 39 .mu.M, the
MMP-9-cleaved peptide to 737 .mu.M, whereas a positive control CPP,
r9-K(DOTA-Gd), reached 504 .mu.M. Thus, MMP-mediated cleavage of
the test peptide increased uptake by a factor of 19-fold over the
positive control. The large absolute uptake values seen in these
experiments suggest that Gd.sup.3+ concentrations adequate for MRI
and neutron capture therapy should be obtainable.
[0302] In vivo MRI imaging on animals was obtained using a 7 Tesla
small animal imager. Animals are anesthetized with circa 1.5%
isoflurane and undergo a series of scans totaling about 2 hours in
length. The first scan is a twenty minute anatomical T1-weighted
scan. The second is a fat saturated sequence, which is also T1
weighted. A third set of scans is employed to determine tissue T1's
and totals around one hour. Finally, a quick three minute T2
weighted scan is done to confirm that the tumors are solid masses.
Animals are monitored for normal breathing throughout the course of
the scanning sequence.
[0303] Following the prescan, animals were anesthetized with circa
1.5% isoflurane and were then injected with approximately 0.2 ml
Gd.sup.3+-loaded ACPP (Suc.sub.9-(70 KDa
dextran)-e.sub.9-XPLGLAG-r.sub.9-K(DOTA-Gd)) in the experiment
shown) through the tail vein. An identical series of scans was run
at 2 h, 6 h, 12 h, 24 h and 48 h. Animals were allowed to wake up
between scanning sessions. In some cases, small amounts of blood
were collected from the tail vein at the same time points.
Following the 48 h scan, animals were sacrificed and either
dissected or perfused with buffered paraformaldehyde. High
resolution anatomical scans and paraffin sections were done on
fixed animals. Nonfixed animals were dissected and organs were
subjected to quantitative gadolinium analysis. Time courses and T1
sequences were analyzed volumetrically using Amira software.
[0304] FIG. 51 shows remarkable positive contrast from lymph nodes
(marked by red arrows) draining the mammary tumors. The axillary
and inguinal lymph nodes were subsequently verified to be positive
for tumor by frozen section. The salivary lymph nodes were not
tested, but their enlarged size makes it likely that they too are
positive for metastasis. The lymph node signal decreased slightly
at 48 h, with a corresponding increase in splenic intensity,
possibly due to recycling of macrophages containing concentrated
peptide. Signals from the primary tumors and the liver (blue arrow)
were more modestly elevated. Bright signals from the gall bladder
and intestine signal (not shown) indicate that the peptide is
cleared via hepatobiliary excretion, similar to fluorescent and
Tc-labeled peptides attached to large carriers. The large bright
spots in the lower right hand side of some images were present
prior to the addition of contrast and most likely reflect stomach
contents (magenta arrow). The bright spots in the lower abdomen are
most likely due to contrast from bile mixing with intestinal
contents. The large objects on both sides of the animal are water
phantoms used to normalize the images.
[0305] While the prime focus of the experiments described above has
been on the use of Gd.sup.3+-loaded ACPPs for MRI, these methods
can be extended allowing for the use of carboranes or .sup.157Gd as
neutron sensitizers. Accordingly, the inventors have examined
peptide uptake of ACPP in Jurkat cells using .sup.157Gd-enriched
material. One of skill in the art would recognize that once in vivo
uptake into tumor-related tissues has been optimized by MRI
monitoring, the same conditions can be tested for neutron capture
therapy. To generate an additional reagent for neutron
sensitization, .sup.10B-enriched sulfhydryl borane
Na.sub.2.sup.10B.sub.12H.sub.11SH can be attaching to ACPPs via a
thiol group.
EXAMPLE 24
[0306] Optimization of Tumor:Background Contrast Ratios
[0307] The significant uptake of ACPPs into liver and kidney
suggested to the inventors that non-MMP proteases in those tissues
might also be cleaving the ACPP substrates. Thus, the inventors
tested the susceptibility of a substrate containing the sequence
PLGLAG to meprin, a metalloprotease distantly related to MMPs and
highly expressed in the kidney. Cy5-labeled substrates were treated
with meprin (gift of Prof. Judith Bond, Penn State). The
proteolytic fragments were analyzed by LC-MS, which showed that
cleavage occurs after the P, whereas MMP-2 and -9 cut between G and
L. This difference in cleavage site should facilitate redesign of
the peptide sequence to retain MMP sensitivity while decreasing
meprin sensitivity by, for example, removing the non-tumor cell
cleavage site while retaining sequences susceptible to proteases
enriched in tumor cells.
[0308] Electrophoresis in tricine gradient gels is currently our
best technique for distinguishing cleaved from uncleaved peptides
recovered from tissues. This assay shows that at 2 hr after
injection, the tumor contains a mixture of cleaved and uncleaved
peptide. By 6 hr after injection, all peptide remaining in tumor,
liver, and kidney has been cleaved. Peptides in other normal
tissues such as skin, pancreas, muscle, or lung are below the
detection limit of these gels.
[0309] Mass spectrometry would be advantageous because it could
determine the cleavage site and because it should not be limited to
fluorescently-labeled peptides. The challenge is to ionize the
highly charged peptides from the complex tissue matrix into the
vacuum. Attempts were made to mount thin slices of tissue on the
sample plate of a matrix-assisted laser desorption ionization
(MALDI) mass spectrometer. Very weak signals were detected, which
were too large in mass to be related to the injected ACPP peptide.
Spiking the mounted samples with a large excess of cleaved peptide
showed that the limit of detection was much too high to permit
analysis of tissue samples in this manner.
[0310] Alternatively, disruption and lysis of tissue cells releases
a complicated background matrix of compounds which appear to
interact strongly with the polyarginine part of the peptide. The
resulting signal suppression requires that sample preparation
methods be developed to purify the peptide from the cell extraction
by-products. Weak cation exchange (WCX), using a stationary phase
consisting of polyaspartic acids, has been found to be effective
for accomplishing this purification. The peptide can be loaded and
extracted by WCX using volatile solvents, which are compatible with
subsequent reverse phase separation and mass spectrometry. Further
studies are in progress to improve the limit of detection and
ensure that the purified peptide is representative of the make-up
of the original tissue sample.]
EXAMPLE 25
[0311] Cyclic Peptides
[0312] To generate cyclic peptides, the inventors synthesized
cyclic[succinoyl-PLGLAG-c(11
KDa-mPEG)-e.sub.9-XPLGLAG-r.sub.9-K]-k(Cy5), which contains two
cleavage sites. This cyclic ACCP was tested in xenografted mice.
Compared to a linear peptide with a single cleavage site, the
inventors found that signal from the tumor decreased by a factor of
2, while signal from the kidney remained approximately the same and
signal from the liver increased by a factor of almost 10. This
result was not unexpected, given that the liver is considerably
faster than the tumor at cleaving a single PLGLAG on an 11 KDa
support. Thus, the cyclic substrate requiring two cleavages should
favor the liver to an even greater extent. One of skill in the art
would recognize that once peptide sequences (preferably two
distinct ones) were obtained that individually are preferentially
cleaved in the tumor over the liver, then the two-cut ACPPs would
become advantageous for the practice of this invention.
EXAMPLE 26
[0313] ACPPs with Self-Quenching Fluorophores or FRET Pairs
[0314] ACPPs with self-quenching fluorophores
[Cy5-X-e.sub.6-XPLGLAG-r.sub.9-Xk(Cy5)] or FRET pairs
[Cy7-X-e.sub.6-XPLGLAG-r.sub.9-Xk(Cy5)] were synthesized. The first
substrate increased fluorescence intensity 3.9 fold upon cleavage
by MMP-9, whereas the FRET substrate demonstrated a 6.6-fold
increase in ratio of 650 to 770 nm emissions. The inventors have
found these two substrates to be poorly soluble in physiological
media, even though the inventors decreased the number of glutamate
residues from nine to six so that the molecules would carry more
net charge. One of skill in the art would recognize based on these
results the advantage of synthesizing more water soluble analogues
of these ACPPs.
EXAMPLE 27
[0315] Increasing the Molecular Size of Carriers
[0316] The inventors have discovered that increasing the size of
the macromolecular carrier from 11 KDa mPEG to 70 KDa dextran, 60
KDa serum albumin, or a 35 KDa PAMAM dendrimer (generation 5)
results in substantially improved optical contrast in transgenic
MMTV-PyMT mice (FIG. 52). Without being bound by any particular
theory or mechanism, the inventors believe that this effect is
achieved by decreasing glomerular filtration of the substrate and
reducing its exposure to proteases such as meprin in the proximal
tubules of the kidney. Also increased half life in the circulation
allows more time for the tumor enzymes to cleave a significant
fraction of the ACPP. One of skill in the art would recognize that
the use of larger macromolecular carriers would require that more
time after injection must elapse (24-48 hr) before the uncleaved
ACPP is eliminated and maximal contrast is obtained. The inventors
have found that liver uptake remains relatively constant provided
that the polymeric support carries a sufficient overall negative
charge. Without being bound by any particular theory, the inventors
believe that enhanced permeability and retention (EPR), while
contributing, is not entirely responsible for the improved
contrast. The inventors have found that control ACPPs with poorly
cleavable plglag instead of PLGLAG showed very little contrast,
although both peptides should have the same EPR effect.
Furthermore, the inventors have found that increasing the size of
the dextran to 250 KDa largely destroyed tumor contrast even though
the larger polymer should possess an even greater EPR effect.
EXAMPLE 28
[0317] Use of Fluorescent ACPPs to Detect Lymph Nodes Containing
Metastatic Tumor Cells
[0318] As shown in FIG. 53, the fluorescent ACPPs also show
prominent lymph node uptake similar to the MR images described
above. However, the lymph nodes are too small and deeply buried to
be visible from outside the mice. Instead, the lymph nodes become
apparent when the body cavity is opened. Not all lymph nodes light
up, as shown by the periaortic cluster in the far right panel of
this figure. The inventors believe that these are normal nodes not
yet containing metastases. Again, much less contrast is observed
with the control peptide containing plglag instead of PLGLAG. One
of skill in the art would recognize that if such optical contrast
could reliably indicate the presence of metastatic tumor cells in
lymph nodes, it could be a valuable guide during surgery.
EXAMPLE 29
[0319] Combinatorial Approaches to Screen Large Libraries of
Synthetic or Phage-Displayed Peptides for Peptide Sequences that
Maximize Accumulation in Tumors Relative to Normal Tissue
[0320] In-vitro and in-vivo selection strategies have been used in
combination with combinatorial phage display libraries to identify
ACPPs that are specifically taken up in tumor cells. Initial
experiments were done by linking control ACPP peptides to either
the N-terminus of the pIII coat protein of M13 phage, or the
C-terminus of the 10B capsid protein of T7 phage (T7select 10-3,
Novagen). The libraries were either
H.sub.6-E.sub.9-Z.sub.6-R.sub.9-(M13 phage) or (T7
phage)-R.sub.9-Z.sub.6-E.sub.9-H.sub.6 (Z denotes a random amino
acid to generate the library of cleavage sites). Thus cleavage
releases the poly-glutamate and hexahistidine domains whether they
located at the N-terminus (M13) or C-terminus (T7). Controls for
both T7 and M13 phage contain either: a His-tag only, or MMP
cleavable PLGLAG or MMP-resistant LALGPG in place of Z.sub.6. Phage
with PLGLAG accumulated in HT1080 tumor xenografts to about 4-fold
higher titer than those with LALGPG, whereas uptake into liver and
kidney was lower and not much different for the two sequences
(FIGS. 37 and 52). This preliminary result suggested that
reasonable contrast was obtainable despite the use of L-amino acids
for the polyglutamate and polyarginine domains. For the T7 phage, a
small test library (10.sup.4 diversity) was generated, while the
M13 phage library had a diversity to 2.5.times.10.sup.6 unique
sequences.
[0321] In-vivo selections were performed by injection of phage
libraries into mice either containing tumor xenografts (HT1080
cells) or mammary tumors in transgenic mice (MMTV-PyMT). Tumor
tissues were removed 3-24 hrs after injection. The rescued phage
were re-amplified in bacteria and reinjected into another mouse for
up to 7 iterative cycles of selection. Representative phage were
isolated at each round and sequenced to monitor the selection
process. Sequences that were isolated multiple times were noted for
further analysis.
[0322] In-vitro selection strategies involved screening for phage
that are specifically cleaved by purified enzymes or tissue
extracts associated with tumor tissue while also selecting against
cleavage by enzymes and extracts associated with normal tissue. The
benefit of this type of selection strategy is that it does not
require that the cleaved phage be bound and retained by the tumor
as required in the in-vivo selection. The inventors have employed
two different in-vitro selection paradigms. In the initial
selection, the 2.5.times.10.sup.6 diversity M13 library was treated
with the kidney proteases meprin and neprilysin for 3 hours. Ni-NTA
agarose (Qiagen) was then added and allowed to bind to the phage
for 5-15 minutes, then the Ni-NTA agarose carrying the phage was
resuspended with MMP-9 and incubated at 37 for 2-3 hours. After
removing the beads by centrifugation, phage released into the
supernatant were plated with fresh bacteria. This procedure selects
for phage that remain uncleaved by kidney proteases but are then
released by MMP-9. This procedure was repeated for as many as six
rounds with representative phage being isolated and sequenced to
monitor the selection process. The same selection procedure was
also performed with liver/kidney extract instead of
meprin/neprilysin and tumor extract instead of MMP-9. The resulting
sequences are tabulated below. The most promising peptides were
chemically resynthesized with flanking e.sub.9 and r.sub.9
sequences and a C-terminal fluorescein tag. They were then assayed
by tricine gel electrophoresis for cleavage by tumor, liver, kidney
extracts and MMP-9. TABLE-US-00004 # times # times # times Total #
identified identified in identified in Initial crude
e.sub.9-Z.sub.6-r.sub.9 Peptide times in in-vivo Nep/Mep/MMP-9
liver/kidney/tumor peptide cleavage by Seq. (X.sub.6) isolated
screen screen screen Tumor/Kidney/Liver/MMP-9 RLQLKL 12 1 11
T+/K+/L+/M- GLWQGP 7 7 QCTGRF 6 1 1 4 Dimer? LPGMMG 5 5 DVGTTE 5 5
No Cleavage TDLGAM 5 5 No Cleavage GMMYRS 4 1 3 T+/K+/L+/M+/-
DSNAES 4 3 1 No Cleavage ITDMAA 4 1 3 RWRTNF 4 4 T+/K+/L+/M- WRPCES
3 1 2 WRNTIA 3 3 IDKQLE 3 3 FMEIET 3 3 HEVVAG 2 2 TSAVRT 2 2 GGHTRQ
2 2 INGKVT 2 2 ARKRSQ 2 2 Note: Since synthetic peptides have
D-amino acids flanking the Z.sub.6 site, their cleavage may not
match phage data.
[0323] The inventors note that no peptide has been confirmed with
the desired T+/K-/L- profile. One of skill in the art would
recognize that among the possible ways to improve the in vivo assay
would be to use mutant phage (developed by E. Ruoslahti's lab)
whose nonspecific filtration by the reticuloendothelial system has
been reduced. The increased half-life in the circulation should
decrease the background of nonspecific adsorption and allow more
time for protease-based discrimination. Also the phage recovered
from the tumor could be adsorbed with Ni-NTA agarose to remove
uncleaved phage.
EXAMPLE 30
[0324] Additional Models for Testing ACPP
[0325] The inventors have found the transgenic MMTV-PyMT breast
cancer model to be an advantageous murine model for several
reasons: (1) it is a more realistic tumor model for breast cancer
than xenografts, (2) it allows examination of interactions between
the tumor and the stroma, (3) the animals are immunocompetent,
allowing one to study uptake in lymph nodes for the large carriers,
(4) a single mouse may carry several tumors at different stages,
allowing an investigator to determine the efficacy of test peptides
in a model more relevant to the progression of cancer in patients.
The pattern of staining in the tumors was observed to begin as a
smooth stain in the stroma. This finding is consistent with
quantitative PCR analysis done by Pedersen et al (2005) who
microdissected tumors from polyoma mice and found that tumor stroma
is enriched in MMP's relative to tumor epithelia. Over a period of
hours, the smooth stain becomes punctate as the label is taken up
in dendritic cells or macrophages near blood vessels and the edges
of tumors. The identity of these phagocytosing cells has been
confirmed by antibody stains using F4/80 as well as by
hematoxylin/eosin staining. Interestingly, the lymph nodes are
quite fluorescent at long time points. It is currently unknown
whether this is due to tumor associated macrophages that have
phagocytosed fluorescent tumor debris and have then traveled to
lymph nodes, or whether this is due to activated macrophages in
reactive lymph nodes taking up dye as it travels through the blood.
The inventors have found that all detectable peptide in the tumors
at these late time points (24 and 48 hours) is cleaved. Other
transgenic mouse models such as MMTV-neu may be used in the
practice of this invention.
[0326] 3D tissue cultures in matrigel (FIG. 53) may also be used in
the practice of this invention. Such 3D cultures are more realistic
models than isolated tumor cells, and offer higher-resolution
analysis than is possible in live animals. A limitation associated
with isolated cells is that they do not accumulate enough
extracellular MMPs to cleave ACPPs, so that exogenous pre-activated
MMPs must be supplied. In contrast, the 3D cultures take up ACPPs
by a cleavage-dependent mechanism without the help of exogenous
enzyme (FIG. 55). The use of 3D gels allows the uptake and
subcellular distribution in individual cells to be resolved in 3D
culture. Such resolution is not currently available using
histological sections from animals. Cell lines that may be of use
in the practice of this aspect of the invention include: MDA-MB-231
and MCF-7 human mammary adenocarcinomas and HT1080 human
fibrosarcoma cell lines. Primary organoids from PyMT transgenic
mice and other less invasive cell lines may also be used in the
practice of this invention.
[0327] For these studies, tricine gels, fluorescence imaging, and
flow cytometry protocols were used to quantify cleavage and uptake
of ACPPs into the 3D cultures. Fluorescence imaging allowed
detection of total fluorescence of surface and intracellular
peptide, whereas flow cytometry measured fluorescence remaining
inside cells after clusters have been digested into single cells
and stripped of surface binding via trypsinization. In MDA-MB-231
cells after 24 hr incubation, ACPPs with PLGLAG cleavage sequences
show up to 5 fold greater uptake than controls with D-amino acids,
i.e., plglag. In addition, the inventors were able to inhibit
uptake of the PLGLAG-containing ACPP roughly 50% with 0.1 mM
GM6001, a broad spectrum MMP inhibitor. A cocktail of inhibitors of
cysteine proteases, serine proteases, aspartic proteases, and
cathepsin D was found to not inhibit substrate cleavage/activation.
HT1080 cells gave comparable results, whereas MCF-7 cells showed no
detectable uptake relative to the D-amino acid control, correlating
with their relative lack of invasiveness. Other cells, such as less
invasive and/or normal cell lines and MMP2/MMP9 double knockout
PyMT cells, can be used in the practice of this invention. The 3D
model disclosed in this invention can be used as a screening assay
for protease specificity as new protease substrates are
synthesized. In addition, this 3D culture model will find use as a
model to assess delivery and uptake of ACPPs carrying therapeutic
cargoes such as cisplatin or doxorubicin.
EXAMPLE 31
[0328] Testing for Potential Side Effects Associated with ACPP
[0329] Test for Immunogenicity of ACPP
[0330] The immunogenicity of a typical ACPP, 11 KDa
mPEG-e.sub.9-PLGLAG-r.sub.9 was tested in comparison with
e.sub.9-PLGLAG-r.sub.9 conjugated to keyhole limpet hemocyanin, a
strongly immunogenic carrier serving as a positive control. The KLH
and mPEG conjugated antigens were suspended in buffer (1 mg/ml) and
emulsified by mixing with an equal volume of Complete Freund's
Adjuvant, for a total volume of 1.0 ml. This sample was then
injected into three to four subcutaneous dorsal sites for the
primary immunization. Subsequent immunizations were performed using
Incomplete Freund's Adjuvant at a dose of 0.5 mg/rabbit. Rabbits
were injected at day 1, 7, 28 and 42. Blood was collected through
the auricular artery from each rabbit on day 21, 35, 49 and 56. The
blood was allowed to clot and serum was collected by
centrifugation. The serum was stored at -20.degree. C. until all
tests were done together after day 56. The anti-peptide antibody
titer was determined with an enzyme linked immunosorbent assay
(ELISA) with (5 .mu.g/well) of BSA-conjugated
e.sub.9-PLGLAG-r.sub.9 as antigen. Results in the Table are
expressed by the reciprocal of the serum dilution that results in
an OD at 492 nm of 0.1 (detection with HRP-anti rabbit IgG
conjugate and OPD). TABLE-US-00005 Rabbit Pre-Bleed Day 21 Bleed
Day 35 Bleed Day 49 Bleed Day 56 Bleed # titer ml Titer ml Titer ml
titer ml titer mPEG <1:200 24.0 <1:200 24.0 <1:200 24.0
<1:200 23.0 <1:200 conjugate-1 mPEG <1:200 20.0 <1:200
23.0 <1:200 23.0 <1:200 21.0 <1:200 conjugate-2 KLH-
<1:200 29.0 1:300 25.0 1:600 27.0 1:1000 24.0 1:800 conjugate-1
KLH- <1:200 24.0 1:1300 25.0 1:4800 25.0 1:10000 24.0 1:11300
conjuate-2
[0331] For the mPEG-conjugated peptide, immune responses were
undetectable, i.e., indistinguishable from the pre-bleed negative
control up to the last sampling at 56 days. In contrast, the
KLH-conjugated peptide generated medium to strong antibody
responses in both rabbits. These results demonstrate that the
peptide itself does not seem to be immunogenic when presented on an
inert carrier. One of skill in the art, of course, would recognize
that testing in humans of these and newly developed ACPPs will be
required if human use is contemplated because antigenicity can vary
between species and individuals.
Test for Furin Inhibition
[0332] The furin assay was performed with Boc-Arg-Val-Arg-Arg-MCA
substrate (Bachem) at a final concentration of 200 .mu.M in 100
.mu.l of 100 mM MES/NaOH, pH 7.0, containing 1 mM CaCl.sub.2.
Inhibitory peptides were pre-incubated with 1 unit furin enzyme
(NEB) for 30 min prior to addition of substrate. Assays were
performed in triplicate by incubation at 37.degree. C. for 4 hours
in a 96-well clear-bottom black-wall Costar plate. Release of MCA
was measured by excitation at 380 nm and emission at 460 nm. IC50's
were determined as the concentration of inhibitor required to
inhibit 50% of the emission increase at 460 nm over the no-enzyme
control. A control inhibitor, decanoyl-RVKR-chloromethylketone
(Calbiochem) gave an IC50 of 27 nM, consistent with published
values, thus validating the assay. The experimental IC50s were:
TABLE-US-00006 Structure IC50 (.mu.M) Ac-r.sub.9-k-NH.sub.2 3.4
mPEG(11kd)-e.sub.9-XPLGLAG-r.sub.9-Xk-NH.sub.2 >50
e.sub.9-XPLGLAG-r.sub.9-Xk-NH.sub.2 uncleaved 27
e.sub.9-XPLGLAG-r.sub.9-Xk-NH.sub.2 cleaved (2.5 hours with MMP-9)
42
Thus, furin inhibition is undetectable for the ACPP with a
macromolecular carrier and very weak for ACPPs of simple structure
before or after cleavage.
EXAMPLE 32
[0333] TABLE-US-00007 Additional ACPP molecules Additional ACPP
molecules are provided below: Macro- Poly- Poly- C- Code Cap
molecule anion P4 P3 P2 P1 P1' P2' P3' . . . Pn' cation Cargo
terminal 1810 Suc9 Dextran e9 X P L G L A G r9 K[DOTA(Gd)] NH2 70
KDa 1809A Suc -- e9 X P L G C(Me) A X r9 DPK NH2 1809B Suc -- e9 X
P ThienylAla G C(Me) A X r9 DPK NH2 1809C Suc -- e9 X P F(4-Cl) G
C(Me) A X r9 DPK NH2 1804 = Suc -- e8 X P L G L A G r9 c[Cy5] NH2
1559 1778a Suc -- e8 X P F(4-Cl) G C(Me) M X r9 c[Cy5] NH2 1778b
Suc -- e8 X P F(4-Cl) G C(Me) Y X r9 c[Cy5] NH2 1778c Suc -- e8 X P
F(4-Cl) G C(Me) R X r9 c[Cy5] NH2 1778d Suc -- e8 X P F(4-Cl) G
C(Me) PhGly X r9 c[Cy5] NH2 1778e Suc -- e8 X P F(4-Cl) G C(Me)
C(Me) X r9 c[Cy5] NH2 1766 Albumin e9 X P L G L A X r9 DPK NH2
1763A Suc e8 X P C(Me) G C(Me) A X r9 c[Cy5] NH2 1763B Suc e8 X P
ThienylAla G C(Me) A X r9 c[Cy5] NH2 1763C Suc e8 X P F(4-Cl) G
C(Me) A X r9 c[Cy5] NH2 1763D Suc e8 X P K(Dnp) G C(Me) A X r9
c[Cy5] NH2 1762 Albumin e9 X P L G L A X r9 DPK NH2 1754A Suc -- e8
X P L G C(Me) M X r9 c[Cy5] NH2 1754B Suc -- e8 X P L G C(Me) Y X
r9 c[Cy5] NH2 1748 Suc127 PAMAM-Gen5 e9 X P L G L A X r9 DPK NH2
1747 Suc -- e8 X P L G C(Me) A X r9 c[Cy5] NH2 1746 Suc9 Dextran e9
X P L G L A G r9 K[DOTA(Gd)] NH2 70 KDa 1743 Suc127 PAMAM-Gen5 e9 X
P L G L A X r9 k[Cy5] NH2 1734 -- -- -- -- -- -- -- -- -- -- r9
Xc[Cy5] NH2 1727 Ac127 PAMAM-Gen5 e9 X P L G L A X r9 k{Cy5] NH2
1726 Suc -- e8 X P L G L F(4-NO2) A Xr9 k[Cy5] NH2 1725 Suc127
PAMAM-Gen5 e9 X P L G L A X r9 k[Cy5] NH2 1712 Suc63 PAMAM-Gen4 e9
X P L G L A X r9 k[Cy5] NH2 1711 Albumin e9 X P L G L A G r9 DPK
NH2 1709 Suc136 Dextran e9 X P L G L A X r9 k[Cy5] NH2 86 KDa 1707
= Suc e8 X P L G L A X r9 k[Cy5] NH2 1589 1702 = Suc9 Dextran e9 X
P L G L A X r9 K[DOTA(Gd)] NH2 1666 70 KDa 1666 Suc9 Dextran e9 X P
L G L A X r9 K[DOTA(Gd)] NH2 70 KDa 1664 = Suc9 Dextran e9 X P L G
L A G r9 k[Cy5] NH2 1608 70 KDa 1652 Suc9 Dextran e9 X P L G L A G
r9 DPK NH2 70 KDa 1647 Suc -- e8 X p I g l a g r9 k[Cy5] NH2 1641
Albumin e9 X p I g l a g r9 k[Cy5] NH2 1640 Suc9 Dextran e9 X p l g
I a g r9 k[Cy5] NH2 1630 Suc97 Dextran e9 X P L G L A G r9 k[Cy5]
NH2 500 KDa 1613 Albumin e9 X P L G L A G r9 k[Cy5] NH2 1608 = Suc9
Dextran e9 X P L G L A G r9 k[Cy5] NH2 1584 70 KDa 1603 Albumin e9
X P L G L A G r9 k[Cy5] NH2 1589 Suc e8 X P L G L A X r9 k[Cy5] NH2
1584 = Suc9 Dextran e9 X P L G L A G r9 k[Cy5] NH2 1568 70 KDa 1581
= Albumin e9 X P L G L A G r9 k[Cy5] NH2 1576 1576 = Albumin e9 X P
L G L A G r9 k[Cy5] NH2 1571 1571 Albumin e9 X P L G L A G r9
k[Cy5] NH2 1568 Suc9 Dextran e9 X P L G L A G r9 k{Cy5] NH2 70 KDa
1562 Suc nonconj. e8 X P L G L A G r9X k[Cy5] NH2 Albumin 1559 Suc
-- e8 X P L G L A G r9X k[Cy5] NH2 1554 Albumin e9 X P L G L A G r9
k{Cy5] NH2 1548 mPEG 5 KDa e9 x p I g I a g r9X k{Cy5] NH2 1531
mPEG 11 KDa e9 X P L G L A G r9 K[DOTA(Gd)] NH2 1530 mPEG 11 KDa
e10 X P L G F(4-NO2) A Q Xr9 k{Cy5] NH2 1506 mPEG 11 KDa e10 X P L
G C(Me) W A Qr9 k{Cy5] NH2 1503 mPEG 11 KDa e9 X P L G C(Me) W A
Qr9 k{Cy5] NH2
[0334]
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 108 <210>
SEQ ID NO 1 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence:linker X
cleavable by matrix metalloproteinase-2 (MMP-2) and meprin
<400> SEQUENCE: 1 Pro Leu Gly Leu Ala Gly 1 5 <210> SEQ
ID NO 2 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence:cleavable
linker <400> SEQUENCE: 2 Glu Asp Asp Asp Asp Lys Ala 1 5
<210> SEQ ID NO 3 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
Sequence:peptide designed to be substrate for enterokinase
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (8) <223> OTHER INFORMATION: Xaa = aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (18) <223> OTHER INFORMATION:
Xaa = aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (19)
<223> OTHER INFORMATION: Xaa = cysteinamide modified by
fluorescein (Fl) <400> SEQUENCE: 3 Glu Asp Asp Asp Asp Lys
Ala Xaa Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Xaa Xaa
<210> SEQ ID NO 4 <400> SEQUENCE: 4 000 <210> SEQ
ID NO 5 <211> LENGTH: 22 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence:peptide 5-47,
oligoglutamates veto oligoarginine-mediated cellular uptake
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1) <223> OTHER INFORMATION: Xaa = aminohexanoic
acid (aminocaproic acid) modified by fluorescein (Fl) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (12)
<223> OTHER INFORMATION: Xaa = aminohexanoic acid
(aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (22) <223> OTHER INFORMATION:
Xaa = cysteinamide <400> SEQUENCE: 5 Xaa Cys Arg Arg Arg Arg
Arg Arg Arg Arg Arg Xaa Glu Glu Glu Glu 1 5 10 15 Glu Glu Glu Glu
Glu Xaa 20 <210> SEQ ID NO 6 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:peptide 6-10, oligoglutamates veto
oligoarginine-mediated cellular uptake <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223>
OTHER INFORMATION: Xaa = aminohexanoic acid (aminocaproic acid)
modified by fluorescein (Fl) <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (7) <223> OTHER
INFORMATION: Xaa = aminohexanoic acid (aminocaproic acid)
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (17) <223> OTHER INFORMATION: Xaa = cysteinamide
<400> SEQUENCE: 6 Xaa Cys Glu Glu Glu Glu Xaa Arg Arg Arg Arg
Arg Arg Arg Arg Arg 1 5 10 15 Xaa <210> SEQ ID NO 7
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:peptide 6-14,
enterokinase substrate cleavage-dependent cellular uptake
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12) <223> OTHER INFORMATION: Xaa = aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (22) <223> OTHER INFORMATION:
Xaa = aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (23)
<223> OTHER INFORMATION: Xaa = cysteinamide modified by
fluorescein (Fl) <400> SEQUENCE: 7 Glu Glu Glu Glu Glu Asp
Asp Asp Asp Lys Ala Xaa Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg
Arg Xaa Xaa 20 <210> SEQ ID NO 8 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:peptide 6-16, enterokinase substrate
cleavage-dependent cellular uptake <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (8) <223> OTHER
INFORMATION: Xaa = aminohexanoic acid (aminocaproic acid)
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (18) <223> OTHER INFORMATION: Xaa = aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (19) <223> OTHER INFORMATION:
Xaa = cysteinamide modified by fluorescein (Fl) <400>
SEQUENCE: 8 Glu Asp Asp Asp Asp Lys Ala Xaa Arg Arg Arg Arg Arg Arg
Arg Arg 1 5 10 15 Arg Xaa Xaa <210> SEQ ID NO 9 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence:peptide 6-27, enterokinase
substrate cleavage-dependent cellular uptake <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (21)
<223> OTHER INFORMATION: Xaa = aminohexanoic acid
(aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (22) <223> OTHER INFORMATION:
Xaa = cysteinamide modified by fluorescein (Fl) <400>
SEQUENCE: 9 Glu Glu Glu Glu Glu Asp Asp Asp Asp Lys Ala Arg Arg Arg
Arg Arg 1 5 10 15 Arg Arg Arg Arg Xaa Xaa 20 <210> SEQ ID NO
10 <400> SEQUENCE: 10 000 <210> SEQ ID NO 11
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:peptide 7-2,
enterokinase substrate cleavage-dependent cellular uptake
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (18) <223> OTHER INFORMATION: Xaa = aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (19) <223> OTHER INFORMATION:
Xaa = cysteinamide modified by fluorescein (Fl) <400>
SEQUENCE: 11 Asp Asp Asp Asp Asp Asp Lys Ala Arg Arg Arg Arg Arg
Arg Arg Arg 1 5 10 15 Arg Xaa Xaa <210> SEQ ID NO 12
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:peptide 7-4,
enterokinase substrate cleavage-dependent cellular uptake
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (10) <223> OTHER INFORMATION: Xaa = aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (13) <223> OTHER INFORMATION:
Xaa = aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (16)
<223> OTHER INFORMATION: Xaa = aminohexanoic acid
(aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (19) <223> OTHER INFORMATION:
Xaa = aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (22)
<223> OTHER INFORMATION: Xaa = aminohexanoic acid
(aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (23) <223> OTHER INFORMATION:
Xaa = cysteinamide modified by fluorescein (Fl) <400>
SEQUENCE: 12 Glu Glu Asp Asp Asp Asp Lys Ala Arg Xaa Arg Arg Xaa
Arg Arg Xaa 1 5 10 15 Arg Arg Xaa Arg Arg Xaa Xaa 20 <210>
SEQ ID NO 13 <400> SEQUENCE: 13 000 <210> SEQ ID NO 14
<400> SEQUENCE: 14 000 <210> SEQ ID NO 15 <400>
SEQUENCE: 15 000 <210> SEQ ID NO 16 <400> SEQUENCE: 16
000 <210> SEQ ID NO 17 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:membrane- translocating sequence (MTS) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4)
<223> OTHER INFORMATION: Xaa = aminohexanoic acid
(aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (10) <223> OTHER INFORMATION:
Xaa = aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (11)
<223> OTHER INFORMATION: Xaa = cysteinamide modified by
fluorescein (Fl) <400> SEQUENCE: 17 Glu Asp Ala Xaa Arg Arg
Arg Arg Arg Xaa Xaa 1 5 10 <210> SEQ ID NO 18 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence:membrane- translocating sequence
(MTS) <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (8) <223> OTHER INFORMATION: Xaa =
aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (15)
<223> OTHER INFORMATION: Xaa = aminohexanoic acid
(aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (16) <223> OTHER INFORMATION:
Xaa = cysteinamide modified by doxorubicin (DOX) <400>
SEQUENCE: 18 Glu Asp Asp Asp Asp Lys Ala Xaa Arg Arg Arg Arg Arg
Arg Xaa Xaa 1 5 10 15 <210> SEQ ID NO 19 <211> LENGTH:
23 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:membrane- translocating sequence (MTS)
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (12) <223> OTHER INFORMATION: Xaa = aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (22) <223> OTHER INFORMATION:
Xaa = aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (23)
<223> OTHER INFORMATION: Xaa = tyrosinamide modified by 125-I
<400> SEQUENCE: 19 Glu Glu Glu Asp Asp Asp Glu Glu Glu Asp
Ala Xaa Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg Xaa Xaa 20
<210> SEQ ID NO 20 <400> SEQUENCE: 20 000 <210>
SEQ ID NO 21 <400> SEQUENCE: 21 000 <210> SEQ ID NO 22
<400> SEQUENCE: 22 000 <210> SEQ ID NO 23 <400>
SEQUENCE: 23 000 <210> SEQ ID NO 24 <400> SEQUENCE: 24
000 <210> SEQ ID NO 25 <400> SEQUENCE: 25 000
<210> SEQ ID NO 26 <400> SEQUENCE: 26 000 <210>
SEQ ID NO 27 <400> SEQUENCE: 27 000 <210> SEQ ID NO 28
<400> SEQUENCE: 28
000 <210> SEQ ID NO 29 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:peptide linker cleavable by matrix metalloproteinase-9
(MMP-9) <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (3) <223> OTHER INFORMATION: Xaa = Ser
or Thr <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (4) <223> OTHER INFORMATION: Xaa = Leu
or Ile <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (5) <223> OTHER INFORMATION: Xaa = Ser
or Thr <400> SEQUENCE: 29 Pro Arg Xaa Xaa Xaa 1 5 <210>
SEQ ID NO 30 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
Sequence:peptide linker cleavable by matrix metalloproteinase-11
(MMP-11) <400> SEQUENCE: 30 Gly Gly Ala Ala Asn Leu Val Arg
Gly Gly 1 5 10 <210> SEQ ID NO 31 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:peptide linker cleavable by matrix
metalloproteinase-14 (MMP-14) <400> SEQUENCE: 31 Ser Gly Arg
Ile Gly Phe Leu Arg Thr Ala 1 5 10 <210> SEQ ID NO 32
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:peptide linker
cleavable by urokinase plasminogen activator (uPA) <400>
SEQUENCE: 32 Ser Gly Arg Ser Ala 1 5 <210> SEQ ID NO 33
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:peptide linker
cleavable by lysosomal enzymes <400> SEQUENCE: 33 Gly Phe Leu
Gly 1 <210> SEQ ID NO 34 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:peptide linker cleavable by lysosomal enzymes <400>
SEQUENCE: 34 Ala Leu Ala Leu 1 <210> SEQ ID NO 35 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence:peptide linker cleavable by
cathepsin D <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (3) <223> OTHER INFORMATION: Xaa =
S-ethylcysteine <400> SEQUENCE: 35 Pro Ile Xaa Phe Phe 1 5
<210> SEQ ID NO 36 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
Sequence:peptide linker cleavable by cathepsin K <400>
SEQUENCE: 36 Gly Gly Pro Arg Gly Leu Pro Gly 1 5 <210> SEQ ID
NO 37 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence:peptide
linker cleavable by prostate-specific antigen <400> SEQUENCE:
37 His Ser Ser Lys Leu Gln 1 5 <210> SEQ ID NO 38 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence:peptide linker cleavable by
Herpes simplex virus protease <400> SEQUENCE: 38 Leu Val Leu
Ala Ser Ser Ser Phe Gly Tyr 1 5 10 <210> SEQ ID NO 39
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:peptide linker
cleavable by HIV protease <400> SEQUENCE: 39 Gly Val Ser Gln
Asn Tyr Pro Ile Val Gly 1 5 10 <210> SEQ ID NO 40 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence:peptide linker cleavable by
Cytomegalovirus protease <400> SEQUENCE: 40 Gly Val Val Gln
Ala Ser Cys Arg Leu Ala 1 5 10 <210> SEQ ID NO 41 <400>
SEQUENCE: 41 000 <210> SEQ ID NO 42 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:peptide linker cleavable by caspase-3
<400> SEQUENCE: 42 Asp Glu Val Asp 1 <210> SEQ ID NO 43
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:peptide linker
cleavable by interleukin 1beta converting enzyme <400>
SEQUENCE: 43 Gly Trp Glu His Asp Gly 1 5 <210> SEQ ID NO 44
<400> SEQUENCE: 44 000 <210> SEQ ID NO 45 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence:amino acid portion of compond
(d) of Figure 17
<400> SEQUENCE: 45 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Gly Tyr 1 5 10 <210> SEQ ID NO 46 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:amino acid portion of compond (e) of Figure 17
<400> SEQUENCE: 46 Ile Arg Arg Arg Lys Lys Leu Arg Arg Leu
Lys 1 5 10 <210> SEQ ID NO 47 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:amino acid portion of compond (f) of Figure 17,
R9, Arg9, peptide portion B, uptake sequence <400> SEQUENCE:
47 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> SEQ ID NO 48
<400> SEQUENCE: 48 000 <210> SEQ ID NO 49 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence:fluorescent positive control
membrane-translocating sequence (MTS) for uptake, abbreviated "R10"
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1) <223> OTHER INFORMATION: Xaa = Gly modified by
fluorescein (Fl) <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (12) <223> OTHER INFORMATION: Xaa =
argininamide <400> SEQUENCE: 49 Xaa Gly Arg Arg Arg Arg Arg
Arg Arg Arg Arg Xaa 1 5 10 <210> SEQ ID NO 50 <400>
SEQUENCE: 50 000 <210> SEQ ID NO 51 <400> SEQUENCE: 51
000 <210> SEQ ID NO 52 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:activatable cell-penetrating peptide (ACPP) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (12)
<223> OTHER INFORMATION: Xaa = 6-aminohexanoic acid
(aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (22) <223> OTHER INFORMATION:
Xaa = 6-aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (23)
<223> OTHER INFORMATION: Xaa = cysteinamide modified by
fluorescein (Fl) <400> SEQUENCE: 52 Glu Glu Glu Glu Glu Asp
Asp Asp Asp Lys Ala Xaa Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg
Arg Xaa Xaa 20 <210> SEQ ID NO 53 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:activatable cell-penetrating peptide (ACPP)
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (21) <223> OTHER INFORMATION: Xaa = 6-aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (22) <223> OTHER INFORMATION:
Xaa = cysteinamide modified by fluorescein (Fl) <400>
SEQUENCE: 53 Glu Glu Glu Glu Glu Asp Asp Asp Asp Lys Ala Arg Arg
Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Xaa Xaa 20 <210> SEQ ID
NO 54 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence:activatable
cell-penetrating peptide (ACPP) <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (8) <223> OTHER
INFORMATION: Xaa = 6-aminohexanoic acid (aminocaproic acid)
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (18) <223> OTHER INFORMATION: Xaa = 6-aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (19) <223> OTHER INFORMATION:
Xaa = cysteinamide modified by fluorescein (Fl) <400>
SEQUENCE: 54 Glu Asp Asp Asp Asp Lys Ala Xaa Arg Arg Arg Arg Arg
Arg Arg Arg 1 5 10 15 Arg Xaa Xaa <210> SEQ ID NO 55
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:activatable
cell-penetrating peptide (ACPP) <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (10) <223> OTHER
INFORMATION: Xaa = 6-aminohexanoic acid (aminocaproic acid)
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13) <223> OTHER INFORMATION: Xaa = 6-aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (16) <223> OTHER INFORMATION:
Xaa = 6-aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (19)
<223> OTHER INFORMATION: Xaa = 6-aminohexanoic acid
(aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (22) <223> OTHER INFORMATION:
Xaa = 6-aminohexanoic acid (aminocaproic acid) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (23)
<223> OTHER INFORMATION: Xaa = cysteinamide modified by
fluorescein (Fl) <400> SEQUENCE: 55 Glu Glu Asp Asp Asp Asp
Lys Ala Arg Xaa Arg Arg Xaa Arg Arg Xaa 1 5 10 15 Arg Arg Xaa Arg
Arg Xaa Xaa 20 <210> SEQ ID NO 56 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:activatable cell-penetrating peptide (ACPP)
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (18) <223> OTHER INFORMATION: Xaa = 6-aminohexanoic
acid (aminocaproic acid) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (19) <223> OTHER INFORMATION:
Xaa = cysteinamide modified by fluorescein (Fl) <400>
SEQUENCE: 56 Asp Asp Asp Asp Asp Asp Lys Ala Arg Arg Arg Arg Arg
Arg Arg Arg 1 5 10 15 Arg Xaa Xaa
<210> SEQ ID NO 57 <400> SEQUENCE: 57 000 <210>
SEQ ID NO 58 <400> SEQUENCE: 58 000 <210> SEQ ID NO 59
<400> SEQUENCE: 59 000 <210> SEQ ID NO 60 <400>
SEQUENCE: 60 000 <210> SEQ ID NO 61 <400> SEQUENCE: 61
000 <210> SEQ ID NO 62 <400> SEQUENCE: 62 000
<210> SEQ ID NO 63 <400> SEQUENCE: 63 000 <210>
SEQ ID NO 64 <400> SEQUENCE: 64 000 <210> SEQ ID NO 65
<400> SEQUENCE: 65 000 <210> SEQ ID NO 66 <400>
SEQUENCE: 66 000 <210> SEQ ID NO 67 <400> SEQUENCE: 67
000 <210> SEQ ID NO 68 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:fluorescence normalization calibration reference peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1) <223> OTHER INFORMATION: Xaa = Gly modified by
fluorescein (Fl) <400> SEQUENCE: 68 Xaa Gly Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg 1 5 10 <210> SEQ ID NO 69 <400>
SEQUENCE: 69 000 <210> SEQ ID NO 70 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence:linker region <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER
INFORMATION: Xaa = aminohexanoic acid (aminocaproic acid)
<400> SEQUENCE: 70 Xaa Pro Leu Gly Leu Ala Gly 1 5
<210> SEQ ID NO 71 <400> SEQUENCE: 71 000 <210>
SEQ ID NO 72 <400> SEQUENCE: 72 000 <210> SEQ ID NO 73
<400> SEQUENCE: 73 000 <210> SEQ ID NO 74 <400>
SEQUENCE: 74 000 <210> SEQ ID NO 75 <400> SEQUENCE: 75
000 <210> SEQ ID NO 76 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:uncleavable control scrambled peptide <400>
SEQUENCE: 76 Leu Ala Leu Gly Pro Gly 1 5 <210> SEQ ID NO 77
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Glu9,
oligoglutamate acidic portion A <400> SEQUENCE: 77 Glu Glu
Glu Glu Glu Glu Glu Glu Glu 1 5 <210> SEQ ID NO 78
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:enterokinase
substrate cleavage site <400> SEQUENCE: 78 Asp Asp Asp Asp
Lys 1 5 <210> SEQ ID NO 79 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:basic portion B <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (10)..(16) <223> OTHER
INFORMATION: Arg at positions 10-16 may be present or absent
<400> SEQUENCE: 79 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg 1 5 10 15 <210> SEQ ID NO 80
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:membrane-
translocating sequence (MTS), multicationic oligomer <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(7)..(12) <223> OTHER INFORMATION: Arg at positions 7-12 may
be present or absent <400> SEQUENCE: 80 Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg 1 5 10 <210> SEQ ID NO 81
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:acidic portion A
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (4) <223> OTHER INFORMATION: Xaa = sulfoserine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (9) <223> OTHER INFORMATION: Xaa = glutamatamine
attached to nitrogen-bound cargo molecule <400> SEQUENCE: 81
Glu Glu Glu Xaa Glu Glu Glu Glu Xaa 1 5 <210> SEQ ID NO 82
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:acidic portion A
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (9) <223> OTHER INFORMATION: Xaa = tyrosine
attached to nitrogen-bound cargo molecule <400> SEQUENCE: 82
Glu Glu Glu Glu Glu Glu Glu Glu Xaa 1 5 <210> SEQ ID NO 83
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:acidic portion A
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (3) <223> OTHER INFORMATION: Xaa =
tetrafluorotyrosine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (9) <223> OTHER INFORMATION:
Xaa = glutamic acid attached to nitrogen-bound cargo molecule
<400> SEQUENCE: 83 Glu Glu Xaa Glu Glu Ala Glu Glu Xaa 1 5
<210> SEQ ID NO 84 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
Sequence:basic portion B <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION:
Xaa = 2'-sulfo-acetamido-isoleucine attached to sulfur-linked cargo
molecule <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (11) <223> OTHER INFORMATION: Xaa =
lysine attached to nitrogen-bound cargo molecule <400>
SEQUENCE: 84 Xaa Arg Arg Arg Lys Lys Leu Arg Arg Leu Xaa 1 5 10
<210> SEQ ID NO 85 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
Sequence:basic portion B <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION:
Xaa = lysinamide attached through alpha and epsilon nitrogen-bound
cargo molecule <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (10) <223> OTHER INFORMATION: Xaa =
dodecaheptyl-arginine <400> SEQUENCE: 85 Xaa Arg Arg Arg Arg
Arg Arg Arg Arg Xaa 1 5 10 <210> SEQ ID NO 86 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence:albumin- conjugated activatable
cell-penetrating peptide (ACPP) labeled with technetium-99m
(99m-Tc) <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1) <223> OTHER INFORMATION: Xaa = Pro
conjugated to murine serum albumin (albumin) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (7) <223>
OTHER INFORMATION: Xaa = technetium-99m (99m-Tc) tricarbonyl
(Tc(CO)-3) chelated with N-epsilon-bis(2-pyridylmethyl)lysine (DPK)
<400> SEQUENCE: 86 Xaa Leu Gly Leu Ala Gly Xaa 1 5
<210> SEQ ID NO 87 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
Sequence:M13 phage display library peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (16)..(21)
<223> OTHER INFORMATION: Xaa = any amino acid, random amino
acid to generate library of cleavage sites (Z-6 site) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (30)
<223> OTHER INFORMATION: Xaa = Arg linked to N-terminus of
pIII coat protein of M13 phage <400> SEQUENCE: 87 His His His
His His His Glu Glu Glu Glu Glu Glu Glu Glu Glu Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Arg Arg Arg Arg Arg Arg Arg Arg Xaa 20 25 30
<210> SEQ ID NO 88 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
Sequence:T7 phage display library peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223>
OTHER INFORMATION: Xaa = Arg linked to C-terminus of 10B capsid
protein of T7 phage (T7select 10-3) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (10)..(15)
<223> OTHER INFORMATION: Xaa = any amino acid, random amino
acid to generate library of cleavage sites (Z-6 site) <400>
SEQUENCE: 88 Xaa Arg Arg Arg Arg Arg Arg Arg Arg Xaa Xaa Xaa Xaa
Xaa Xaa Glu 1 5 10 15 Glu Glu Glu Glu Glu Glu Glu Glu His His His
His His His 20 25 30 <210> SEQ ID NO 89 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: hexahistidine domain (H-6) <400>
SEQUENCE: 89 His His His His His His 1 5 <210> SEQ ID NO 90
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 90 Arg Leu Gln Leu Lys
Leu 1 5 <210> SEQ ID NO 91 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 91 Gly Leu Trp Gln Gly Pro 1 5 <210> SEQ ID NO 92
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 92 Gln Cys Thr Gly Arg
Phe
1 5 <210> SEQ ID NO 93 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 93 Leu Pro Gly Met Met Gly 1 5 <210> SEQ ID NO 94
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 94 Asp Val Gly Thr Thr
Glu 1 5 <210> SEQ ID NO 95 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 95 Thr Asp Leu Gly Ala Met 1 5 <210> SEQ ID NO 96
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 96 Gly Met Met Tyr Arg
Ser 1 5 <210> SEQ ID NO 97 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 97 Asp Ser Asn Ala Glu Ser 1 5 <210> SEQ ID NO 98
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 98 Ile Thr Asp Met Ala
Ala 1 5 <210> SEQ ID NO 99 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 99 Arg Trp Arg Thr Asn Phe 1 5 <210> SEQ ID NO 100
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 100 Trp Arg Pro Cys Glu
Ser 1 5 <210> SEQ ID NO 101 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 101 Trp Arg Asn Thr Ile Ala 1 5 <210> SEQ ID NO 102
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 102 Ile Asp Lys Gln Leu
Glu 1 5 <210> SEQ ID NO 103 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 103 Phe Met Glu Ile Glu Thr 1 5 <210> SEQ ID NO 104
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 104 His Glu Val Val Ala
Gly 1 5 <210> SEQ ID NO 105 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 105 Thr Ser Ala Val Arg Thr 1 5 <210> SEQ ID NO 106
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 106 Gly Gly His Thr Arg
Gln 1 5 <210> SEQ ID NO 107 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence:Z-6 phage displayed peptide cleavage site <400>
SEQUENCE: 107 Ile Asn Gly Lys Val Thr 1 5 <210> SEQ ID NO 108
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence:Z-6 phage displayed
peptide cleavage site <400> SEQUENCE: 108 Ala Arg Lys Arg Ser
Gln 1 5
* * * * *