U.S. patent application number 13/224193 was filed with the patent office on 2012-02-09 for truncated car peptides and methods and compositions using truncated car peptides.
This patent application is currently assigned to Sanford-Burnham Medical Research Institute California. Invention is credited to Tero Jarvinen, Masanobu Komatsu, Erkki Ruoslahti.
Application Number | 20120034164 13/224193 |
Document ID | / |
Family ID | 44507619 |
Filed Date | 2012-02-09 |
United States Patent
Application |
20120034164 |
Kind Code |
A1 |
Ruoslahti; Erkki ; et
al. |
February 9, 2012 |
TRUNCATED CAR PEPTIDES AND METHODS AND COMPOSITIONS USING TRUNCATED
CAR PEPTIDES
Abstract
Disclosed are compositions and methods useful for targeting and
internalizing molecules into cells of interest and for penetration
by molecules of tissues of interest. The compositions and methods
are based on peptide sequences, such as truncated CAR peptides,
that are selectively internalized by a cell, penetrate tissue, or
both. The disclosed internalization and tissue penetration is
useful for delivering therapeutic and detectable agents to cells
and tissues of interest.
Inventors: |
Ruoslahti; Erkki; (Buellton,
CA) ; Jarvinen; Tero; (La Jolla, CA) ;
Komatsu; Masanobu; (Orlando, FL) |
Assignee: |
Sanford-Burnham Medical Research
Institute California
|
Family ID: |
44507619 |
Appl. No.: |
13/224193 |
Filed: |
September 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2011/026535 |
Feb 28, 2011 |
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13224193 |
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61308826 |
Feb 26, 2010 |
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Current U.S.
Class: |
424/1.69 ;
424/1.81; 424/450; 424/9.1; 424/93.6; 514/12.2; 514/13.3; 514/15.7;
514/16.5; 514/16.7; 514/19.3; 530/329 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 1/6883 20130101; A61P 37/00 20180101; A61K 38/08 20130101;
C07K 7/06 20130101; A61K 31/551 20130101; A61P 29/00 20180101; A61K
45/06 20130101; A61P 35/00 20180101; A61P 43/00 20180101; A61P 7/00
20180101; A61K 38/00 20130101; A61P 19/02 20180101; A61P 9/12
20180101; A61K 31/506 20130101; A61K 47/64 20170801 |
Class at
Publication: |
424/1.69 ;
530/329; 514/16.5; 514/19.3; 514/13.3; 514/12.2; 514/16.7;
514/15.7; 424/93.6; 424/450; 424/9.1; 424/1.81 |
International
Class: |
A61K 38/08 20060101
A61K038/08; A61P 43/00 20060101 A61P043/00; A61P 35/00 20060101
A61P035/00; A61P 7/00 20060101 A61P007/00; A61P 29/00 20060101
A61P029/00; A61K 51/08 20060101 A61K051/08; A61P 9/12 20060101
A61P009/12; A61P 37/00 20060101 A61P037/00; A61K 35/76 20060101
A61K035/76; A61K 9/127 20060101 A61K009/127; A61K 49/00 20060101
A61K049/00; C07K 7/06 20060101 C07K007/06; A61P 19/02 20060101
A61P019/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
1 R41 HL088771 from the National Heart Lung Blood Institute (NHLBI)
of the National Institutes of Health (NIH). The government has
certain rights in the invention.
Claims
1. An isolated peptide, wherein the C-terminal end of the peptide
consists of the amino acid sequence CARSKNK (SEQ ID NO:4).
2. The peptide of claim 1, wherein the peptide is a modified
peptide.
3-5. (canceled)
6. The peptide of claim 1, wherein the peptide is an activatable
peptide.
7. The peptide of claim 6, wherein the amino acid sequence CARSKNK
(SEQ ID NO:4) at the C-terminal end of the peptide is blocked by a
blocking group coupled to the terminal carboxy group.
8. The peptide of claim 7, wherein the blocking group comprises an
amino acid or an amino acid sequence.
9. A composition comprising the peptide of claim 1.
10. The composition of claim 9 further comprising a co-composition,
wherein the peptide and the co-composition are not covalently
coupled or non-covalently associated with each other.
11. The composition of claim 9 further comprising a cargo
composition, wherein the peptide and the cargo composition are
covalently coupled or non-covalently associated with each
other.
12. The composition of claim 9, wherein the peptide selectively
homes to regenerating tissue, a site of injury, a surgical site, a
tumor, tumor vasculature, a site of tumor angiogenesis, a site of
inflammation, a site of arthritis, lung tissue, pulmonary arterial
hypertension lung vasculature, pulmonary arterial hypertension
lesions, remodeled pulmonary arteries, or interstitial space of
lungs.
13. (canceled)
14. The composition of claim 10, wherein the co-composition
selectively homes to regenerating tissue, a site of injury, a
surgical site, a tumor, tumor vasculature, a site of tumor
angiogenesis, a site of inflammation, a site of arthritis, lung
tissue, pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries, or
interstitial space of lungs.
15. The composition of claim 10, wherein the peptide and the
co-composition are not bound to each other.
16-18. (canceled)
19. The composition of claim 10, wherein the co-composition
comprises a therapeutic agent, a therapeutic protein, a therapeutic
compound, a therapeutic composition, a chemotherapeutic agent, a
cancer chemotherapeutic agent, a toxin, a cytotoxic agent,
Abraxane, paclitaxel, taxol, imatinib, an anti-angiogenic agent, a
pro-angiogenic agent, an anti-inflammatory agent, an anti-arthritic
agent, a TGF-.beta. inhibitor, decorin, a systemic vasodilator, an
anti-coagulant, tissue factor pathway inhibitor (TFPI),
site-inactivated factor VIIa, a .beta.-2 agonist, salmeterol,
formoterol, N-Acetylcysteine (NAC), Superoxide Dismutase (SOD), a
superoxide dismutase mimetic, EUK-8, an endothelin (ET-1) receptor
antagonist, a prostacyclin derivative, a phosphodiesterase type 5
inhibitor, Ketoconazole, a small interfering RNA (siRNA), a
microRNA (miRNA), a polypeptide, a nucleic acid molecule, a small
molecule, a carrier, a vehicle, a virus, a phage, a viral particle,
a phage particle, a viral capsid, a phage capsid, a virus-like
particle, a liposome, a micelle, a bead, a nanoparticle, a
microparticle, a detectable agent, a contrast agent, an imaging
agent, a label, a labeling agent, a fluorophore, fluorescein,
rhodamine, FAM, a radionuclide, indium-111, technetium-99,
carbon-11, carbon-13, or a combination.
20. The composition of claim 11, wherein the peptide is comprised
in a tCAR composition.
21. The composition of claim 20, wherein the tCAR composition
comprises one or more cargo compositions.
22. The composition of claim 20, wherein the tCAR composition
comprises one or more homing molecules.
23. The composition of claim 11, wherein the peptide is comprised
in a tCAR conjugate.
24. The composition of claim 23, wherein the tCAR conjugate
comprises one or more cargo compositions.
25. The composition of claim 10, wherein the tCAR conjugate
comprises one or more homing molecules.
26. The composition of claim 10, wherein the composition comprises
a plurality of cargo compositions.
27. The composition of claim 10, wherein the composition comprises
a plurality of copies of the peptide.
28. The composition of claim 10, wherein the composition comprises
a plurality of co-compositions.
29. The composition of claim 9, wherein the composition further
comprises a surface molecule and a plurality of membrane perturbing
molecules.
30. The composition of claim 29, wherein the composition further
comprises one or more homing molecules, wherein the homing
molecules selectively home to tumor vasculature, lung vasculature,
regenerating tissue, wounded tissue, angiogenic tissue, cancer
issue, lung tissue, pulmonary arterial hypertension lung
vasculature, pulmonary arterial hypertension lesions, remodeled
pulmonary arteries, interstitial space of lungs, a site of injury,
a surgical site, a tumor, a site of angiogenesis, a site of tumor
angiogenesis, a site of inflammation, or a site of arthritis.
31. The composition of claim 10, wherein the co-composition
comprises a surface molecule, one or more homing molecules, and a
plurality of membrane perturbing molecules, wherein the homing
molecules selectively home to tumor vasculature, lung vasculature,
regenerating tissue, wounded tissue, angiogenic tissue, cancer
issue, lung tissue, pulmonary arterial hypertension lung
vasculature, pulmonary arterial hypertension lesions, remodeled
pulmonary arteries, interstitial space of lungs, a site of injury,
a surgical site, a tumor, a site of angiogenesis, a site of tumor
angiogenesis, a site of inflammation, or a site of arthritis.
32. The composition of claim 11, wherein the cargo composition
comprises a surface molecule, one or more homing molecules, and a
plurality of membrane perturbing molecules, wherein the homing
molecules selectively home to tumor vasculature, lung vasculature,
regenerating tissue, wounded tissue, angiogenic tissue, cancer
issue, lung tissue, pulmonary arterial hypertension lung
vasculature, pulmonary arterial hypertension lesions, remodeled
pulmonary arteries, interstitial space of lungs, a site of injury,
a surgical site, a tumor, a site of angiogenesis, a site of tumor
angiogenesis, a site of inflammation, or a site of arthritis.
33-39. (canceled)
40. The composition of claim 9, wherein the composition is
internalized in cells.
41. The composition of claim 9, wherein the composition penetrates
tissue.
42-55. (canceled)
56. The composition of claim 9 further comprising one or more
moieties.
57. The composition of claim 56, wherein the moieties are
independently selected from the group consisting of a therapeutic
agent, a therapeutic protein, a therapeutic compound, a therapeutic
composition, a chemotherapeutic agent, a cancer chemotherapeutic
agent, a toxin, a cytotoxic agent, Abraxane, paclitaxel, taxol,
imatinib, an anti-angiogenic agent, a pro-angiogenic agent, an
anti-inflammatory agent, an anti-arthritic agent, a TGF-.beta.
inhibitor, decorin, a systemic vasodilator, an anti-coagulant,
tissue factor pathway inhibitor (TFPI), site-inactivated factor
VIIa, a .beta.-2 agonist, salmeterol, formoterol, N-Acetylcysteine
(NAC), Superoxide Dismutase (SOD), a superoxide dismutase mimetic,
EUK-8, an endothelin (ET-1) receptor antagonist, a prostacyclin
derivative, a phosphodiesterase type 5 inhibitor, Ketoconazole, a
small interfering RNA (siRNA), a microRNA (miRNA), a polypeptide, a
nucleic acid molecule, a small molecule, a carrier, a vehicle, a
virus, a phage, a viral particle, a phage particle, a viral capsid,
a phage capsid, a virus-like particle, a liposome, a micelle, a
bead, a nanoparticle, a microparticle, a detectable agent, a
contrast agent, an imaging agent, a label, a labeling agent, a
fluorophore, fluorescein, rhodamine, FAM, a radionuclide,
indium-111, technetium-99, carbon-11, carbon-13, or a
combination.
58. The composition of claim 9, wherein the tCAR composition has a
therapeutic effect.
59. The composition of claim 10, wherein the co-composition has a
therapeutic effect.
60. The composition of claim 58, wherein the therapeutic effect is
a slowing in the increase of or a reduction of tumor burden.
61. The composition of claim 58, wherein the therapeutic effect is
a slowing of the increase of or reduction of tumor size.
62. The composition of claim 58, wherein the therapeutic effect
comprises a reduction in inflammation, an increase in speed of
wound healing, reduction in amounts of scar tissue, decrease in
pain, decrease in swelling, or decrease in necrosis.
63. The composition of claim 58, wherein the therapeutic effect
comprises pulmonary vasodilation, decrease in pulmonary pressure,
anti-coagulation, airway smooth muscle relaxation, increase in
glutathione (GSH), decrease in inflammatory immune response,
inhibition of thromboxane synthesis, or inhibition of leukotriene
synthesis.
64. A method of enhancing internalization, penetration, or both
into or through a cell, tissue, or both, the method comprising:
exposing the cell, tissue, or both to a tCAR composition, thereby
enhancing internalization, penetration, or both into or through the
cell, tissue, or both, wherein the tCAR composition comprises the
peptide of claim 1.
65. A method of enhancing internalization, penetration, or both
into or through a cell, tissue, or both, the method comprising:
exposing the cell, tissue, or both to a tCAR composition, thereby
enhancing internalization, penetration, or both into or through the
cell, tissue, or both, wherein the tCAR composition comprises the
composition of claim 9.
66. The method of claim 64 further comprising exposing the cell,
tissue, or both to a co-composition, thereby enhancing
internalization, penetration, or both of the co-composition into or
through the cell, tissue, or both, wherein, prior to exposing the
cell, tissue, or both, the tCAR composition and the co-composition
are not covalently coupled or non-covalently associated with each
other.
67. The method of claim 64 further comprising exposing the cell,
tissue, or both to a cargo composition, thereby enhancing
internalization, penetration, or both of the cargo composition into
or through the cell, tissue, or both, wherein the tCAR composition
and the cargo composition are covalently coupled or non-covalently
associated with each other.
68. The method of claim 65, wherein the cell, tissue, or both is in
a subject.
69. The method of claim 68, wherein the cell, tissue, or both are
exposed to the tCAR composition by administering the tCAR
composition to the subject.
70. The method of claim 66, wherein the cell, tissue, or both is in
a subject, wherein the cell, tissue, or both are exposed to the
tCAR composition and the co-composition by administering the tCAR
composition and the co-composition to the subject.
71. The method of claim 67, wherein the cell, tissue, or both is in
a subject, wherein the cell, tissue, or both are exposed to the
tCAR composition and the cargo composition by administering the
tCAR composition and the cargo composition to the subject.
72-75. (canceled)
76. The method of claim 70, wherein the tCAR composition and the
co-composition are administered to the subject simultaneously.
77. The method of claim 76, wherein the tCAR composition and the
co-composition are administered to the subject in a single
composition comprising the tCAR composition and the
co-composition.
78. The method of claim 70, wherein the tCAR composition and the
co-composition are administered to the subject in separate
compositions.
79. The method of claim 70, wherein the tCAR composition and the
co-composition are administered to the subject at different
times.
80. The method of claim 79, wherein the tCAR composition and the
co-composition are administered to the subject in separate
compositions.
81. The method of claim 78, wherein the tCAR composition and the
co-composition are administered to the subject by separate
routes.
82. The method of claim 66, wherein the tCAR composition and the
co-composition are not bound to each other.
83-94. (canceled)
95. The method of claim 68, wherein the subject has a disease or
condition.
96. The method of claim 95, wherein the disease is pulmonary or
fibrotic.
97. The method of claim 96, wherein the disease or condition is
pulmonary arterial hypertension (PAH).
98. The method of claim 95, wherein the disease or condition is
cancer.
99. The method of claim 95, wherein the disease or condition is an
autoimmune disease.
100. The method of claim 95, wherein the disease or condition is an
inflammatory disease.
101. The method of claim 68, wherein the subject has one or more
sites to be targeted, wherein the tCAR composition homes to one or
more of the sites to be targeted.
102. The method of claim 68, wherein the subject has one or more
sites to be targeted, wherein the co-composition or cargo
composition homes to one or more of the sites to be targeted.
103. The method of claim 64, wherein the peptide selectively homes
to tumor vasculature, lung vasculature, regenerating tissue,
wounded tissue, angiogenic tissue, cancer issue, lung tissue,
pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries,
interstitial space of lungs, a site of injury, a surgical site, a
tumor, a site of angiogenesis, a site of tumor angiogenesis, a site
of inflammation, or a site of arthritis.
104. The method of claim 64, wherein the tCAR composition
selectively homes to tumor vasculature, lung vasculature,
regenerating tissue, wounded tissue, angiogenic tissue, cancer
issue, lung tissue, pulmonary arterial hypertension lung
vasculature, pulmonary arterial hypertension lesions, remodeled
pulmonary arteries, interstitial space of lungs, a site of injury,
a surgical site, a tumor, a site of angiogenesis, a site of tumor
angiogenesis, a site of inflammation, or a site of arthritis.
105. The method of claim 66, wherein the co-composition or cargo
composition selectively homes to tumor vasculature, lung
vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of PCT
Application No. PCT/US2011/026535, filed Feb. 28, 2011, which
claims benefit of U.S. Provisional Application Ser. No. 61/308,826,
filed Feb. 26, 2010. PCT/US2011/026535, filed Feb. 28, 2011, and
U.S. Provisional Application Ser. No. 61/308,826, filed Feb. 26,
2010, are hereby incorporated herein by reference in their
entirety.
REFERENCE TO SEQUENCE LISTING
[0003] The Sequence Listing submitted Sep. 1, 2011, as a text file
named
"SBMRI.sub.--57.sub.--8401_AMD_AFD_Sequence_Lisiting_Text_File.txt,"
created on Sep. 1, 2011, and having a size of 37,865 bytes is
hereby incorporated by reference pursuant to 37 C.F.R.
.sctn.1.52(e)(5).
FIELD OF THE INVENTION
[0004] The present invention relates generally to the field of
molecular medicine, more specifically, to cell and tissue-targeting
peptides.
BACKGROUND OF THE INVENTION
[0005] Peptides that are internalized into cells are commonly
referred to as cell-penetrating peptides. There are two main
classes of such peptides: hydrophobic and cationic (Zorko and
Langel, 2005). The cationic peptides, which are commonly used to
introduce nucleic acids, proteins into cells, include the
prototypic cell-penetrating peptides (CPP), Tat, and penetratin
(Derossi et al., 1998; Meade and Dowdy, 2007). A herpes virus
protein, VP22, is capable of both entering and exiting cells and
carrying a payload with it (Elliott and O'Hare, 1997; Brewis et
al., 2003). A major limitation of these peptides as delivery
vehicles is that they are not selective; they enter into all
cells.
[0006] Tissue penetration is a serious limitation in the delivery
of compositions to cells. Comparison of the distribution of
fluorescein-labeled peptides to that of iron oxide particles coated
with the same peptide shows that the particles remain close to the
tumor blood vessels, whereas the fluorescent peptide reaches all
areas of the tumor. The frequently cited "leakiness" of tumor
vessels does not appear to substantially mitigate this problem.
Moreover, anti-angiogenic treatments that cause "normalization" of
tumor vasculature (Jain, 2005), creating a need to target tumors
whose vasculature is not leaky. Thus, it is important to find new
ways of improving the passage of diverse compositions into the
extravascular space. A number of proteins are known to translocate
through the endothelium of blood vessels, including the blood-brain
barrier. A prime example is transferrin, which is carried across
the blood-brain barrier by the transferrin receptor. This system
has been used to bring other payloads into the brain (Li et al.,
2002; Fenart and Cecchelli, 2003). Peptide signals for endothelial
transcytosis that can mediate translocation of compositions from
the circulation into tissues is useful.
[0007] Tissue regeneration, inflammation and tumors induce the
growth of new blood vessels from pre-existing ones. This process,
angiogenesis, is a vital requirement for wound healing as the
formation of new blood vessels allows a variety of mediators,
nutrients, and oxygen to reach the healing tissue (Martin 1997,
Singer & Clark 1999, Falanga 2006, Folkman 2006). Newly formed
blood vessels differ in structure from pre-existing vasculature.
Such differences have been extensively characterized by comparing
tumor vasculature to normal vessels (Ruoslahti, 2002). Angiogenic
vessels in non-malignant tissues and in pre-malignant lesions share
markers with tumor vessels (Gerlag et al, 2001), but distinct
markers also exist (Hoffman et al., 2003; Joyce et al., 2003).
[0008] Regarding tissue injuries, substantive basic science and
clinical research have been conducted to evaluate the mechanisms of
wound healing, the efficacy of various modalities for treatment of
wounds, and the best methods for diagnosing wound infection. Tissue
injuries caused by trauma, medical procedures, and inflammation are
a major medical problem. Systemic medication is available for most
major medical conditions, but therapeutic options in promoting
tissue regeneration are largely limited to local intervention. As
deep injuries and multiple sites of injury often limit the
usefulness of local treatment, systemic approaches to tissue
regeneration are valuable.
[0009] A major problem limiting tissue regeneration is scar
formation. The response to tissue injury in adult mammals seems to
be mainly focused on quick sealing on the injury. Fibroblast
(astrocyte, smooth muscle cell) proliferation and enhanced
extracellular matrix production are the main element of the scar
formation, and the scar prevents tissue regeneration. In contrast,
fetal tissues heal by a process that restores the original tissue
architecture with no scarring. Transforming growth factor-beta
(TGF-beta) is a major factor responsible for impaired tissue
regeneration, scar formation and fibrosis (Werner and Grose 2002;
Brunner and Blakytny 2004; Leask and Abraham 2004).
[0010] A major hurdle to advances in treating cancer is the
relative lack of agents that can selectively target the cancer
while sparing normal tissue. For example, radiation therapy and
surgery, which generally are localized treatments, can cause
substantial damage to normal tissue in the treatment field,
resulting in scarring and loss of normal tissue. Chemotherapy, in
comparison, which generally is administered systemically, can cause
substantial damage to organs such as the bone marrow, mucosae, skin
and small intestine, which undergo rapid cell turnover and
continuous cell division. As a result, undesirable side effects
such as nausea, loss of hair and drop in blood cell count often
occur when a cancer patient is treated intravenously with a
chemotherapeutic drug. Such undesirable side effects can limit the
amount of a drug that can be safely administered, thereby hampering
survival rate and impacting the quality of patient life. For
decades, researchers have examined avenues to increase targeted
specificity of therapeutics against only the disease, thereby
preserving normal cellular integrity.
[0011] One manner by which therapeutic specificity may be increased
is by targeting diseases at the cellular level. More specifically,
therapeutics may be enhanced by interacting directly with those
components at the level of the cell surface or membrane. These
components include, among others, laminin, collagen, fibronectin
and other proteoglycans. Proteoglycans are proteins classified by a
posttranslational attachment of polysaccharide glycosaminoglycan
(GAG) moieties each comprised of repeating disaccharide units. One
monosaccharide of the disaccharide repeat is an amino sugar with
D-glucosamine or galactosamine, and the other unit is typically,
but not always, a uronic acid residue of either D-glucuronic acid
or iduronic acid. Both units are variably N- and O-sulfated, which
adds to the heterogeneity of these complex macromolecules. They can
be found associated with both the extracellular matrix and plasma
membranes. The most common GAG structures are dermatan sulfate
(DS), chondroitin sulfate (CS), heparan sulfate (HS), keratan
sulfate (KS), hyaluronic acid (HA), and heparin; representative
structures for each disaccharide are shown below.
##STR00001## ##STR00002##
[0012] These unbranched sulfated GAGs are defined by the repeating
disaccharide units that comprise their chains, by their specific
sites of sulfation, and by their susceptibility to bacterial
enzymes known to cleave distinct GAG linkages. All have various
degrees of sulfation which result in a high density of negative
charge. Proteoglycans can be modified by more than one type of GAG
and have a diversity of functions, including roles in cellular
adhesion, differentiation, and growth. In addition, cell surface
proteoglycans are known to act as cellular receptors for some
bacteria and several animal viruses, including; foot-and-mouth
disease type O virus, HSV types 1 and 2 and dengue virus.
Accordingly, it would be advantageous from a therapeutic
perspective to design agents which may be used at the cell surface
level.
[0013] A major function of cell surface proteoglycans is in cell
adhesion and migration, dynamic processes that are mediated through
interactions between the proteoglycan GAG chains and extracellular
matrix (ECM) components, such as laminin, collagen, and
fibronectin. Proteoglycans also occur as integral components of
basement membranes in most mammalian tissues. Interactions of these
macromolecules with other ECM constituents contribute to the
general architecture and permeability properties of the basement
membrane, and thus these GAGs play a structural role. Proteoglycans
and GAGs play a critical role in the pathophysiology of basement
membrane-related diseases, including diabetes, atherosclerosis, and
metastasis. In addition, cell-specific growth factors and enzymes
are immobilized in the ECM and at the cell surface are bound to
GAGs. As such, GAGs localize proteins and enzymes at their site of
action to facilitate their physiological functions and in some
cases prevent their proteolytic degradation. Proteoglycans and GAGs
have been shown to regulate protein secretion and gene expression
in certain tissues by mechanisms involving both membrane and
nuclear events, including the binding of GAGs to transcription
factors (Jackson, R. L. 1991). Limited information is available on
the factors that regulate the expression of proteoglycans and their
associated GAGs. There is a need in the art to develop
cell-penetrating agents which bind to cell surface proteoglycans in
order to have disease-specific efficacy.
[0014] HS2ST1 (heparan sulfate 2-O-sulfotransferase 1) catalyzes
the transfer of sulfate to the C2-position of selected hexuronic
acid residues within the maturing heparan sulfate. EXT1 (exostosin
1) is an endoplasmic reticulum-resident type II transmembrane
glycosyltransferase involved in the chain elongation step of
heparan sulfate biosynthesis. GLT8D2 (glycosyltransferase 8 domain
containing 2) is an enzyme involved in HSPG biosynthesis. NDST1
(Heparan sulfate N-deacetylase/N-sulfotransferase) is a HSPG
biosynthetic enzyme. OGT (O-linked N-acetylglucosamine (O-GlcNAc)
transferase) catalyzes the addition of a single N-acetylglucosamine
in O-glycosidic linkage to serine or threonine residues of
intracellular proteins including HSPGs.
[0015] US Patent Application Publication No. 20090036349 discloses
a novel composition that selectively binds to regenerating tissue,
wound sites and tumors in animals. In vivo screening of
phage-displayed peptide libraries was used to probe vascular
specialization. This screening method resulted in the
identification of several peptides that selectively target phage to
skin and tendon wounds. One peptide in particular was identified
and contains the following sequence: CARSKNKDC (CAR) (SEQ ID
NO:147). CAR displays homology to heparin-binding sites in various
proteins, and binds to cell surface heparan sulfate and heparin.
More specifically, CAR binds to glycosaminoglycan moieties in cell
surface heparan sulfate proteoglycans (HSPGs) (Jarvinen and
Ruoslahti 2007), and other cell-penetrating peptides have also
mediated their entry into cells through binding to HSPGs (Poon and
Gariepy 2007). HSPGs fine-tune mammalian physiology and orchestrate
metabolism, transport, information transfer, support and regulation
at the systemic level, as well as the cellular level (Bishop,
Schuksz and Esko 2007). Overexpression of HSPG biosynthetic enzymes
result in distinct heparan sulfate sulfation patterns (Pikas,
Erikson and Kjellen 2000). The overexpression of HSPG biosynthetic
enzymes have not been previously detected in a disease in which the
co-administration of a cell penetrating peptide along with a
bioactive agent which results in the disease-selective action of
the co-administration of the peptide/agent combination.
[0016] Thus, there is a need for new therapeutic strategies for
selectively targeting various types of cells, and for internalizing
proteins and peptides into those cells and penetration of tissue by
proteins and peptides. There is also a need for increasing the
delivery of compounds and compositions to and into cells and
tissues. The present invention satisfies these needs by providing
peptides that can be selectively targeted, and selectively
internalized, by cells and/or can penetrate tissue. Related
advantages also are provided.
BRIEF SUMMARY OF THE INVENTION
[0017] Disclosed are peptides that target tumor vascularture,
regenerating tissue, wounded tissue, pulmonary tissue, fibrotic
tissue, and related tissue, that are readily internalized into
adjacent cells, and that extensively penetrate and invade tumor
tissue. The disclosed peptides can also mediate targeting,
internalization, and tissue pentration of compounds and
compositions coupled to, associated with, conjugated to, or even
co-administered with the peptide. Examples of the disclosed
peptides include peptides where the C-terminal end of the peptide
consists of the amino acid sequence CARSKNK (SEQ ID NO:4) and
peptides consisting of CARSKNK (SEQ ID NO:4). The disclosed
peptides can be used in and with a variety of compositions and
methods to, for example, enhancing internalization, penetration, or
both of such compositions into or through a cell, tissue, or both.
Such compositions and methods are also disclosed herein.
[0018] Disclosed are compounds, compositions, and methods
comprising and using truncated CAR peptides. Truncated CAR peptides
are peptides where the C-terminal end of the peptide consists of
the amino acid sequence CARSKNK (SEQ ID NO:4). Thus, disclosed
herein are isolated peptides, where the C-terminal end of the
peptide consists of the amino acid sequence CARSKNK (SEQ ID
NO:4).
[0019] In some forms, the peptide can be a modified peptide. In
some forms, the peptide can be a methylated peptide. In some forms,
one or more of the methylated peptide can comprise a methylated
amino acid segment. In some forms, the peptide can be N- or
C-methylated in at least one position. In some forms, the peptide
can be an activatable peptide. In some forms, the amino acid
sequence CARSKNK (SEQ ID NO:4) at the C-terminal end of the peptide
can be blocked by a blocking group coupled to the terminal carboxy
group. In some forms, the blocking group can comprise an amino acid
or an amino acid sequence.
[0020] Also disclosed are compositions comprising the disclosed
peptide. In some forms, the composition can further comprise a
co-composition, where the peptide and the co-composition are not
covalently coupled or non-covalently associated with each other. In
some forms, the composition can further comprise a cargo
composition, where the peptide and the cargo composition are
covalently coupled or non-covalently associated with each other. In
some forms, the peptide can selectively home to regenerating
tissue, a site of injury, a surgical site, a tumor, tumor
vasculature, a site of tumor angiogenesis, a site of inflammation,
a site of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs.
[0021] In some forms, the composition can selectively home to
regenerating tissue, a site of injury, a surgical site, a tumor,
tumor vasculature, a site of tumor angiogenesis, a site of
inflammation, a site of arthritis, lung tissue, pulmonary arterial
hypertension lung vasculature, pulmonary arterial hypertension
lesions, remodeled pulmonary arteries, or interstitial space of
lungs. In some forms, the co-composition or cargo composition can
selectively home to regenerating tissue, a site of injury, a
surgical site, a tumor, tumor vasculature, a site of tumor
angiogenesis, a site of inflammation, a site of arthritis, lung
tissue, pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries, or
interstitial space of lungs.
[0022] In some forms, the peptide and the co-composition are not
bound to each other. In some forms, the co-composition or cargo
composition can comprise a therapeutic agent. In some forms, the
co-composition or cargo composition can comprise a detection agent.
In some forms, the co-composition or cargo composition can comprise
a carrier, vehicle, or both. In some forms, the co-composition or
cargo composition can comprise a therapeutic agent, a therapeutic
protein, a therapeutic compound, a therapeutic composition, a
chemotherapeutic agent, a cancer chemotherapeutic agent, a toxin, a
cytotoxic agent, Abraxane, paclitaxel, taxol, imatinib, an
anti-angiogenic agent, a pro-angiogenic agent, an anti-inflammatory
agent, an anti-arthritic agent, a TGF-.beta. inhibitor, decorin, a
systemic vasodilator, an anti-coagulant, tissue factor pathway
inhibitor (TFPI), site-inactivated factor VIIa, a .beta.-2 agonist,
salmeterol, formoterol, N-Acetylcysteine (NAC), Superoxide
Dismutase (SOD), a superoxide dismutase mimetic, EUK-8, an
endothelin (ET-1) receptor antagonist, a prostacyclin derivative, a
phosphodiesterase type 5 inhibitor, Ketoconazole, a small
interfering RNA (siRNA), a microRNA (miRNA), a polypeptide, a
nucleic acid molecule, a small molecule, a carrier, a vehicle, a
virus, a phage, a viral particle, a phage particle, a viral capsid,
a phage capsid, a virus-like particle, a liposome, a micelle, a
bead, a nanoparticle, a microparticle, a detectable agent, a
contrast agent, an imaging agent, a label, a labeling agent, a
fluorophore, fluorescein, rhodamine, FAM, a radionuclide,
indium-111, technetium-99, carbon-11, carbon-13, or a
combination.
[0023] In some forms, the peptide can be comprised in a tCAR
composition. In some forms, the tCAR composition can comprise one
or more cargo compositions. In some forms, the tCAR composition can
comprise one or more homing molecules. In some forms, the peptide
can be comprised in a tCAR conjugate. In some forms, the tCAR
conjugate can comprise one or more cargo compositions. In some
forms, the tCAR conjugate can comprise one or more homing
molecules.
[0024] In some forms, the composition comprises a plurality of
cargo compositions. In some forms, the composition can comprise a
plurality of copies of the peptide. In some forms, the composition
can comprise a plurality of co-compositions. In some forms, the
composition can further comprise a surface molecule and a plurality
of membrane perturbing molecules. In some forms, the composition
can further comprise one or more homing molecules, wherein the
homing molecules selectively home to tumor vasculature, lung
vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0025] In some forms, the co-composition can comprise a surface
molecule, one or more homing molecules, and a plurality of membrane
perturbing molecules, wherein the homing molecules selectively home
to tumor vasculature, lung vasculature, regenerating tissue,
wounded tissue, angiogenic tissue, cancer issue, lung tissue,
pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries,
interstitial space of lungs, a site of injury, a surgical site, a
tumor, a site of angiogenesis, a site of tumor angiogenesis, a site
of inflammation, or a site of arthritis. In some forms, the cargo
composition can comprise a surface molecule, one or more homing
molecules, and a plurality of membrane perturbing molecules,
wherein the homing molecules selectively home to tumor vasculature,
lung vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0026] In some forms, one or more of the membrane perturbing
molecules can be conjugated to one or more of the homing molecules.
In some forms, the homing molecules can be conjugated with the
surface molecule. In some forms, the membrane perturbing molecules
can be conjugated with the surface molecule. In some forms, one or
more of the conjugated homing molecules can be indirectly
conjugated to the surface molecule via a linker, one or more of the
conjugated membrane perturbing molecules are indirectly conjugated
to the surface molecule via a linker, or both. In some forms, the
composition can further comprise a plurality of linkers. In some
forms, at least one of the linkers can comprise polyethylene
glycol.
[0027] In some forms, the composition can bind inside tumor blood
vessels, blood vessels of regenerating tissue, blood vessels of
wounded tissue, or lung blood vessels. In some forms, the
composition can be internalized in cells. In some forms, the
composition can penetrate tissue.
[0028] In some forms, the surface molecule can comprise a
nanoparticle, a nanoworm, an iron oxide nanoworm, an iron oxide
nanoparticle, an albumin nanoparticle, a liposome, a micelle, a
phospholipid, a polymer, a microparticle, or a fluorocarbon
microbubble. In some forms, the composition can comprise at least
100 homing molecules, at least 1000 homing molecules, or at least
10,000 homing molecules. In some forms, the composition can
comprise at least 100 membrane perturbing molecules, at least 1000
membrane perturbing molecules, or at least 10,000 membrane
perturbing molecules.
[0029] In some forms, one or more of the homing molecules can be
modified homing molecules. In some forms, one or more of the homing
molecules can comprise a methylated homing molecule. In some forms,
one or more of the methylated homing molecules can comprise a
methylated amino acid segment. In some forms, one or more of the
membrane perturbing molecules can be modified membrane perturbing
molecules. In some forms, one or more of the membrane perturbing
molecules can comprise a methylated membrane perturbing molecule.
In some forms, one or more of the methylated membrane perturbing
molecules can comprise a methylated amino acid segment. In some
forms, the amino acid segment can be N- or C-methylated in at least
one position.
[0030] In some forms, the composition can further comprise one or
more moieties. In some forms, the moieties can be independently
selected from the group consisting of a therapeutic agent, a
therapeutic protein, a therapeutic compound, a therapeutic
composition, a chemotherapeutic agent, a cancer chemotherapeutic
agent, a toxin, a cytotoxic agent, Abraxane, paclitaxel, taxol,
imatinib, an anti-angiogenic agent, a pro-angiogenic agent, an
anti-inflammatory agent, an anti-arthritic agent, a TGF-.beta.
inhibitor, decorin, a systemic vasodilator, an anti-coagulant,
tissue factor pathway inhibitor (TFPI), site-inactivated factor
VIIa, a .beta.-2 agonist, salmeterol, formoterol, N-Acetylcysteine
(NAC), Superoxide Dismutase (SOD), a superoxide dismutase mimetic,
EUK-8, an endothelin (ET-1) receptor antagonist, a prostacyclin
derivative, a phosphodiesterase type 5 inhibitor, Ketoconazole, a
small interfering RNA (siRNA), a microRNA (miRNA), a polypeptide, a
nucleic acid molecule, a small molecule, a carrier, a vehicle, a
virus, a phage, a viral particle, a phage particle, a viral capsid,
a phage capsid, a virus-like particle, a liposome, a micelle, a
bead, a nanoparticle, a microparticle, a detectable agent, a
contrast agent, an imaging agent, a label, a labeling agent, a
fluorophore, fluorescein, rhodamine, FAM, a radionuclide,
indium-111, technetium-99, carbon-11, carbon-13, or a
combination.
[0031] In some forms, the tCAR composition can have a therapeutic
effect. In some forms, the co-composition or cargo composition can
have a therapeutic effect. In some forms, the therapeutic effect
can be a slowing in the increase of or a reduction of tumor burden.
In some forms, the therapeutic effect can be a slowing of the
increase of or reduction of tumor size. In some forms, the
therapeutic effect can comprise a reduction in inflammation, an
increase in speed of wound healing, reduction in amounts of scar
tissue, decrease in pain, decrease in swelling, or decrease in
necrosis. In some forms, the therapeutic effect can comprise
pulmonary vasodilation, decrease in pulmonary pressure,
anti-coagulation, airway smooth muscle relaxation, increase in
glutathione (GSH), decrease in inflammatory immune response,
inhibition of thromboxane synthesis, or inhibition of leukotriene
synthesis.
[0032] Also disclosed are methods of using truncated CAR peptides.
For example, disclosed are methods of enhancing internalization,
penetration, or both into or through a cell, tissue, or both. In
some forms, the method can comprise exposing the cell, tissue, or
both to a tCAR composition, thereby enhancing internalization,
penetration, or both into or through the cell, tissue, or both. The
tCAR composition can comprise any of the disclosed peptides or any
of the disclosed compositions.
[0033] In some forms, the method can further comprise exposing the
cell, tissue, or both to a co-composition, thereby enhancing
internalization, penetration, or both of the co-composition into or
through the cell, tissue, or both. In some forms, prior to exposing
the cell, tissue, or both, the tCAR composition and the
co-composition are not covalently coupled or non-covalently
associated with each other. In some forms, the method can further
comprise exposing the cell, tissue, or both to a cargo composition,
thereby enhancing internalization, penetration, or both of the
cargo composition into or through the cell, tissue, or both. In
some forms, the tCAR composition and the cargo composition are
covalently coupled or non-covalently associated with each
other.
[0034] In some forms, the cell, tissue, or both can be in a
subject. In some forms, the cell, tissue, or both can be exposed to
the tCAR composition by administering the tCAR composition to the
subject. In some forms, the cell, tissue, or both can be exposed to
the tCAR composition and the co-composition by administering the
tCAR composition and the co-composition to the subject. In some
forms, the cell, tissue, or both can be exposed to the tCAR
composition and the cargo composition by administering the tCAR
composition and the cargo composition to the subject.
[0035] In some forms, the tCAR composition can penetrate tissue. In
some forms, the tCAR composition can penetrate tumor vasculature,
lung vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0036] In some forms, the co-composition or cargo composition can
penetrate tissue. In some forms, the co-composition or cargo
composition can penetrate tumor vasculature, lung vasculature,
regenerating tissue, wounded tissue, angiogenic tissue, cancer
issue, lung tissue, pulmonary arterial hypertension lung
vasculature, pulmonary arterial hypertension lesions, remodeled
pulmonary arteries, interstitial space of lungs, a site of injury,
a surgical site, a tumor, a site of angiogenesis, a site of tumor
angiogenesis, a site of inflammation, or a site of arthritis.
[0037] In some forms, the tCAR composition and the co-composition
can be administered to the subject simultaneously. In some forms,
the tCAR composition and the co-composition can be administered to
the subject in a single composition comprising the tCAR composition
and the co-composition. In some forms, the tCAR composition and the
co-composition can be administered to the subject in separate
compositions. In some forms, the tCAR composition and the
co-composition can be administered to the subject at different
times. In some forms, the tCAR composition and the co-composition
can be administered to the subject in separate compositions. In
some forms, the tCAR composition and the co-composition can be
administered to the subject by separate routes. In some forms, the
tCAR composition and the co-composition are not bound to each
other.
[0038] In some forms, the co-composition or cargo composition can
comprise a therapeutic agent, a therapeutic protein, a therapeutic
compound, a therapeutic composition, a chemotherapeutic agent, a
cancer chemotherapeutic agent, a toxin, a cytotoxic agent,
Abraxane, paclitaxel, taxol, imatinib, an anti-angiogenic agent, a
pro-angiogenic agent, an anti-inflammatory agent, an anti-arthritic
agent, a TGF-.beta. inhibitor, decorin, a systemic vasodilator, an
anti-coagulant, tissue factor pathway inhibitor (TFPI),
site-inactivated factor VIIa, a .beta.-2 agonist, salmeterol,
formoterol, N-Acetylcysteine (NAC), Superoxide Dismutase (SOD), a
superoxide dismutase mimetic, EUK-8, an endothelin (ET-1) receptor
antagonist, a prostacyclin derivative, a phosphodiesterase type 5
inhibitor, Ketoconazole, a small interfering RNA (siRNA), a
microRNA (miRNA), a polypeptide, a nucleic acid molecule, a small
molecule, a carrier, a vehicle, a virus, a phage, a viral particle,
a phage particle, a viral capsid, a phage capsid, a virus-like
particle, a liposome, a micelle, a bead, a nanoparticle, a
microparticle, a detectable agent, a contrast agent, an imaging
agent, a label, a labeling agent, a fluorophore, fluorescein,
rhodamine, FAM, a radionuclide, indium-111, technetium-99,
carbon-11, carbon-13, or a combination.
[0039] In some forms, the tCAR composition can comprise one or more
accessory molecules. In some forms, the cell, tissue, or both can
be exposed to a plurality of homing molecules. In some forms, the
cell, tissue, or both can be exposed to a plurality of cargo
compositions. In some forms, the cell, tissue, or both can be
exposed to a plurality of tCAR compositions. In some forms, the
cell, tissue, or both can be exposed to a plurality of
co-compositions.
[0040] In some forms, the tCAR composition can have a therapeutic
effect. In some forms, the co-composition or cargo composition can
have a therapeutic effect. In some forms, the therapeutic effect
can be a slowing in the increase of or a reduction of tumor burden.
In some forms, the therapeutic effect can be a slowing of the
increase of or reduction of tumor size. In some forms, the
therapeutic effect can comprise a reduction in inflammation, an
increase in speed of wound healing, reduction in amounts of scar
tissue, decrease in pain, decrease in swelling, or decrease in
necrosis. In some forms, the therapeutic effect can comprise
pulmonary vasodilation, decrease in pulmonary pressure,
anti-coagulation, airway smooth muscle relaxation, increase in
glutathione (GSH), decrease in inflammatory immune response,
inhibition of thromboxane synthesis, or inhibition of leukotriene
synthesis.
[0041] In some forms, the subject has a disease or condition. In
some forms, the disease can be pulmonary or fibrotic. In some
forms, the disease or condition can be pulmonary arterial
hypertension (PAH). In some forms, the disease or condition can be
cancer. In some forms, the disease or condition can be an
autoimmune disease. In some forms, the disease or condition can be
an inflammatory disease.
[0042] In some forms, the subject can have one or more sites to be
targeted, wherein the tCAR composition homes to one or more of the
sites to be targeted. In some forms, the subject can have one or
more sites to be targeted, wherein the co-composition or cargo
composition homes to one or more of the sites to be targeted. In
some forms, the peptide can selectively home to tumor vasculature,
lung vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0043] In some forms, the tCAR composition can selectively home to
tumor vasculature, lung vasculature, regenerating tissue, wounded
tissue, angiogenic tissue, cancer issue, lung tissue, pulmonary
arterial hypertension lung vasculature, pulmonary arterial
hypertension lesions, remodeled pulmonary arteries, interstitial
space of lungs, a site of injury, a surgical site, a tumor, a site
of angiogenesis, a site of tumor angiogenesis, a site of
inflammation, or a site of arthritis. In some forms, the
co-composition or cargo composition can selectively home to tumor
vasculature, lung vasculature, regenerating tissue, wounded tissue,
angiogenic tissue, cancer issue, lung tissue, pulmonary arterial
hypertension lung vasculature, pulmonary arterial hypertension
lesions, remodeled pulmonary arteries, interstitial space of lungs,
a site of injury, a surgical site, a tumor, a site of angiogenesis,
a site of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0044] The peptide can be an activatable peptide. The peptide can
be a protease-activatable peptide. The protein or peptide can be
circular (cyclic) or can contain a loop. The peptide can be at the
C-terminal end of the protein or peptide. The peptide can comprise
a terminal carboxyl group. A blocking group can be coupled to the
terminal carboxyl group. The bond coupling the blocking group and
the terminal carboxyl group can be selected to be cleavable by a
protease, enzyme, cleaving agent, and/or cleaving conditions
present in proximity to the cell of interest. The blocking group
can be coupled to the C-terminal amino acid of the peptide. The
blocking group can be coupled to an amino acid of the peptide other
than the C-terminal amino acid of the peptide. The blocking group
can comprise or consist of an amino acid or an amino acid
sequence.
[0045] Also disclosed are methods of producing an activatable
peptide that can be activated in proximity to a cell of interest,
the method comprising forming an activatable peptide wherein a
blocking group is coupled to a peptide via a cleavable bond,
wherein the cleavable bond is cleavable by an enzyme, cleaving
agent, and/or cleaving conditions present in proximity to the cell
of interest. Also disclosed are methods of producing an activatable
peptide that can be activated in proximity to a cell of interest,
the method comprising forming an activatable peptide wherein a
blocking group is coupled to a peptide via a cleavable bond,
wherein the cleavable bond is cleavable by an enzyme, cleaving
agent, and/or cleaving conditions present in proximity to the cell
of interest. The cell can be in a subject. The enzyme, cleaving
agent, and/or cleaving conditions that is present in proximity to
the cell of interest can be identified. The enzyme, cleaving agent,
and/or cleaving conditions present in proximity to the cell of
interest can be identified prior to forming the activatable
peptide. The cleavable bond can be selected based on the enzyme
that is present in proximity to the cell of interest. The cleavable
bond can be selected based on the cleaving agent present at site
where the peptide is delivered, homes, travels or accumulates, such
as the cell of interest. The cleavable bond can be selected based
on the cleaving conditions present at site where the peptide is
delivered, homes, travels or accumulates, such as the cell of
interest. The cleavable bond can be selected prior to forming the
activatable peptide. The peptide can comprise a terminal carboxyl
group, wherein the blocking group is coupled to the terminal
carboxyl group. Also disclosed are methods of producing an
activatable peptide, the method comprising forming an activatable
peptide wherein a blocking group is coupled to a peptide via a
cleavable bond. The cleavable bond can be cleaved in any suitable
way. For example, the cleavable bond can be cleaved enzymatically
or non-enzymatically. For enzymatic cleavage, the cleaving enzyme
can be supplied or can be present at a site where the peptide is
delivered, homes, travels or accumulates. For example, the enzyme
can be present in proximity to a cell to which the peptide is
delivered, homes, travels, or accumulates. For non-enzymatic
cleavage, the peptide can be brought into contact with a cleaving
agent, can be placed in cleaving conditions, or both. A cleaving
agent is any substance that can mediate or stimulate cleavage of
the cleavable bond. Cleaving conditions can be any solution or
environmental conditions that can mediate or stimulate cleavage of
the cleavable bond.
[0046] Also disclosed are methods of forming an activatable
peptide, the method comprising causing a blocking group to be
covalently coupled to a peptide, wherein a bond coupling the
blocking group and the peptide is cleavable. Also disclosed are
methods of forming an activatable peptide, the method comprising
causing a blocking group to be covalently coupled to an amino acid
sequence, wherein the amino acid sequence comprises a peptide the
peptide, wherein a bond coupling the blocking group and the peptide
is cleavable. Also disclosed are methods of forming an activatable
peptide, the method comprising (a) selecting an amino acid sequence
for internalization into a cell and/or penetration of tissue,
wherein the amino acid sequence comprises a peptide, and (b)
causing a blocking group to be covalently coupled to the peptide,
wherein a bond coupling the blocking group and the peptide is
cleavable. The blocking group covalently coupled to the peptide
reduces or prevents internalization into a cell and/or penetration
of tissue. The blocking group covalently coupled to the peptide can
reduce or prevent internalization into a cell and/or penetration of
tissue compared to the same peptide with no blocking group. For
example, an amino acid sequence comprising tCAR sequence-cleavage
site-homing module can be made and then tested for activatability
(via cleavage of the cleavage site, for example). For example, a
pool of peptides having the amino acid sequence
CARSKNK-XXXXXXXXXXXXXXXXX (SEQ ID NO:8) can be tested for homing
and activatability. That is, such peptides can be identified by
screens using libraries. The activatable peptide can comprise the
selected amino acid sequence and the blocking group. The cell can
be in a subject. The enzyme, cleaving agent, and/or cleaving
conditions present in proximity to the cell of interest can be
identified. The enzyme, cleaving agent, and/or cleaving conditions
present in proximity to the cell of interest can be identified
prior to forming the activatable peptide. The cleavable bond can be
selected based on the enzyme that is present in proximity to the
cell of interest. The cleavable bond can be selected based on the
cleaving agent present at site where the peptide is delivered,
homes, travels or accumulates, such as the cell of interest. The
cleavable bond can be selected based on the cleaving conditions
present at site where the peptide is delivered, homes, travels or
accumulates, such as the cell of interest. The cleavable bond can
be selected prior to forming the activatable peptide. The peptide
can comprise a terminal carboxyl group, wherein the blocking group
is coupled to the terminal carboxyl group.
[0047] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0049] FIG. 1 shows the lung homing efficiency and specificity of
targeting peptides. Immunostained lung sections were analyzed for
MCT-treated and untreated rats using Aperio's Color Deconvolution
to quantify the staining intensity. The peptide-positive area
relative to the total lung cell area is shown. Control, CG7C
peptide. Three detection thresholds were set to determine weak to
strong accumulations of the peptides. Multiple sections were
analyzed for each group. Bar indicates mean.+-.S.D.
[0050] FIG. 2 is a bar graph of the percent of control without
blocking versus different peptides. The graph shows the effect of
blocking neuropilin-1 on cell binding and internalization of CAR
and tCAR phage. Error bars represent mean.+-.SEM.
[0051] FIG. 3 is a bar graph of the fold over control phageshows
the internalization of phage displaying CAR or a truncated CAR
peptide (tCAR) into cells. Error bars represent mean.+-.SEM.
[0052] FIG. 4 shows the effects of acute administration of fasudil
with (+CAR) and without co-administration of CAR (1 mg/300 g rat)
(-CAR) on right (RVSP) and left ventricle systolic pressure (LVSP).
Vasodilator effects were expressed as % reduction of baseline
pressure. Values are means of n=1-2 each.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The disclosed method and compositions can be understood more
readily by reference to the following detailed description of
particular embodiments and the Examples included therein and to the
Figures and their previous and following description.
[0054] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that they are not limited to specific synthetic methods
or specific recombinant biotechnology methods unless otherwise
specified, or to particular reagents unless otherwise specified, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to be limiting.
A. DEFINITIONS
[0055] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a pharmaceutical carrier" includes mixtures of two or
more such carriers, and the like.
[0056] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15. It is
also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0057] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0058] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
[0059] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon.
[0060] It is to be understood that the disclosed method and
compositions are not limited to specific synthetic methods,
specific analytical techniques, or to particular reagents unless
otherwise specified, and, as such, may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting.
B. GENERAL
[0061] Disclosed herein are peptides that enable intracellular
delivery, exit and tissue penetration of compositions. The delivery
can be targeted to cells or tissues of interest, such as tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, and interstitial space of lungs.
Internalization of compositions (including nanoparticles, drugs,
detectable markers, and other compounds) and their payload into
target cells and penetration into target tissue can increase the
efficiency of the targeting and the effectiveness of the
payload.
[0062] A class of peptides was recently described that improves
drug delivery by increasing penetration of drugs into tissues, such
as solid tumors. These peptides contain a C-terminal C-end Rule
(CendR) sequence motif (R/K)XX(R/K), which is responsible for cell
internalization and tissue penetration activity. CendR peptides
contain a cryptic CendR motif that is proteolytically unmasked in
certain tissues. Technology has recently been described that
provides a way to overcome the limited tissue penetration. CendR
peptides induce extravasation and tissue penetration via a
mechanism that involves cell internalization (Teesalu et al, 2009;
Sugahara et al, 2009, Sugahara et al, 2010). CendR peptides are
defined by the presence of the motif R/KXXR/K (X represents any
amino acid), which has to be at the C-terminus for the cell- and
tissue-penetration activity. The receptor for the CendR motif was
shown to be neuropilin 1 (NRP1) (Teesalu et al, 2009).
[0063] NRP1 is a modular transmembrane protein previously
identified as a receptor for various forms and isoforms of VEGF and
members of the class 3 semaphorin family (Takagi et al, 1987; He
and Tessier-Lavigne, 1997, Kolodkin et al, 1997; Soker et al,
1998). Neuropilin 2 (NRP2), the second member of the neuropilin
family, exhibits sequence and structure homology with NRP1, and
shares common ligands NRP-1, VEGFA.sub.165 among them (Chen et al,
1997; Kolodkin et al, 1997; Glutzman-Poltorak et al, 2000).
However, there are also ligands that show selective affinity for
one or the other NRP (Chen et al, 1997; Gluzman-Poltorak et al,
2000). Moreover, NRP1 and NRP2 display different expression
patterns, with NRP2 (but not NRP1) overexpressed in tumor
lymphatics (Caunt et al, 2008). In the CendR pathway, NRP1 appears
to be essential for cell internalization and tissue penetration,
whereas the role of NRP2 has not been investigated (Teesalu et al,
2009). The disclosed tCAR peptides do not rely on the CendR pathway
for cell internalization (Example 2).
[0064] Two peptides that selectively target phage to skin and
tendon wounds were identified: CARSKNKDC (CAR, SEQ ID NO:147) and
CRKDKC (CRK, SEQ ID NO:2). CAR displays homology to heparin-binding
sites in various proteins, and binds to cell surface heparan
sulfate and heparin. CRK is homologous to a segment in
thrombospondin type 1 repeat. Intravenously injected CAR and CRK
phage, and the fluorescein-labeled free peptides selectively
accumulate at wound sites, partially co-localizing with blood
vessels. The CAR peptide shows a preference for early stages of
wound healing, whereas the CRK favors wounds at later stages of
wound healing. The CAR peptide is internalized into the target
cells and delivers the fluorescent label into their nuclei. These
results show that the molecular markers in the vasculature of wound
tissues change as healing progresses. CAR peptides are described in
U.S. Patent Application Publication No. 2009-0036349.
[0065] Described herein is a truncated form of CAR (CARSKNK; tCAR;
SEQ ID NO:4). It was discovered that the truncated peptide is more
potent for cell internalization and tissue penetration than the
parent peptide CAR. These properties make tCAR a useful tool for
targeted delivery of therapeutic and diagnostic agents to breast
cancers and perhaps other types of tumors as well.
[0066] Targeted delivery of therapeutic or diagnostic agents to
tissue of interest constitutes a major goal in treatment. By
increasing the amount of a drug reaching the tissue, efficacy is
improved while side effects are reduced. This strategy relies on
the identification of the molecular signature of tissues, and
development of specific affinity ligands to carry payloads to the
tissue (Ruoslahti, 2002). Nanoparticles can be used to further
improve drug delivery and efficacy by incorporating multiple
functions and increasing the payload (Ruoslahti et al, 2010).
However, dysfunctional tumor blood vessels and high interstitial
pressure tend to prevent penetration of drugs and nanoparticles
into the tumor tissue, limiting the efficacy of the treatments
(Jain et al, 1999; Heldin et al, 2004).
[0067] iRGD is a tumor penetrating peptide, which contains a RGD
motif for recruitment to angiogenic blood vessels and a cryptic
CendR motif that is proteolytically unmasked in tumor to trigger
extravasation and tissue penetration (Sugahara et al, 2009,
Sugahara et al, 2010). As a result of proteolytic cleavage, iRGD
loses its affinity for the integrins, acquires NRP1-binding
capacity, and induces extravasation (Sugahara et al, 2009).
Importantly, co-injected drugs or particles penetrate inside the
tumor parenchyma along with iRGD, allowing an increase of treatment
efficacy in a number of different cancer models (Sugahara et al,
2010).
[0068] LyP-1 is a tumor-homing cyclic nonapeptide (sequence:
CGNKRTRGC; SEQ ID NO:10) identified by phage display (Laakkonen et
al, 2002). LyP-1 homes to tumor lymphatics, tumor cells, and tumor
macrophages by specifically binding to its receptor p32, a
mitochondrial protein expressed on the surface of these cells
(Laakkonen et al, 2002; Fogal et al, 2008). LyP-1 also homes to
atherosclerotic plaques and penetrates into their interior (Hamzah
et al, 2011, Uchida et al, 2011). Similar to iRGD, LyP-1 contains a
cryptic CendR motif, which is consistent with secondary binding to
NRP1 (and perhaps to NRP2 in the lymphatics) and involvement of the
CendR pathway. This is also consistent with previous studies
showing that LyP-1 is able to extravasate and penetrate the tumor
parenchyma (Laakkonen et al, 2002; Von Maltzahn et al, 2008;
Karmali et al, 2009). Characterization of the LyP-1 internalization
pathway is described in U.S. Pat. No. 7,919,466.
[0069] The previously identified cryptic CendR peptide, iRGD,
looses its affinity for the primary tumor receptor .alpha.v
integrin after proteolytic cleavage, and acquires affinity for NRP1
(Sugahara et al, 2009). Similarly, LyP-1 CendR fragment exhibited a
weak affinity for the primary receptor p32, indicating that cryptic
CendR peptides follow a general pattern involving loss of affinity
for the primary receptor after cleavage, and acquisition of an
affinity for NRP1. It was realized that the full inhibition of
LyP-1 internalization by the CendR fragment of LyP-1 indicates that
internalization occurs through NRP, and not through p32, even
though this cannot entirely be ruled out the participation of other
binding molecules.
[0070] VEGFA.sub.165, which induces vascular permeability through
its interaction with NRP1 (Becker et al, 2005; Mamluk et al, 2005,
Acevedo et al, 2008), binds to NRP2 as well (Soker et al, 1998;
Glutzman-Poltorak et al, 2000). Interestingly, it was discovered
that NRP2 is also a receptor for CendR peptides, although with a
lower binding capacity compared to NRP1. Because NRP2 is expressed
in tissues and cells where NRP1 is absent (Caunt et al, 2008). An
ability to bind NRP2 might be crucial for penetration of CendR
peptides in these specific tissues, an example of which may be
tumor lymphatics, which express high levels of NRP2 and are a
specific target of LyP-1 (Laakknone et al., 2002; 2004). Thus, the
distinct properties of various CendR peptides increase the
targeting possibilities offered by this technology.
[0071] In vivo screening of phage-displayed peptide libraries was
used to probe vascular specialization. This method has revealed a
large degree of heterogeneity in the vasculature; and
tissue-specific homing peptides have been identified for a large
number of normal organs and tissues (Rajotte at al., 1998; Zhang et
al., 2005; Kolonin et al., 2006), and tumors and atherosclerotic
lesions have been shown to carry their own vascular markers, both
in the blood vessels and in lymphatics (Ruoslahti, 2002; Liu et
al., 2003; Zhang et al., 2006). It was reasoned that surveying
non-malignant angiogenesis could reveal a different repertoire of
markers than has been gleaned from studies with tumor. Wounds were
chosen as the target, as wounds are one of the few the locations
where angiogenesis takes place in an adult organism.
[0072] The disclosed tCAR peptides can be specific for a particular
pathological lesion or an individual tissue. Examples include
tumors, wounded tissue, diseased lung tissued, and fibrotic tissue.
The ability of compositions to penetrate into the extravascular
space is a major factor limiting the targeting efficacy of
compositions in vivo. It has been discovered that a truncated form
of the CAR homing peptide mediates highly efficient internalization
of phage and free peptides into cells.
[0073] Various compositions can be internalized through this
mechanism. The internalization mechanism can also be used for exit
of compositions of interest from the vasculature and their spread
into tissue. The tCAR peptide can cause spread of compositions from
the vasculature (and thus can be spread into tumor tissue from an
intravenous injection, for example). tCAR peptides can also be used
to mediate passage of compositions of interest through other
suitable membranes, such as mucous membranes and the blood-brain
barrier. As used herein, "tissue penetration" and "penetration of
tissue" refer to passage into or through a tissue beyond or through
the outer or a first layer of cells or through a tissue membrane.
Such passage or penetration through tissue (which can also be
referred to as extravasation and tissue penetration) can be a
function of, for example, cell internalization and passage between
cells in the tissue. Throughout this application, when the term
"tissue penetration" is used, it is understood that such
penetration can also extend to other barriers and suitable
membranes found throughout the body, such as the blood brain
barrier.
[0074] In the case of the disclosed tCAR peptides, the sequence of
the peptides provides both homing to particular vasculature,
tissues, and cells and cell internalization and tissue pentration
at the site of accumulation. Unlike prior cell-penetrating
peptides, the disclosed tCAR internalizing element is
position-dependent--it is less active when present in positions
other than the C-terminus of the peptide.
[0075] Co-compositions and cargos of various sizes can be used with
the tCAR peptides. Including a tCAR peptide with a drug can result
in a higher concentration of the drug in, for example, a tumor
without affecting its concentration in non-tumor tissues. The
disclosed methods and compositions can also result in a broader
distribution of the drug within the targeted tissue. As a result,
drug activity can be enhanced. tCAR peptides can be combined with
numerous other elements, such as accessory molecules and homing
motifs, as well as components to be delivered and internalized,
such as co-compositions and cargo compositions.
[0076] Penetration into tumor tissue is an issue with all
anti-cancer drugs because of the high intra-tumor fluid pressure
that forces tissue fluid to flow out of the tumor, which works
against diffusion of drugs into the extravascular tumor tissue
(Jain et al., 2007). The presumed reasons are that the blood
vessels tend to be leaky and the lymphatic vessels are poorly
functional in tumors. If a drug were completely tumor-specific and
innocuous in normal tissues (and if cost were not an issue), it
would be possible to administer so much of that drug that it would
overwhelm any barriers to the delivery of sufficient doses to all
parts of the tumor. This obviously is not the case with anti-cancer
agents; drug toxicity limits the dosing, and tumor penetration is a
major obstacle. The disclosed methods and compositions can have the
highest impact on drugs that either have penetration problems, or
that are effective but highly toxic even at the standard
therapeutic doses. Essentially all anti-cancer drugs have one or
both of these problems.
[0077] It has been discovered that tCAR peptides specifically
increase the penetration of drugs into tumors, wounded or injured
tissue, regenerating tissue, injured, diseased, or fibrotic lung
tissue, and other cells and tissues. Disclosed are homing peptides
that specifically increase the penetration of compounds and
compositions into vasculatures, tissues, and cells targeted by tCAR
peptides. These peptides specifically home to target tissues,
penetrate tissue, and internalize into cells. Drug, fluorophore,
nanoparticle, etc., payloads attached to these peptides accumulate
in targeted tissues and penetrate deep into the extravascular
tissues, such as extravascular tumor tissues. However, it has also
been discovered that the payload does not need to be coupled to or
associated with the tCAR peptide. The free tCAR peptide
specifically induces tissue permeability in the targeted tissues,
allowing a co-injected drug, nanoparticle, etc., to extravasate and
penetrate into the targeted tissue tissue. This same effect can be
achieved with any cells and tissue suitable for tCAR
internalization.
[0078] Disclosed are peptides that target tumor vascularture,
regenerating tissue, wounded tissue, pulmonary tissue, fibrotic
tissue, and related tissue, that are readily internalized into
adjacent cells, and that extensively penetrate and invade tumor
tissue. The disclosed peptides can also mediate targeting,
internalization, and tissue pentration of compounds and
compositions coupled to, associated with, conjugated to, or even
co-administered with the peptide. Examples of the disclosed
peptides include peptides where the C-terminal end of the peptide
consists of the amino acid sequence CARSKNK (SEQ ID NO:4) and
peptides consisting of CARSKNK (SEQ ID NO:4). The disclosed
peptides can be used in and with a variety of compositions and
methods to, for example, enhancing internalization, penetration, or
both of such compositions into or through a cell, tissue, or both.
Such compositions and methods are also disclosed herein.
[0079] Disclosed are compounds, compositions, and methods
comprising and using truncated CAR peptides. Truncated CAR peptides
are peptides where the C-terminal end of the peptide consists of
the amino acid sequence CARSKNK (SEQ ID NO:4). Thus, disclosed
herein are isolated peptides, where the C-terminal end of the
peptide consists of the amino acid sequence CARSKNK (SEQ ID
NO:4).
[0080] In some forms, the peptide can be a modified peptide. In
some forms, the peptide can be a methylated peptide. In some forms,
one or more of the methylated peptide can comprise a methylated
amino acid segment. In some forms, the peptide can be N- or
C-methylated in at least one position. In some forms, the peptide
can be an activatable peptide. In some forms, the amino acid
sequence CARSKNK (SEQ ID NO:4) at the C-terminal end of the peptide
can be blocked by a blocking group coupled to the terminal carboxy
group. In some forms, the blocking group can comprise an amino acid
or an amino acid sequence.
[0081] Also disclosed are compositions comprising the disclosed
peptide. In some forms, the composition can further comprise a
co-composition, where the peptide and the co-composition are not
covalently coupled or non-covalently associated with each other. In
some forms, the composition can further comprise a cargo
composition, where the peptide and the cargo composition are
covalently coupled or non-covalently associated with each other. In
some forms, the peptide can selectively home to regenerating
tissue, a site of injury, a surgical site, a tumor, tumor
vasculature, a site of tumor angiogenesis, a site of inflammation,
a site of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs.
[0082] In some forms, the composition can selectively home to
regenerating tissue, a site of injury, a surgical site, a tumor,
tumor vasculature, a site of tumor angiogenesis, a site of
inflammation, a site of arthritis, lung tissue, pulmonary arterial
hypertension lung vasculature, pulmonary arterial hypertension
lesions, remodeled pulmonary arteries, or interstitial space of
lungs. In some forms, the co-composition or cargo composition can
selectively home to regenerating tissue, a site of injury, a
surgical site, a tumor, tumor vasculature, a site of tumor
angiogenesis, a site of inflammation, a site of arthritis, lung
tissue, pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries, or
interstitial space of lungs.
[0083] In some forms, the peptide and the co-composition are not
bound to each other. In some forms, the co-composition or cargo
composition can comprise a therapeutic agent. In some forms, the
co-composition or cargo composition can comprise a detection agent.
In some forms, the co-composition or cargo composition can comprise
a carrier, vehicle, or both. In some forms, the co-composition or
cargo composition can comprise a therapeutic agent, a therapeutic
protein, a therapeutic compound, a therapeutic composition, a
chemotherapeutic agent, a cancer chemotherapeutic agent, a toxin, a
cytotoxic agent, Abraxane, paclitaxel, taxol, imatinib, an
anti-angiogenic agent, a pro-angiogenic agent, an anti-inflammatory
agent, an anti-arthritic agent, a TGF-.beta. inhibitor, decorin, a
systemic vasodilator, an anti-coagulant, tissue factor pathway
inhibitor (TFPI), site-inactivated factor VIIa, a .beta.-2 agonist,
salmeterol, formoterol, N-Acetylcysteine (NAC), Superoxide
Dismutase (SOD), a superoxide dismutase mimetic, EUK-8, an
endothelin (ET-1) receptor antagonist, a prostacyclin derivative, a
phosphodiesterase type 5 inhibitor, Ketoconazole, a small
interfering RNA (siRNA), a microRNA (miRNA), a polypeptide, a
nucleic acid molecule, a small molecule, a carrier, a vehicle, a
virus, a phage, a viral particle, a phage particle, a viral capsid,
a phage capsid, a virus-like particle, a liposome, a micelle, a
bead, a nanoparticle, a microparticle, a detectable agent, a
contrast agent, an imaging agent, a label, a labeling agent, a
fluorophore, fluorescein, rhodamine, FAM, a radionuclide,
indium-111, technetium-99, carbon-11, carbon-13, or a
combination.
[0084] In some forms, the peptide can be comprised in a tCAR
composition. In some forms, the tCAR composition can comprise one
or more cargo compositions. In some forms, the tCAR composition can
comprise one or more homing molecules. In some forms, the peptide
can be comprised in a tCAR conjugate. In some forms, the tCAR
conjugate can comprise one or more cargo compositions. In some
forms, the tCAR conjugate can comprise one or more homing
molecules.
[0085] In some forms, the composition comprises a plurality of
cargo compositions. In some forms, the composition can comprise a
plurality of copies of the peptide. In some forms, the composition
can comprise a plurality of co-compositions. In some forms, the
composition can further comprise a surface molecule and a plurality
of membrane perturbing molecules. In some forms, the composition
can further comprise one or more homing molecules, wherein the
homing molecules selectively home to tumor vasculature, lung
vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0086] In some forms, the co-composition can comprise a surface
molecule, one or more homing molecules, and a plurality of membrane
perturbing molecules, wherein the homing molecules selectively home
to tumor vasculature, lung vasculature, regenerating tissue,
wounded tissue, angiogenic tissue, cancer issue, lung tissue,
pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries,
interstitial space of lungs, a site of injury, a surgical site, a
tumor, a site of angiogenesis, a site of tumor angiogenesis, a site
of inflammation, or a site of arthritis. In some forms, the cargo
composition can comprise a surface molecule, one or more homing
molecules, and a plurality of membrane perturbing molecules,
wherein the homing molecules selectively home to tumor vasculature,
lung vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0087] In some forms, one or more of the membrane perturbing
molecules can be conjugated to one or more of the homing molecules.
In some forms, the homing molecules can be conjugated with the
surface molecule. In some forms, the membrane perturbing molecules
can be conjugated with the surface molecule. In some forms, one or
more of the conjugated homing molecules can be indirectly
conjugated to the surface molecule via a linker, one or more of the
conjugated membrane perturbing molecules are indirectly conjugated
to the surface molecule via a linker, or both. In some forms, the
composition can further comprise a plurality of linkers. In some
forms, at least one of the linkers can comprise polyethylene
glycol.
[0088] In some forms, the composition can bind inside tumor blood
vessels, blood vessels of regenerating tissue, blood vessels of
wounded tissue, or lung blood vessels. In some forms, the
composition can be internalized in cells. In some forms, the
composition can penetrate tissue.
[0089] In some forms, the surface molecule can comprise a
nanoparticle, a nanoworm, an iron oxide nanoworm, an iron oxide
nanoparticle, an albumin nanoparticle, a liposome, a micelle, a
phospholipid, a polymer, a microparticle, or a fluorocarbon
microbubble. In some forms, the composition can comprise at least
100 homing molecules, at least 1000 homing molecules, or at least
10,000 homing molecules. In some forms, the composition can
comprise at least 100 membrane perturbing molecules, at least 1000
membrane perturbing molecules, or at least 10,000 membrane
perturbing molecules.
[0090] In some forms, one or more of the homing molecules can be
modified homing molecules. In some forms, one or more of the homing
molecules can comprise a methylated homing molecule. In some forms,
one or more of the methylated homing molecules can comprise a
methylated amino acid segment. In some forms, one or more of the
membrane perturbing molecules can be modified membrane perturbing
molecules. In some forms, one or more of the membrane perturbing
molecules can comprise a methylated membrane perturbing molecule.
In some forms, one or more of the methylated membrane perturbing
molecules can comprise a methylated amino acid segment. In some
forms, the amino acid segment can be N- or C-methylated in at least
one position.
[0091] In some forms, the composition can further comprise one or
more moieties. In some forms, the moieties can be independently
selected from the group consisting of a therapeutic agent, a
therapeutic protein, a therapeutic compound, a therapeutic
composition, a chemotherapeutic agent, a cancer chemotherapeutic
agent, a toxin, a cytotoxic agent, Abraxane, paclitaxel, taxol,
imatinib, an anti-angiogenic agent, a pro-angiogenic agent, an
anti-inflammatory agent, an anti-arthritic agent, a TGF-.beta.
inhibitor, decorin, a systemic vasodilator, an anti-coagulant,
tissue factor pathway inhibitor (TFPI), site-inactivated factor
VIIa, a .beta.-2 agonist, salmeterol, formoterol, N-Acetylcysteine
(NAC), Superoxide Dismutase (SOD), a superoxide dismutase mimetic,
EUK-8, an endothelin (ET-1) receptor antagonist, a prostacyclin
derivative, a phosphodiesterase type 5 inhibitor, Ketoconazole, a
small interfering RNA (siRNA), a microRNA (miRNA), a polypeptide, a
nucleic acid molecule, a small molecule, a carrier, a vehicle, a
virus, a phage, a viral particle, a phage particle, a viral capsid,
a phage capsid, a virus-like particle, a liposome, a micelle, a
bead, a nanoparticle, a microparticle, a detectable agent, a
contrast agent, an imaging agent, a label, a labeling agent, a
fluorophore, fluorescein, rhodamine, FAM, a radionuclide,
indium-111, technetium-99, carbon-11, carbon-13, or a
combination.
[0092] In some forms, the tCAR composition can have a therapeutic
effect. In some forms, the co-composition or cargo composition can
have a therapeutic effect. In some forms, the therapeutic effect
can be a slowing in the increase of or a reduction of tumor burden.
In some forms, the therapeutic effect can be a slowing of the
increase of or reduction of tumor size. In some forms, the
therapeutic effect can comprise a reduction in inflammation, an
increase in speed of wound healing, reduction in amounts of scar
tissue, decrease in pain, decrease in swelling, or decrease in
necrosis. In some forms, the therapeutic effect can comprise
pulmonary vasodilation, decrease in pulmonary pressure,
anti-coagulation, airway smooth muscle relaxation, increase in
glutathione (GSH), decrease in inflammatory immune response,
inhibition of thromboxane synthesis, or inhibition of leukotriene
synthesis.
[0093] Also disclosed are methods of using truncated CAR peptides.
For example, disclosed are methods of enhancing internalization,
penetration, or both into or through a cell, tissue, or both. In
some forms, the method can comprise exposing the cell, tissue, or
both to a tCAR composition, thereby enhancing internalization,
penetration, or both into or through the cell, tissue, or both. The
tCAR composition can comprise any of the disclosed peptides or any
of the disclosed compositions.
[0094] In some forms, the method can further comprise exposing the
cell, tissue, or both to a co-composition, thereby enhancing
internalization, penetration, or both of the co-composition into or
through the cell, tissue, or both. In some forms, prior to exposing
the cell, tissue, or both, the tCAR composition and the
co-composition are not covalently coupled or non-covalently
associated with each other. In some forms, the method can further
comprise exposing the cell, tissue, or both to a cargo composition,
thereby enhancing internalization, penetration, or both of the
cargo composition into or through the cell, tissue, or both. In
some forms, the tCAR composition and the cargo composition are
covalently coupled or non-covalently associated with each
other.
[0095] In some forms, the cell, tissue, or both can be in a
subject. In some forms, the cell, tissue, or both can be exposed to
the tCAR composition by administering the tCAR composition to the
subject. In some forms, the cell, tissue, or both can be exposed to
the tCAR composition and the co-composition by administering the
tCAR composition and the co-composition to the subject. In some
forms, the cell, tissue, or both can be exposed to the tCAR
composition and the cargo composition by administering the tCAR
composition and the cargo composition to the subject.
[0096] In some forms, the tCAR composition can penetrate tissue. In
some forms, the tCAR composition can penetrate tumor vasculature,
lung vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0097] In some forms, the co-composition or cargo composition can
penetrate tissue. In some forms, the co-composition or cargo
composition can penetrate tumor vasculature, lung vasculature,
regenerating tissue, wounded tissue, angiogenic tissue, cancer
issue, lung tissue, pulmonary arterial hypertension lung
vasculature, pulmonary arterial hypertension lesions, remodeled
pulmonary arteries, interstitial space of lungs, a site of injury,
a surgical site, a tumor, a site of angiogenesis, a site of tumor
angiogenesis, a site of inflammation, or a site of arthritis.
[0098] In some forms, the tCAR composition and the co-composition
can be administered to the subject simultaneously. In some forms,
the tCAR composition and the co-composition can be administered to
the subject in a single composition comprising the tCAR composition
and the co-composition. In some forms, the tCAR composition and the
co-composition can be administered to the subject in separate
compositions. In some forms, the tCAR composition and the
co-composition can be administered to the subject at different
times. In some forms, the tCAR composition and the co-composition
can be administered to the subject in separate compositions. In
some forms, the tCAR composition and the co-composition can be
administered to the subject by separate routes. In some forms, the
tCAR composition and the co-composition are not bound to each
other.
[0099] In some forms, the co-composition or cargo composition can
comprise a therapeutic agent, a therapeutic protein, a therapeutic
compound, a therapeutic composition, a chemotherapeutic agent, a
cancer chemotherapeutic agent, a toxin, a cytotoxic agent,
Abraxane, paclitaxel, taxol, imatinib, an anti-angiogenic agent, a
pro-angiogenic agent, an anti-inflammatory agent, an anti-arthritic
agent, a TGF-.beta. inhibitor, decorin, a systemic vasodilator, an
anti-coagulant, tissue factor pathway inhibitor (TFPI),
site-inactivated factor VIIa, a .beta.-2 agonist, salmeterol,
formoterol, N-Acetylcysteine (NAC), Superoxide Dismutase (SOD), a
superoxide dismutase mimetic, EUK-8, an endothelin (ET-1) receptor
antagonist, a prostacyclin derivative, a phosphodiesterase type 5
inhibitor, Ketoconazole, a small interfering RNA (siRNA), a
microRNA (miRNA), a polypeptide, a nucleic acid molecule, a small
molecule, a carrier, a vehicle, a virus, a phage, a viral particle,
a phage particle, a viral capsid, a phage capsid, a virus-like
particle, a liposome, a micelle, a bead, a nanoparticle, a
microparticle, a detectable agent, a contrast agent, an imaging
agent, a label, a labeling agent, a fluorophore, fluorescein,
rhodamine, FAM, a radionuclide, indium-111, technetium-99,
carbon-11, carbon-13, or a combination.
[0100] In some forms, the tCAR composition can comprise one or more
accessory molecules. In some forms, the cell, tissue, or both can
be exposed to a plurality of homing molecules. In some forms, the
cell, tissue, or both can be exposed to a plurality of cargo
compositions. In some forms, the cell, tissue, or both can be
exposed to a plurality of tCAR compositions. In some forms, the
cell, tissue, or both can be exposed to a plurality of
co-compositions.
[0101] In some forms, the tCAR composition can have a therapeutic
effect. In some forms, the co-composition or cargo composition can
have a therapeutic effect. In some forms, the therapeutic effect
can be a slowing in the increase of or a reduction of tumor burden.
In some forms, the therapeutic effect can be a slowing of the
increase of or reduction of tumor size. In some forms, the
therapeutic effect can comprise a reduction in inflammation, an
increase in speed of wound healing, reduction in amounts of scar
tissue, decrease in pain, decrease in swelling, or decrease in
necrosis. In some forms, the therapeutic effect can comprise
pulmonary vasodilation, decrease in pulmonary pressure,
anti-coagulation, airway smooth muscle relaxation, increase in
glutathione (GSH), decrease in inflammatory immune response,
inhibition of thromboxane synthesis, or inhibition of leukotriene
synthesis.
[0102] In some forms, the subject has a disease or condition. In
some forms, the disease can be pulmonary or fibrotic. In some
forms, the disease or condition can be pulmonary arterial
hypertension (PAH). In some forms, the disease or condition can be
cancer. In some forms, the disease or condition can be an
autoimmune disease. In some forms, the disease or condition can be
an inflammatory disease.
[0103] In some forms, the subject can have one or more sites to be
targeted, wherein the tCAR composition homes to one or more of the
sites to be targeted. In some forms, the subject can have one or
more sites to be targeted, wherein the co-composition or cargo
composition homes to one or more of the sites to be targeted. In
some forms, the peptide can selectively home to tumor vasculature,
lung vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0104] In some forms, the tCAR composition can selectively home to
tumor vasculature, lung vasculature, regenerating tissue, wounded
tissue, angiogenic tissue, cancer issue, lung tissue, pulmonary
arterial hypertension lung vasculature, pulmonary arterial
hypertension lesions, remodeled pulmonary arteries, interstitial
space of lungs, a site of injury, a surgical site, a tumor, a site
of angiogenesis, a site of tumor angiogenesis, a site of
inflammation, or a site of arthritis. In some forms, the
co-composition or cargo composition can selectively home to tumor
vasculature, lung vasculature, regenerating tissue, wounded tissue,
angiogenic tissue, cancer issue, lung tissue, pulmonary arterial
hypertension lung vasculature, pulmonary arterial hypertension
lesions, remodeled pulmonary arteries, interstitial space of lungs,
a site of injury, a surgical site, a tumor, a site of angiogenesis,
a site of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0105] The disease can be selected from the group consisting of
pulmonary hypertension, interstitial lung disease, acute lung
injury (ALI), acute respiratory distress syndrome (ARDS), sepsis,
septic shock, sarcoidosis of the lung, pulmonary manifestations of
connective tissue diseases, including systemic lupus erythematosus,
rheumatoid arthritis, scleroderma, and polymyositis,
dermatomyositis, bronchiectasis, asbestosis, berylliosis,
silicosis, Histiocytosis X, pneumotitis, smoker's lung,
bronchiolitis obliterans, the prevention of lung scarring due to
tuberculosis and pulmonary fibrosis, other fibrotic diseases such
as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, angiogenic diseases, wound healing, infections, trauma
injuries and systemic connective tissue diseases including systemic
lupus erythematosus, rheumatoid arthritis, scleroderma,
polymyositis, and dermatomyositis.
[0106] Tissue-penetrating tCAR peptides can be used, for example,
to augment, tissue imaging and treatment of targeted tissues. For
example, tumor imaging and tumor treatment with anti-cancer drugs.
FDA-approved imaging agents, such as iron oxide nanoparticle MRI
contrast agent, can be injected into subjects with a tCAR peptide
followed by imaging. Any known or future drug can be used with tCAR
peptides to affect targeted tissues. For example, the
co-composition can be any clinically used drugs. For example, the
co-composition can be any clinically used anti-cancer drugs. Drug
accumulation and distribution in tumor tissue, as well as
anti-tumor efficacy can be determined using known techniques
(examples of such are described herein). As another example, the
co-composition can be any clinically used drugs for improving wound
healing, reducing scar formation, etc.
[0107] The disclosed enhancement of internalization and tissue
penetration has broad application. Using the disclosed tCAR
peptides, the effective targeting, delivery, and penetration of any
drug, compound or composition can be augmented and enhanced. The
effect of tCAR peptides has several significant implications.
First, drugs and other compounds and compositions can be delivered
to cells and tissues of interest at higher concentrations than is
possible in standard therapy. This is a result of the increased
internalization and tissue penetration mediated by the tCAR
peptide. This is particularly significant because the amount of
drug that can be administered is generally limited by side effects.
Increasing the drug concentration at the target without increasing
the amount of drug administered can thus extend and enhance the
effectiveness of any known or future drugs and therapeutics. When
using tCAR peptides, the increase in drug concentration only occurs
in target cells and tissues and not in non-target tissues. In such
cases, the efficacy of the treatment can be increased, while side
effects can remain the same. Second, the dose or amount of drug or
other compound or composition can be reduced without compromising
the efficacy of the treatment. The tCAR peptide would result in the
same drug concentration at the target cell or tissue even though
the amount of drug administered is less. Third, because the
adjuvant tCAR peptide and the drug, imaging agent, or other
compound or composition need not be coupled to one another, a
validated and approved tCAR peptide can be used to augment any
drug, imaging agent, or other compound or composition.
[0108] In another example, the tCAR peptides can be used in
nanomedicine. One of the main goals of nanomedicine is to design
devices that surpass simple drugs by performing multiple functions
in diagnosing, monitoring, and treating disease. New technologies
can be applied to solve some of the main problems in the medical
uses of multifunctional nanoparticles, such as poor penetration
into extravascular tissue.
[0109] Disclosed are tCAR compositions, tCAR conjugates, tCAR
molecules, tCAR proteins, and tCAR peptides. tCAR peptides are the
basic feature of tCAR compositions, tCAR conjugates, tCAR
molecules, tCAR proteins, and the like. tCAR compositions are any
composition, conglomeration, conjugate, molecule, protein, peptide,
etc. that comprises a tCAR peptide. tCAR conjugates are
associations, whether covalent or non-covalent, of a tCAR peptide
and one or more other elements, peptides, proteins, compounds,
molecules, agents, compounds, etc. For example, a tCAR conjugate
can comprise a tCAR peptide, tCAR protein, tCAR compound, tCAR
molecule, etc. tCAR molecules are molecules that comprise a tCAR
peptide. For example, a tCAR molecule can comprise a tCAR protein,
tCAR peptide, etc. In general, tCAR peptides, tCAR proteins, tCAR
molecules, and tCAR conjugates are all forms of tCAR compositions.
tCAR compounds, tCAR peptides and tCAR proteins can be forms of
tCAR molecules. Unless the context indicates otherwise, reference
to a tCAR composition is intended to refer to tCAR compositions,
tCAR molecules, tCAR proteins, tCAR peptides, and the like. A tCAR
component is any molecule, peptide, protein, compound, conjugate,
composition, etc. that comprises a tCAR peptide. Examples of tCAR
components include, for example, tCAR compositions, tCAR molecules,
tCAR proteins, and tCAR peptides.
[0110] tCAR components can comprise one or more tCAR peptides.
Where a tCAR element comprises two or more tCAR peptides, it is
useful for the tCAR component to be designed to allow some or all
of the tCAR peptides to be exposed at the C-terminus of a protein
or peptide. This can be accomplished in numerous ways in, for
example, conjugates and compositions. This can also be accomplished
in, for example, branching peptides and proteins.
[0111] Disclosed are peptides that target tumors, regenerating
tissue, sites of injury, surgical sites, tumor vasculature, sites
of tumor angiogenesis, sites of inflammation, sites of arthritis,
lung tissue, pulmonary arterial hypertension lung vasculature,
pulmonary arterial hypertension lesions, remodeled pulmonary
arteries, and interstitial space of lungs, are readily internalized
into adjacent cells, and extensively penetrate and invade the
targeted tissue. The disclosed peptides can also mediate targeting,
internalization, and tissue pentration of compounds and
compositions coupled to, associated with, conjugated to, or even
co-administered with the peptide. Examples of the disclosed
peptides include peptides where the C-terminal end of the peptide
consists of the amino acid sequence CARSKNK (SEQ ID NO:4) and
peptides consisting of CARSKNK (SEQ ID NO:4). The disclosed
peptides can be used in and with a variety of compositions and
methods to, for example, enhancing internalization, penetration, or
both of such compositions into or through a cell, tissue, or both.
Such compositions and methods are also disclosed herein.
[0112] Disclosed are peptides where the C-terminal end of the
peptide consists of the amino acid sequence CARSKNK (SEQ ID NO:4).
In some forms, the peptide can be a modified peptide. In some
forms, the peptide can be a methylated peptide. In some forms, one
or more of the methylated peptide can comprise a methylated amino
acid segment. In some forms, the peptide can be N- or C-methylated
in at least one position.
[0113] tCAR peptides are peptides consisting of or having a
C-terminal end with the amino acid sequence CARSKNK (SEQ ID NO:4).
tCAR peptides can be composed of standard amino acids with standard
peptide linkages or can be embodied in other than standard amino
acids and/or with other than standard peptide linkages. tCAR
peptides can include modifications to the peptide, amino acids,
and/or linkages. Examples of suitable modifications known to those
in the art and are described elsewhere herein. Variant tCAR
peptides can be used in place of or in addition to tCAR peptides.
Variant tCAR peptides are not tCAR peptides.
[0114] Disclosed are compositions comprising the disclosed tCAR
peptide. In some forms, the composition can further comprise a
co-composition, where the peptide and the co-composition are not
covalently coupled or non-covalently associated with each other. In
some forms, the composition can further comprise a cargo
composition, where the peptide and the cargo composition are
covalently coupled or non-covalently associated with each
other.
[0115] In some forms, the peptide can selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. In
some forms, the peptide can selectively home to tumor vasculature.
In some forms, the peptide and the co-composition are not bound to
each other. In some forms, the co-composition and/or cargo
composition can comprise a therapeutic agent. In some forms, the
co-composition and/or cargo composition can comprise a detection
agent. In some forms, the co-composition and/or cargo composition
can comprise a carrier, vehicle, or both. In some forms, the
co-composition and/or cargo composition can comprise a therapeutic
protein, a therapeutic compound, a therapeutic composition, a
cancer chemotherapeutic agent, a toxin, a cytotoxic agent, a virus,
a phage, a viral particle, a phage particle, a viral capsid, a
phage capsid, a virus-like particle, a liposome, a micelle, a bead,
a nanoparticle, a microparticle, a chemotherapeutic agent, a
contrast agent, an imaging agent, a label, a labeling agent, an
anti-angiogenic agent, or a combination.
[0116] In some forms, the peptide can be comprised in a tCAR
composition. In some forms, the tCAR composition can comprise one
or more cargo compositions. In some forms, the tCAR composition can
comprise one or more homing molecules. In some forms, the peptide
can be comprised in a tCAR conjugate. In some forms, the tCAR
conjugate can comprise one or more cargo compositions. In some
forms, the tCAR conjugate can comprise one or more homing
molecules. In some forms, the composition can comprise a plurality
of cargo compositions. In some forms, the composition can comprise
a plurality of copies of the peptide. In some forms, the
composition can comprise a plurality of co-compositions.
[0117] In some forms, the composition can further comprise a
surface molecule and a plurality of membrane perturbing molecules.
In some forms, the composition can further comprise one or more
homing molecules, wherein the homing molecules selectively home to
tumors, regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. In
some forms, the co-composition can comprise a surface molecule, one
or more homing molecules, and a plurality of membrane perturbing
molecules, wherein the homing molecules selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. In
some forms, the cargo composition can comprise a surface molecule,
one or more homing molecules, and a plurality of membrane
perturbing molecules, wherein the homing molecules selectively home
to tumors, regenerating tissue, sites of injury, surgical sites,
tumor vasculature, sites of tumor angiogenesis, sites of
inflammation, sites of arthritis, lung tissue, pulmonary arterial
hypertension lung vasculature, pulmonary arterial hypertension
lesions, remodeled pulmonary arteries, or interstitial space of
lungs.
[0118] Pulmonary arterial hypertension (PAH) is a disorder of the
pulmonary vasculature associated with elevated pulmonary vascular
resistance. Despite recent advances in the treatment of PAH, with
eight approved clinical therapies and additional therapies
undergoing clinical trials, PAH remains a serious, life-threatening
condition. The lack of pulmonary vascular selectivity and
associated systemic adverse effects of these therapies remain the
main obstacles to successful treatment. Peptide-mediated drug
delivery that specifically targets the vasculature of PAH lungs may
offer a solution to the lack of drug selectivity. Here, highly
selective targeting of rat PAH lesions by a novel cyclic peptide,
CARSKNKDC (CAR; SEQ ID NO:147) is shown. Intravenous administration
of CAR peptide resulted in intense accumulation of the peptide in
monocrotaline-induced and SU5416/hypoxia-induced hypertensive lungs
but not in the normal healthy lungs or in other organs of the PAH
rats. CAR homed to all layers of remodeled pulmonary arteries, i.e.
endothelium, neointima, medial smooth muscle, and adventitia, in
the hypertensive lungs. CAR also homed to capillary vessels and
accumulated in the interstitial space of the PAH lungs, manifesting
its extravasation activity. The results demonstrated a remarkable
ability of CAR to selectively target PAH lung vasculature,
effectively penetrate, and spread throughout the diseased lung
tissue. These results indicate clinical utility of CAR in targeted
delivery of therapeutic compounds and imaging probes to the PAH
lungs.
[0119] In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative derivative thereof, the amino acid sequence CRKDKC
(SEQ ID NO:2) or a conservative derivative thereof, or a
combination. In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative variant thereof. In some forms, one or more of the
homing molecules can comprise the amino acid sequence CGKRK (SEQ ID
NO:1). In some forms, all of the one or more homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1) or a
conservative derivative thereof, the amino acid sequence CRKDKC
(SEQ ED NO:2) or a conservative derivative thereof, or a
combination.
[0120] In some forms, one or more of the membrane perturbing
molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative variant
thereof, (KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative variant
thereof, (KLAKKLA).sub.2 (SEQ ID NO:5) or a conservative variant
thereof, (KAAKKAA).sub.2 (SEQ ID NO:6) or a conservative variant
thereof, or (KLGKKLG).sub.3 (SEQ ID NO:7) or a conservative variant
thereof, or a combination. In some forms, one or more of the
membrane perturbing molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3), (KLAKLAK).sub.2 (SEQ ID NO:3),
(KLAKKLA).sub.2 (SEQ ID NO:5), (KAAKKAA).sub.2 (SEQ ID NO:6), or
(KLGKKLG).sub.3 (SEQ ID NO:7), or a combination. In some forms, one
or more of the membrane perturbing molecules can comprise the amino
acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative
variant thereof. In some forms, one or more of the membrane
perturbing molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3).
[0121] In some forms, one or more of the membrane perturbing
molecules can be conjugated to one or more of the homing molecules.
In some forms, the homing molecules can be conjugated with the
surface molecule. In some forms, the membrane perturbing molecules
can be conjugated with the surface molecule. In some forms, one or
more of the conjugated homing molecules can be indirectly
conjugated to the surface molecule via a linker, one or more of the
conjugated membrane perturbing molecules can be indirectly
conjugated to the surface molecule via a linker, or both. In some
forms, the composition can further comprise a plurality of linkers.
In some forms, at least one of the linkers can comprise
polyethylene glycol.
[0122] In some forms, the composition can bind inside tumor blood
vessels. In some forms, the composition can be internalized in
cells. In some forms, the composition can penetrate tissue. In some
forms, the composition can reduce tumor growth.
[0123] In some forms, the surface molecule can comprise a
nanoparticle, a nanoworm, an iron oxide nanoworm, an iron oxide
nanoparticle, an albumin nanoparticle, a liposome, a micelle, a
phospholipid, a polymer, a microparticle, or a fluorocarbon
microbubble. In some forms, the composition can comprise at least
100 homing molecules. In some forms, the composition can comprise
at least 1000 homing molecules. In some forms, the composition can
comprise at least 10,000 homing molecules. In some forms, the
composition can comprise at least 100 membrane perturbing
molecules. In some forms, the composition can comprise at least
1000 membrane perturbing molecules. In some forms, the composition
can comprise at least 10,000 membrane perturbing molecules. In some
forms, the composition can comprise at least 100 copies of the
peptide. In some forms, the composition can comprise at least 1000
copies of the peptide. In some forms, the composition can comprise
at least 10,000 copies of the peptide.
[0124] In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise a plurality of
surface molecules, a plurality of homing molecules and a plurality
of cargo molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise one
or more surface molecules, a plurality of homing molecules and a
plurality of cargo molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise a
plurality of surface molecules, one or more homing molecules and a
plurality of cargo molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise a
plurality of surface molecules, a plurality of homing molecules and
one or more cargo molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise one
or more surface molecules, one or more homing molecules and a
plurality of cargo molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise one
or more surface molecules, a plurality of homing molecules and one
or more cargo molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise a
plurality of surface molecules, one or more homing molecules and
one or more cargo molecules.
[0125] In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise a surface
molecule, a plurality of homing molecules and a plurality of cargo
molecules, wherein one or more of the homing molecules and one or
more of the cargo molecules are associated with the surface
molecule. In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise a surface
molecule, a plurality of homing molecules and a plurality of cargo
molecules, wherein a plurality of the plurality of homing molecules
and a plurality of the plurality of cargo molecules are associated
with the surface molecule. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise a
surface molecule, a plurality of homing molecules and a plurality
of cargo molecules, wherein the homing molecules and the cargo
molecules are associated with the surface molecule.
[0126] In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise a surface
molecule, wherein the surface molecule is multivalent for homing
molecules and cargo molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise a
surface molecule, wherein the surface molecule is multivalent for
homing molecules and comprises one or more cargo molecules. In some
forms, the composition, tCAR composition, co-composition, or cargo
composition can comprise a surface molecule, wherein the surface
molecule is multivalent for cargo molecules and comprises one or
more homing molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise a
surface molecule, wherein the surface molecule is multivalent for
conjugates, wherein one or more of the conjugates comprise one or
more homing molecules and one or more cargo molecules. In some
forms, the composition, tCAR composition, co-composition, or cargo
composition can comprise a surface molecule, wherein the surface
molecule is multivalent for conjugates, wherein one or more of the
conjugates comprise a plurality of homing molecules and a plurality
cargo molecules. In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise a surface
molecule, wherein the surface molecule is multivalent for
conjugates, wherein one or more of the conjugates comprise a homing
molecule and a cargo molecule. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise a
surface molecule, wherein the surface molecule is multivalent for
conjugates, wherein each of the conjugates comprises a plurality of
homing molecules and a plurality cargo molecules. In some forms,
the composition, tCAR composition, co-composition, or cargo
composition can comprise a surface molecule, wherein the surface
molecule is multivalent for conjugates, wherein each of the
conjugates comprises a homing molecule and a cargo molecule. As
used herein, a component that is stated to be "multivalent for" one
or more other components refers to a component that has a plurality
of the other components associated with, conjugated to and/or
covalent coupled to the first component.
[0127] In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise a surface
molecule, wherein the surface molecule comprises one or more
conjugates, wherein one or more of the conjugates comprise one or
more homing molecules and one or more cargo molecules. In some
forms, the composition, tCAR composition, co-composition, or cargo
composition can comprise a surface molecule, wherein the surface
molecule comprises one or more conjugates, wherein one or more of
the conjugates comprise a plurality of homing molecules and a
plurality cargo molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise a
surface molecule, wherein the surface molecule comprises one or
more conjugates, wherein one or more of the conjugates comprise a
homing molecule and a cargo molecule. In some forms, the
composition, tCAR composition, co-composition, or cargo composition
can comprise a surface molecule, wherein the surface molecule
comprises one or more conjugates, wherein each of the conjugates
comprises a plurality of homing molecules and a plurality cargo
molecules. In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise a surface
molecule, wherein the surface molecule comprises one or more
conjugates, wherein each of the conjugates comprises a homing
molecule and a cargo molecule.
[0128] In some forms, one or more of the membrane perturbing
molecules can be conjugated to one or more of the homing molecules.
In some forms, one or more of the conjugated membrane perturbing
molecules and homing molecules can be covalently coupled. In some
forms, one or more of the covalently coupled membrane perturbing
molecules and homing molecules can comprise fusion peptides. In
some forms, the homing molecules can be conjugated with the surface
molecule. In some forms, one or more of the conjugated homing
molecules can be directly conjugated to the surface molecule. In
some forms, one or more of the conjugated homing molecules can be
indirectly conjugated to the surface molecule. In some forms, one
or more of the homing molecules can be covalently coupled to the
surface molecule. In some forms, one or more of the covalently
coupled homing molecules can be directly covalently coupled to the
surface molecule. In some forms, one or more of the covalently
coupled homing molecules can be indirectly covalently coupled to
the surface molecule. In some forms, the membrane perturbing
molecules can be conjugated with the surface molecule. In some
forms, one or more of the conjugated membrane perturbing molecules
are directly conjugated to the surface molecule. In some forms, one
or more of the conjugated membrane perturbing molecules can be
indirectly conjugated to the surface molecule. In some forms, one
or more of the membrane perturbing molecules can be covalently
coupled to the surface molecule. In some forms, one or more of the
covalently coupled membrane perturbing molecules can be directly
covalently coupled to the surface molecule. In some forms, one or
more of the covalently coupled membrane perturbing molecules can be
indirectly covalently coupled to the surface molecule.
[0129] In some forms, the surface molecule can comprise a
nanoparticle. In some forms, the surface molecule can comprise a
nanoworm. In some forms, the surface molecule can comprise an iron
oxide nanoworm. In some forms, the surface molecule can comprise an
iron oxide nanoparticle. In some forms, the surface molecule can
comprise an albumin nanoparticle. In some forms, the surface
molecule can comprise a liposome. In some forms, the surface
molecule can comprise a micelle. In some forms, the surface
molecule comprises a phospholipid. In some forms, the surface
molecule comprises a polymer. In some forms, the surface molecule
can comprise a microparticle. In some forms, the surface molecule
can comprise a fluorocarbon microbubble.
[0130] In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise at least 100
homing molecules. In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise at least 1000
homing molecules. In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise at least 10,000
homing molecules. In some forms, the composition, tCAR composition,
co-composition, or cargo composition can comprise at least 100
membrane perturbing molecules. In some forms, the composition, tCAR
composition, co-composition, or cargo composition can comprise at
least 1000 membrane perturbing molecules. In some forms, the
composition, tCAR composition, co-composition, or cargo composition
can comprise at least 10,000 membrane perturbing molecules. In some
forms, the composition, tCAR composition, co-composition, or cargo
composition can comprise at least 100 tCAR peptides. In some forms,
the composition, tCAR composition, co-composition, or cargo
composition can comprise at least 1000 tCAR peptides. In some
forms, the composition, tCAR composition, co-composition, or cargo
composition can comprise at least 10,000 tCAR peptides.
[0131] In some forms, one or more of the homing molecules can be
modified homing molecules. In some forms, one or more of the homing
molecules can comprise a methylated homing molecule. In some forms,
one or more of the methylated homing molecules can comprise a
methylated amino acid segment. In some forms, one or more of the
membrane perturbing molecules can be modified membrane perturbing
molecules. In some forms, one or more of the membrane perturbing
molecules can comprise a methylated membrane perturbing molecule.
In some forms, one or more of the methylated membrane perturbing
molecules can comprise a methylated amino acid segment. In some
forms, the amino acid segment can be N- or C-methylated in at least
one position.
[0132] In some forms, the composition can further comprise one or
more moieties. In some forms, the moieties can be independently
selected from the group consisting of, for example, an
anti-angiogenic agent, a pro-angiogenic agent, a cancer
chemotherapeutic agent, a cytotoxic agent, a polypeptide, a nucleic
acid molecule, a small molecule, an image contrast agent, a
fluorophore, fluorescein, rhodamine, a radionuclide, indium-111,
technetium-99, carbon-11, and carbon-13. In some forms, at least
one of the moieties can be a therapeutic agent. In some forms, the
therapeutic agent can be Abraxane. In some forms, the therapeutic
agent can be paclitaxel. In some forms, the therapeutic agent can
be taxol. In some forms, at least one of the moieties can be a
detectable agent. In some forms, the detectable agent can be
FAM.
[0133] The term "bioactive agent" refers to a substance which is
used in connection with an application that is therapeutic or
diagnostic in nature, such as in methods for diagnosing the
presence or absence of a disease in a patient and/or in methods for
treating a disease in a patient. Therapeutic agents and detectable
agents are examples of bioactive agents. As to compatible bioactive
agents, those skilled in the art will appreciate that any
therapeutic or diagnostic agent may be incorporated in the
stabilized dispersions of the disclosed compositions. For example,
the bioactive agent may be selected from the group consisting of
antiallergics, bronchodilators, vasodilators, antihypertensive
agents, bronchoconstrictors, pulmonary lung surfactants,
analgesics, antibiotics, leukotriene inhibitors or antagonists,
anticholinergics, mast cell inhibitors, antihistamines,
anti-inflammatories, anti-neoplastics, anesthetics,
anti-tuberculars, imaging agents, cardiovascular agents, enzymes,
steroids, genetic material, viral vectors, antisense agents, small
molecule drugs, proteins, peptides and combinations thereof.
Particularly preferred bioactive agents comprise compounds which
are to be administered systemically (i.e. to the systemic
circulation of a patient) such as small molecule drugs, imaging
agents, peptides, proteins or polynucleotides. As is disclosed in
more detail elsewhere herein, the bioactive agent can be
incorporated, blended in, coated on or otherwise associated with
the targeting peptide disclosed herein. Particularly preferred
bioactive agents for use in the disclosed compositions and methods
include anti-allergics, peptides and proteins, bronchodilators,
anti-inflammatory agents and anti-cancer compounds for use in the
treatment of disorders involving diseased tissue reflecting altered
heparan sulfate variants specific to said disease. Yet another
associated advantage of the disclosed compositions and methods is
the effective delivery of bioactive agents administered or combined
with a targeting peptide.
[0134] In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1), where one or
more of the membrane perturbing molecules can comprise the amino
acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID NO:3), where one or
more of the homing molecules can be indirectly conjugated to the
surface molecule via a linker, and where one or more of the
membrane perturbing molecules can be indirectly conjugated to the
surface molecule via a linker. In some forms, at least one of the
linkers can comprise polyethylene glycol.
[0135] Also disclosed are methods of enhancing internalization,
penetration, or both of a co-composition into or through a cell,
tissue, or both. In some forms, the method can comprising exposing
the cell, tissue, or both to the co-composition and a tCAR
composition, thereby enhancing internalization, penetration, or
both of the co-composition into or through the cell, tissue, or
both. The tCAR composition can comprise any of the disclosed tCAR
peptides or any of the disclosed compositions that comprise a tCAR
peptide. In some forms, the tCAR composition and the co-composition
are not covalently coupled or non-covalently associated with each
other prior to exposing the cell, tissue, or both.
[0136] Also disclosed are methods of enhancing internalization,
penetration, or both of a cargo composition into or through a cell,
tissue, or both. In some forms, the method can comprise exposing
the cell, tissue, or both to the cargo composition and a tCAR
composition, thereby enhancing internalization, penetration, or
both of the cargo composition into or through the cell, tissue, or
both. The tCAR composition can comprise any of the disclosed tCAR
peptides or any of the disclosed compositions that comprise a tCAR
peptide. In some forms, the tCAR composition and the cargo
composition can be covalently coupled or non-covalently associated
with each other.
[0137] Also disclosed are methods of enhancing internalization,
penetration, or both into or through a cell, tissue, or both. In
some forms, the method can comprise exposing the cell, tissue, or
both to a tCAR composition, thereby enhancing internalization,
penetration, or both into or through the cell, tissue, or both. The
tCAR composition can comprise any of the disclosed tCAR peptides or
any of the disclosed compositions that comprise a tCAR peptide.
[0138] In some forms, the cell, tissue, or both can be in a
subject. In some forms, the cell, tissue, or both can be exposed to
the tCAR composition and the co-composition by administering the
tCAR composition and the co-composition to the subject. In some
forms, the cell, tissue, or both can be exposed to the tCAR
composition and the cargo composition by administering the tCAR
composition and the cargo composition to the subject. In some
forms, the cell, tissue, or both can be exposed to the tCAR
composition by administering the tCAR composition to the
subject.
[0139] In some forms, the tCAR composition can selectively home to
tumors, regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. In
some forms, the tCAR composition can selectively home to tumor
vasculature. In some forms, the tCAR composition and the
co-composition can be administered to the subject simultaneously.
In some forms, the tCAR composition and the co-composition can be
administered to the subject in a single composition comprising the
tCAR composition and the co-composition. In some forms, the tCAR
composition and the co-composition can be administered to the
subject in separate compositions. In some forms, the tCAR
composition and the co-composition can be administered to the
subject at different times. In some forms, the tCAR composition and
the co-composition can be administered to the subject in separate
compositions. In some forms, the tCAR composition and the
co-composition can be administered to the subject by separate
routes.
[0140] In some forms, the tCAR composition and the co-composition
are not bound to each other. In some forms, the tCAR composition,
co-composition, and/or cargo composition can comprise a therapeutic
agent. In some forms, the tCAR composition, co-composition, and/or
cargo composition can comprise a detection agent. In some forms,
the tCAR composition, co-composition, and/or cargo composition can
comprise a carrier, vehicle, or both. In some forms, the tCAR
composition, co-composition, and/or cargo composition can comprise
a therapeutic protein, a therapeutic compound, a therapeutic
composition, an anti-angiogenic agent, a pro-angiogenic agent, a
cancer chemotherapeutic agent, a toxin, a cytotoxic agent, an
anti-inflammatory agent, an anti-arthritic agent, a growth factor,
a cytokine, a chemokine, a compound that modulates one or more
signaling pathways, an antibody, a nucleic acid, a nucleic acid
analog, a cell, a virus, a phage, a viral particle, a phage
particle, a viral capsid, a phage capsid, a virus-like particle, a
liposome, a micelle, a bead, a nanoparticle, a microparticle, a
chemotherapeutic agent, a contrast agent, an imaging agent, a
label, a labeling agent, or a combination.
[0141] In some forms, the tCAR composition can comprise one or more
accessory molecules. In some forms, the cell, tissue, or both can
be exposed to a plurality of homing molecules. In some forms, the
cell, tissue, or both can be exposed to a plurality of cargo
compositions. In some forms, the cell, tissue, or both can be
exposed to a plurality of tCAR compositions. In some forms, the
cell, tissue, or both can be exposed to a plurality of
co-compositions.
[0142] In some forms, the tCAR composition, co-composition, and/or
cargo composition can have a therapeutic effect. In some forms, the
therapeutic effect can be a slowing in the increase of or a
reduction of tumor burden. In some forms, the therapeutic effect
can be a slowing of the increase of or reduction of tumor size. In
some forms, the subject can have one or more sites to be targeted,
where the tCAR composition, co-composition, and/or cargo
composition homes to one or more of the sites to be targeted. In
some forms, the subject can have a tumor, where the tCAR
composition, co-composition, and/or cargo composition has a
therapeutic effect on the tumor. In some forms, the tCAR
composition, co-composition, and/or cargo composition can penetrate
tissue. In some forms, the tCAR composition, co-composition, and/or
cargo composition can penetrate tumor vasculature, lung
vasculature, regenerating tissue, wounded tissue, angiogenic
tissue, cancer issue, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, interstitial space of lungs, a site
of injury, a surgical site, a tumor, a site of angiogenesis, a site
of tumor angiogenesis, a site of inflammation, or a site of
arthritis.
[0143] Multiple different tCAR peptides, tCAR compounds, tCAR
conjugates, tCAR compositions, or a combination can be used
together. Similarly, multiple different co-compositions, multiple
different cargo compositions, or a combination can be used
together. Where such multiple different tCAR peptides, tCAR
compounds, tCAR conjugates, tCAR compositions, or a combination are
used together, they can be used with a single type of
co-composition, a single type of cargo composition, multiple
different co-compositions, multiple different cargo compositions,
or a combination. Similarly, when multiple different
co-compositions, multiple different cargo compositions, or a
combination can be used together, they can be used with a single
type of tCAR peptide, tCAR compound, tCAR conjugate, tCAR
composition, or with multiple different tCAR peptides, tCAR
compounds, tCAR conjugates, tCAR compositions, or a
combination.
[0144] For example, a CARSKNK peptide (SEQ ID NO:4) can be used
together with one or multiple different tCAR peptides, tCAR
compounds, tCAR conjugates, tCAR compositions, or a combination,
one or multiple different co-compositions, multiple different cargo
compositions, or a combination, or any combination of these. In
such combinations, the CARSKNK peptide (SEQ ID NO:4) itself can be
combined in the same conjugate or composition with one or more
cargo compositions, one or more accessory molecules, one or more
homing molecules, etc.
[0145] The cell, tissue, or both can be exposed to combinations of
different tCAR components and combinations of different
co-compositions by administering the tCAR components and the
co-compositions to the subject. One or more of the tCAR components
and one or more of the co-compositions can be administered to the
subject simultaneously. One or more of the tCAR components and one
or more of the co-compositions can be administered to the subject
in one or more single compositions comprising the tCAR component(s)
and the co-composition(s). One or more of the tCAR components and
one or more of the co-compositions can be administered to the
subject in one or more separate compositions. One or more of the
tCAR components and one or more of the co-compositions can be
administered to the subject at different times. The tCAR
composition and the co-composition can be administered to the
subject in one or more separate compositions. One or more of the
tCAR components and one or more of the co-compositions can be
administered to the subject by one or more separate routes. In some
forms, the tCAR composition and the co-composition are not bound to
each other.
[0146] The cell, tissue, or both can be exposed to combinations of
different tCAR components and combinations of different cargo
compositions by administering the tCAR components and the cargo
compositions to the subject. One or more of the tCAR components and
one or more of the cargo compositions can be administered to the
subject simultaneously. One or more of the tCAR components and one
or more of the cargo compositions can be administered to the
subject in one or more single compositions comprising the tCAR
component(s) and the cargo composition(s). One or more of the tCAR
components and one or more of the cargo compositions can be
administered to the subject in one or more separate compositions.
One or more of the tCAR components and one or more of the cargo
compositions can be administered to the subject at different times.
The tCAR composition and the cargo composition can be administered
to the subject in one or more separate compositions. One or more of
the tCAR components and one or more of the cargo compositions can
be administered to the subject by one or more separate routes.
[0147] The tCAR peptide can be comprised in an amino acid sequence
in a protein or peptide. In some forms, the protein or peptide can
be internalized into a cell, penetrate tissue, or both when the
amino acid sequence is present in the protein or peptide but not
when the amino acid sequence is not present in the protein or
peptide. In some forms, the protein or peptide can penetrate tissue
when the amino acid sequence is present in the protein or peptide
but not when the amino acid sequence is not present in the protein
or peptide. In some forms, the protein or peptide can be
internalized into a cell and penetrate tissue when the amino acid
sequence is present in the protein or peptide but not when the
amino acid sequence is not present in the protein or peptide. In
some forms, the amino acid sequence can be internalized into a
cell, penetrate tissue, or both without being associated with the
co-composition. In some forms, the amino acid sequence can
penetrate tissue without being associated with the co-composition.
In some forms, the amino acid sequence can be internalized into a
cell and penetrate tissue without being associated with the
co-composition. In some forms, the amino acid sequence is the only
functional internalization element in the protein or peptide.
[0148] In some forms, the internalization, penetration, or both of
the co-composition into or through a cell, tissue, or both is
enhanced when the cell, tissue, or both is exposed to the tCAR
peptide but not when the cell, tissue, or both is not exposed to
the tCAR peptide. In some forms, the penetration of the
co-composition into or through tissue is enhanced when the tissue
is exposed to the tCAR peptide but not when the tissue is not
exposed to the tCAR peptide. In some forms, the internalization and
penetration of the co-composition into or through a cell and tissue
is enhanced when the cell and tissue are exposed to the tCAR
peptide but not when the cell and tissue is not exposed to the tCAR
peptide. In some forms, the internalization, penetration, or both
of the co-composition into or through a cell, tissue, or both is
enhanced when the amino acid sequence is present in the protein or
peptide but not when the amino acid sequence is not present in the
protein or peptide. In some forms, the penetration of the
co-composition into or through tissue is enhanced when the amino
acid sequence is present in the protein or peptide but not when the
amino acid sequence is not present in the protein or peptide. In
some forms, the internalization and penetration of the
co-composition into or through a cell and tissue is enhanced when
the amino acid sequence is present in the protein or peptide but
not when the amino acid sequence is not present in the protein or
peptide.
[0149] In some forms, the internalization, penetration, or both of
the cargo composition into or through a cell, tissue, or both is
enhanced when the cell, tissue, or both is exposed to the tCAR
peptide but not when the cell, tissue, or both is not exposed to
the tCAR peptide. In some forms, the penetration of the cargo
composition into or through tissue is enhanced when the tissue is
exposed to the tCAR peptide but not when the tissue is not exposed
to the tCAR peptide. In some forms, the internalization and
penetration of the cargo composition into or through a cell and
tissue is enhanced when the cell and tissue are exposed to the tCAR
peptide but not when the cell and tissue is not exposed to the tCAR
peptide. In some forms, the internalization, penetration, or both
of the cargo composition into or through a cell, tissue, or both is
enhanced when the amino acid sequence is present in the protein or
peptide but not when the amino acid sequence is not present in the
protein or peptide. In some forms, the penetration of the cargo
composition into or through tissue is enhanced when the amino acid
sequence is present in the protein or peptide but not when the
amino acid sequence is not present in the protein or peptide. In
some forms, the internalization and penetration of the cargo
composition into or through a cell and tissue is enhanced when the
amino acid sequence is present in the protein or peptide but not
when the amino acid sequence is not present in the protein or
peptide.
[0150] The tCAR peptide can be associated with one or more
accessory molecules. For example, an accessory molecule can be a
part of an amino acid sequence, a protein, or a peptide that
comprises the tCAR peptide. As another example, the accessory
molecule can be covalently coupled or non-covalently associated
with the tCAR peptide or an amino acid sequence, a protein, or a
peptide that comprises the tCAR peptide. The accessory molecule can
be separate from or overlapping with the tCAR peptide. For example,
some accessory molecules are amino acid sequences. This can allow
the amino acid sequence consisting of the tCAR peptide to overlap
the amino acid sequence that consists of the accessory amino acid
sequence. Alternatively the accessory peptide can be a separate
entity that does not overlap with the tCAR peptide. For example, a
HER2 binding peptide, CREKA peptide, NGR peptide, or an RGD peptide
that is not a tCAR peptide can consist of amino acid sequence that
does not overlap with a tCAR peptide. In some forms, the accessory
molecule can comprise a sequence in, for example, a tCAR peptide
that binds to a specific receptor distinct from the receptor for
the tCAR peptide.
[0151] The amino acid sequence can comprise one or more accessory
peptides. The protein or peptide can comprise one or more accessory
peptides. In some forms, the co-composition does not comprise an
accessory molecule. The co-composition can comprise one or more
accessory molecules. In some forms, the co-composition does not
comprise an accessory peptide. The co-composition can comprise one
or more accessory peptides. The co-composition can selectively home
to tumors, regenerating tissue, sites of injury, surgical sites,
tumor vasculature, sites of tumor angiogenesis, sites of
inflammation, sites of arthritis, lung tissue, pulmonary arterial
hypertension lung vasculature, pulmonary arterial hypertension
lesions, remodeled pulmonary arteries, or interstitial space of
lungs. In some forms, the co-composition does not selectively home
to tumor vasculature. The co-composition can selectively home to
tumor vasculature. In some forms, the cargo composition does not
comprise an accessory molecule. The cargo composition can comprise
one or more accessory molecules. In some forms, the cargo
composition does not comprise an accessory peptide. The cargo
composition can comprise one or more accessory peptides. The cargo
composition can selectively home to tumors, regenerating tissue,
sites of injury, surgical sites, tumor vasculature, sites of tumor
angiogenesis, sites of inflammation, sites of arthritis, lung
tissue, pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries, or
interstitial space of lungs. In some forms, the cargo composition
does not selectively home to tumor vasculature. The cargo
composition can selectively home to tumor vasculature.
[0152] The peptide can be an activatable peptide. The activatable
peptide can be a protease-activatable peptide. The tCAR peptide can
be an activatable tCAR peptide. The activatable tCAR peptide can be
a protease-activatable tCAR peptide. The tCAR peptide can be at the
C-terminal end of the protein or peptide. The tCAR conjugate can be
an activatable tCAR conjugate. The activatable tCAR conjugate can
be a protease-activatable tCAR conjugate. The tCAR conjugate can be
at the C-terminal end of the protein or peptide. The tCAR
composition can be an activatable tCAR composition. The activatable
tCAR composition can be a protease-activatable tCAR composition.
The tCAR composition can be at the C-terminal end of the protein or
peptide.
[0153] The tCAR peptide can be associated with one or more homing
molecules. For example, a homing molecule can be a part of an amino
acid sequence, a protein, or a peptide that comprises the tCAR
peptide. As another example, the homing molecule can be covalently
coupled or non-covalently associated with the tCAR peptide or an
amino acid sequence, a protein, or a peptide that comprises the
tCAR peptide. The homing molecule can be separate from or
overlapping with the tCAR peptide. For example, some homing
molecules are amino acid sequences. This can allow the amino acid
sequence consisting of the tCAR peptide to overlap the amino acid
sequence that consists of the homing amino acid sequence.
Alternatively the homing peptide can be a separate entity that does
not overlap with the tCAR peptide. For example, a HER2 binding
peptide, CREKA peptide, NGR peptide, or an RGD peptide that is not
a tCAR peptide can consist of amino acid sequence that does not
overlap with a tCAR peptide. In some forms, the homing molecule can
comprise a sequence in, for example, a peptide that binds to a
specific receptor distinct from the receptor for the tCAR
peptide.
[0154] Many homing molecules and homing peptides home to the
vasculature of the target tissue. However, for the sake of
convenience homing is referred to in some places herein as homing
to the tissue associated with the vasculature to which the homing
molecule or homing peptide may actually home. Thus, for example, a
homing peptide that homes to tumor vasculature can be referred to
herein as homing to tumor tissue or to tumor cells. By including or
associating a homing molecule or homing peptide with, for example,
a protein, peptide, amino acid sequence, co-composition, or cargo
composition, the protein, peptide, amino acid sequence,
co-composition, or cargo composition can be targeted or can home to
the target of the homing molecule or homing peptide. In this way,
the protein, peptide, amino acid sequence, co-composition, or cargo
composition, or can be said to home to the target of the homing
molecule or homing peptide. For convenience and unless otherwise
indicated, reference to homing of a protein, peptide, amino acid
sequence, co-composition, cargo composition, etc. is intended to
indicate that the protein, peptide, amino acid sequence,
co-composition, cargo composition, etc. includes or is associated
with an appropriate homing molecule or homing peptide.
[0155] The amino acid sequence can be selected for internalization
into a cell. The amino acid sequence can be selected for tissue
penetration. The amino acid sequence can be selected for
internalization into a cell and tissue penetration. The amino acid
sequence can comprise one or more homing peptides. The amino acid
sequence can comprise a tCAR peptide.
[0156] In some forms, the tCAR peptide and the co-composition are
not covalently coupled or non-covalently associated with each
other. In some forms, the co-composition does not comprise a
functional internalization element. The co-composition can comprise
a functional internalization element. In some forms, the
co-composition does not comprise a homing molecule. The
co-composition can comprise one or more homing molecules. In some
forms, the co-composition does not comprise a homing peptide. The
co-composition can comprise one or more homing peptides. The
co-composition can selectively home to tumors, regenerating tissue,
sites of injury, surgical sites, tumor vasculature, sites of tumor
angiogenesis, sites of inflammation, sites of arthritis, lung
tissue, pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries, or
interstitial space of lungs. In some forms, the co-composition does
not selectively home to tumor vasculature. The co-composition can
selectively home to tumor vasculature.
[0157] In some forms, the tCAR peptide and the cargo composition
are not covalently coupled or non-covalently associated with each
other. In some forms, the cargo composition does not comprise a
functional internalization element. The cargo composition can
comprise a functional internalization element. In some forms, the
cargo composition does not comprise a homing molecule. The cargo
composition can comprise one or more homing molecules. In some
forms, the cargo composition does not comprise a homing peptide.
The cargo composition can comprise one or more homing peptides. The
cargo composition can selectively home to tumors, regenerating
tissue, sites of injury, surgical sites, tumor vasculature, sites
of tumor angiogenesis, sites of inflammation, sites of arthritis,
lung tissue, pulmonary arterial hypertension lung vasculature,
pulmonary arterial hypertension lesions, remodeled pulmonary
arteries, or interstitial space of lungs. In some forms, the cargo
composition does not selectively home to tumor vasculature. The
cargo composition can selectively home to tumor vasculature.
[0158] The tCAR peptide can be the only functional internalization
element in the tCAR composition, conjugate, molecule, protein,
peptide, etc., the tCAR peptide can be the only functional tissue
penetration element in the tCAR composition, conjugate, molecule,
protein, peptide, etc., or both. The selected amino acid sequence
can be the only functional internalization element in the tCAR
composition, conjugate, molecule, protein, peptide, etc., the
selected amino acid sequence can be the only functional tissue
penetration element in the tLtP-1 composition, conjugate, molecule,
protein, peptide, etc., or both.
[0159] Disclosed herein is a method of forming a tCAR composition,
the method comprising selecting an amino acid sequence for
internalization into a cell, and causing a tCAR peptide to be
covalently coupled to or non-covalently associated with the
selected amino acid sequence, wherein the tCAR composition
comprises the selected amino acid sequence and the coupled or
associated tCAR peptide.
[0160] Disclosed is a method of making a tCAR composition
comprising: (a) selecting an amino acid sequence for
internalization into a cell, (b) causing a tCAR peptide to be
covalently coupled to or non-covalently associated with the
selected amino acid sequence, wherein the tCAR composition
comprises the selected amino acid sequence and the coupled or
associated tCAR peptide.
[0161] Also disclosed is a method of delivering a co-composition
into a cell, the method comprising: exposing the cell to a tCAR
composition and the co-composition, wherein the tCAR composition
can then enter the cell, thereby delivering the co-composition into
the cell.
[0162] Also disclosed is a method of causing a co-composition to
penetrate tissue, the method comprising: exposing the tissue to a
tCAR composition and the co-composition, wherein the tCAR
composition can then enter and exit cells in the tissue, thereby
causing the co-composition to penetrate the tissue.
[0163] Also disclosed is a method of delivering a cargo composition
into a cell, the method comprising: exposing the cell to a tCAR
composition and the cargo composition, wherein the tCAR composition
can then enter the cell, thereby delivering the cargo composition
into the cell.
[0164] Also disclosed is a method of causing a cargo composition to
penetrate tissue, the method comprising: exposing the tissue to a
tCAR composition and the cargo composition, wherein the tCAR
composition can then enter and exit cells in the tissue, thereby
causing the cargo composition to penetrate the tissue.
[0165] Also disclosed is a method of delivering a cargo composition
into a cell, the method comprising: exposing the cell to a tCAR
composition and the cargo composition, wherein the tCAR composition
comprises the cargo composition, wherein the tCAR composition can
then enter the cell, thereby delivering the cargo composition into
the cell.
[0166] Also disclosed is a method of causing a cargo composition to
penetrate tissue, the method comprising: exposing the tissue to a
tCAR composition and the cargo composition, wherein the tCAR
composition comprises the cargo composition, wherein the tCAR
composition can then enter and exit cells in the tissue, thereby
causing the cargo composition to penetrate the tissue.
[0167] Further disclosed is a method of delivering a composition
into a cell, the method comprising: exposing the cell to the
composition and a tCAR composition comprising an activatable tCAR
peptide, whereupon a cleaving agent activates the activatable tCAR
peptide of the tCAR composition, wherein the tCAR composition can
then enter the cell, thereby delivering the composition into the
cell.
[0168] Further disclosed is a method of causing a composition to
penetrate tissue, the method comprising: exposing the tissue to the
composition and a tCAR composition comprising an activatable tCAR
peptide, whereupon a cleaving agent activates the activatable tCAR
peptide of the tCAR composition, wherein the tCAR composition can
then enter and pass cells in the tissue, thereby causing the
composition to penetrate the tissue.
[0169] Further disclosed is a method of delivering a co-composition
into a cell, the method comprising: exposing the cell to the
co-composition and a tCAR composition comprising an activatable
tCAR peptide, whereupon a cleaving agent activates the activatable
tCAR peptide of the tCAR composition, wherein the tCAR composition
can then enter the cell, thereby delivering the co-composition into
the cell.
[0170] Further disclosed is a method of causing a co-composition to
penetrate tissue, the method comprising: exposing the tissue to the
co-composition and a tCAR composition comprising an activatable
tCAR peptide, whereupon a cleaving agent activates the activatable
tCAR peptide of the tCAR composition, wherein the tCAR composition
can then enter and pass cells in the tissue, thereby causing the
co-composition to penetrate the tissue.
[0171] Further disclosed is a method of delivering a cargo
composition into a cell, the method comprising: exposing the cell
to the cargo composition and a tCAR composition comprising an
activatable tCAR peptide, wherein the tCAR composition comprises
the cargo composition, whereupon a cleaving agent activates the
activatable tCAR peptide of the tCAR composition, wherein the tCAR
composition can then enter the cell, thereby delivering the cargo
composition into the cell.
[0172] Further disclosed is a method of causing a cargo composition
to penetrate tissue, the method comprising: exposing the tissue to
the cargo composition and a tCAR composition comprising an
activatable tCAR peptide, wherein the tCAR composition comprises
the cargo composition, whereupon a cleaving agent activates the
activatable tCAR peptide of the tCAR composition, wherein the tCAR
composition can then enter and pass cells in the tissue, thereby
causing the cargocomposition to penetrate the tissue.
[0173] Cells that can internalize a tCAR peptide can be identified
by (a) exposing a cell to a tCAR peptide; and (b) determining if
the tCAR peptide was internalized. The cell can be in an assay, for
example. Cells that can internalize an activatable peptide can be
identified by (a) exposing a cell to an activatable peptide; (b)
determining if the activatable peptide was internalized. The
activatable peptide can be unblocked before exposure to the cell,
but does not need to be. This can be used to test the blocking
ability of the blocker, for example.
[0174] Cancer cells, or subjects harboring cancer cells, can be
identified as candidates for tCAR-based therapy by (a) exposing the
cancer cell to a tCAR peptide; and (b) determining if the tCAR
peptide was internalized by the cancer cell, wherein an
internalized tCAR peptide identifies the cancer cell or the subject
as being a candidate for tCAR-based therapy. The cell can be in an
assay, or can be in a subject, for example.
[0175] Tumors, or subjects harboring a tumor, can be identified as
a candidate for tCAR-based therapy by (a) exposing tissue from the
tumor to a tCAR peptide; and (b) determining if the tCAR peptide
passed through the tissue or was internalized by cells in the
tissue, wherein a passed-through or internalized tCAR peptide
identifies the tumor or the subject as being a candidate for
tCAR-based therapy.
[0176] In some forms, the tCAR peptide and the co-composition are
not covalently coupled or non-covalently associated with each
other. In some forms, the tCAR conjugate and the co-composition are
not covalently coupled or non-covalently associated with each
other. In some forms, the tCAR composition and the co-composition
are not covalently coupled or non-covalently associated with each
other. In some forms, the tCAR peptide and the cargo composition
are covalently coupled or non-covalently associated with each
other. In some forms, the tCAR conjugate and the cargo composition
are covalently coupled or non-covalently associated with each
other. In some forms, the tCAR composition and the cargo
composition are covalently coupled or non-covalently associated
with each other.
[0177] Disclosed are compositions comprising a tCAR peptide and a
co-composition. Also disclosed are compositions comprising a tCAR
conjugate and a co-composition. Also disclosed are compositions
comprising a tCAR composition and a co-composition. Disclosed are
compositions comprising a tCAR peptide and a co-composition,
wherein the tCAR peptide and the co-composition are not covalently
coupled or non-covalently associated with each other. Also
disclosed are compositions comprising a tCAR conjugate and a
co-composition, wherein the tCAR conjugate and the co-composition
are not covalently coupled or non-covalently associated with each
other. Also disclosed are compositions comprising a tCAR
composition and a co-composition, wherein the tCAR composition and
the co-composition are not covalently coupled or non-covalently
associated with each other.
[0178] Disclosed are compositions comprising a tCAR peptide and one
or more co-compositions. Also disclosed are compositions comprising
a tCAR conjugate and one or more co-composition. Also disclosed are
compositions comprising a tCAR composition and one or more
co-compositions. Disclosed are compositions comprising a tCAR
peptide and one or more co-compositions, wherein the tCAR peptide
and at least one of the co-compositions are not covalently coupled
or non-covalently associated with each other. Also disclosed are
compositions comprising a tCAR conjugate and one or more
co-compositions, wherein the tCAR conjugate and at least one of the
co-compositions are not covalently coupled or non-covalently
associated with each other. Also disclosed are compositions
comprising a tCAR composition and one or more co-compositions,
wherein the tCAR composition and at least one of the
co-compositions are not covalently coupled or non-covalently
associated with each other.
[0179] Disclosed are compositions comprising a tCAR peptide and a
cargo composition. Also disclosed are compositions comprising a
tCAR conjugate and a cargo composition. Also disclosed are
compositions comprising a tCAR composition and a cargo composition.
Disclosed are compositions comprising a tCAR peptide and a cargo
composition, wherein the tCAR peptide and the cargo composition are
covalently coupled or non-covalently associated with each other.
Also disclosed are compositions comprising a tCAR conjugate and a
cargo composition, wherein the tCAR conjugate and the cargo
composition are covalently coupled or non-covalently associated
with each other. Also disclosed are compositions comprising a tCAR
composition and a cargo composition, wherein the tCAR composition
and the cargo composition are covalently coupled or non-covalently
associated with each other.
[0180] Disclosed are compositions comprising a tCAR peptide and one
or more cargo compositions. Also disclosed are compositions
comprising a tCAR conjugate and one or more cargo composition. Also
disclosed are compositions comprising a tCAR composition and one or
more cargo compositions. Disclosed are compositions comprising a
tCAR peptide and one or more cargo compositions, wherein the tCAR
peptide and at least one of the cargo compositions are covalently
coupled or non-covalently associated with each other. Also
disclosed are compositions comprising a tCAR conjugate and one or
more cargo compositions, wherein the tCAR conjugate and at least
one of the cargo compositions are covalently coupled or
non-covalently associated with each other. Also disclosed are
compositions comprising a tCAR composition and one or more cargo
compositions, wherein the tCAR composition and at least one of the
cargo compositions are covalently coupled or non-covalently
associated with each other.
[0181] Also disclosed are compositions comprising a tCAR peptide,
an accessory molecule, and a co-composition, wherein the tCAR
peptide and the co-composition are not covalently coupled or
non-covalently associated with each other, wherein the tCAR peptide
and the accessory molecule are covalently coupled or non-covalently
associate with each other. Also disclosed are compositions
comprising a tCAR conjugate, an accessory molecule, and a
co-composition, wherein the tCAR conjugate and the co-composition
are not covalently coupled or non-covalently associated with each
other, wherein the tCAR conjugate and the accessory molecule are
covalently coupled or non-covalently associate with each other.
Also disclosed are compositions comprising a tCAR composition, an
accessory molecule, and a co-composition, wherein the tCAR
composition and the co-composition are not covalently coupled or
non-covalently associated with each other, wherein the tCAR
composition and the accessory molecule are covalently coupled or
non-covalently associate with each other. Also disclosed are
compositions comprising a tCAR peptide, an accessory molecule, and
a co-composition, wherein the tCAR peptide and the co-composition
are not covalently coupled or non-covalently associated with each
other, wherein the tCAR peptide comprises the accessory molecule.
Also disclosed are compositions comprising a tCAR conjugate, an
accessory molecule, and a co-composition, wherein the tCAR
conjugate and the co-composition are not covalently coupled or
non-covalently associated with each other, wherein the tCAR
conjugate comprises the accessory molecule. Also disclosed are
compositions comprising a tCAR composition, an accessory molecule,
and a co-composition, wherein the tCAR composition and the
co-composition are not covalently coupled or non-covalently
associated with each other, wherein the tCAR composition comprises
the accessory molecule. In these compositions, the accessory
molecule can be or can comprise an accessory peptide. In these
compositions, the composition can comprise one or more
co-compositions and/or one or more accessory molecules, wherein the
tCAR peptide, tCAR conjugate, or tCAR composition and at least one
of the co-compositions are not covalently coupled or non-covalently
associated with each other, wherein the tCAR peptide, tCAR
conjugate, or tCAR composition and at least one of the accessory
molecules are covalently coupled or non-covalently associated with
each other.
[0182] Also disclosed are compositions comprising a tCAR peptide, a
homing molecule, and a co-composition, wherein the tCAR peptide and
the co-composition are not covalently coupled or non-covalently
associated with each other, wherein the tCAR peptide and the homing
molecule are covalently coupled or non-covalently associate with
each other. Also disclosed are compositions comprising a tCAR
conjugate, a homing molecule, and a co-composition, wherein the
tCAR conjugate and the co-composition are not covalently coupled or
non-covalently associated with each other, wherein the tCAR
conjugate and the homing molecule are covalently coupled or
non-covalently associate with each other. Also disclosed are
compositions comprising a tCAR composition, a homing molecule, and
a co-composition, wherein the tCAR composition and the
co-composition are not covalently coupled or non-covalently
associated with each other, wherein the tCAR composition and the
homing molecule are covalently coupled or non-covalently associate
with each other. Also disclosed are compositions comprising a tCAR
peptide, a homing molecule, and a co-composition, wherein the tCAR
peptide and the co-composition are not covalently coupled or
non-covalently associated with each other, wherein the tCAR peptide
comprises the homing molecule. Also disclosed are compositions
comprising a tCAR conjugate, a homing molecule, and a
co-composition, wherein the tCAR conjugate and the co-composition
are not covalently coupled or non-covalently associated with each
other, wherein the tCAR conjugate comprises the homing molecule.
Also disclosed are compositions comprising a tCAR composition, a
homing molecule, and a co-composition, wherein the tCAR composition
and the co-composition are not covalently coupled or non-covalently
associated with each other, wherein the tCAR composition comprises
the homing molecule. In these compositions, the homing molecule can
be or can comprise a homing peptide. In these compositions, the
composition can comprise one or more co-compositions and/or one or
more homing molecules, wherein the tCAR peptide, tCAR conjugate, or
tCAR composition and at least one of the co-compositions are not
covalently coupled or non-covalently associated with each other,
wherein the tCAR peptide, tCAR conjugate, or tCAR composition and
at least one of the homing molecules are covalently coupled or
non-covalently associated with each other.
[0183] Also disclosed are compositions comprising a tCAR peptide,
an accessory molecule, and a cargo composition, wherein the tCAR
peptide and the cargo composition are covalently coupled or
non-covalently associated with each other, wherein the tCAR peptide
and the accessory molecule are covalently coupled or non-covalently
associate with each other. Also disclosed are compositions
comprising a tCAR conjugate, an accessory molecule, and a cargo
composition, wherein the tCAR conjugate and the cargo composition
are covalently coupled or non-covalently associated with each
other, wherein the tCAR conjugate and the accessory molecule are
covalently coupled or non-covalently associate with each other.
Also disclosed are compositions comprising a tCAR composition, an
accessory molecule, and a cargo composition, wherein the tCAR
composition and the cargo composition are covalently coupled or
non-covalently associated with each other, wherein the tCAR
composition and the accessory molecule are covalently coupled or
non-covalently associate with each other. Also disclosed are
compositions comprising a tCAR peptide, an accessory molecule, and
a cargo composition, wherein the tCAR peptide and the cargo
composition are covalently coupled or non-covalently associated
with each other, wherein the tCAR peptide comprises the accessory
molecule. Also disclosed are compositions comprising a tCAR
conjugate, an accessory molecule, and a cargo composition, wherein
the tCAR conjugate and the cargo composition are covalently coupled
or non-covalently associated with each other, wherein the tCAR
conjugate comprises the accessory molecule. Also disclosed are
compositions comprising a tCAR composition, an accessory molecule,
and a cargo composition, wherein the tCAR composition and the cargo
composition are covalently coupled or non-covalently associated
with each other, wherein the tCAR composition comprises the
accessory molecule. In these compositions, the accessory molecule
can be or can comprise an accessory peptide. In these compositions,
the composition can comprise one or more cargo compositions and/or
one or more accessory molecules, wherein the tCAR peptide, tCAR
conjugate, or tCAR composition and at least one of the cargo
compositions are covalently coupled or non-covalently associated
with each other, wherein the tCAR peptide, tCAR conjugate, or tCAR
composition and at least one of the accessory molecules are
covalently coupled or non-covalently associated with each
other.
[0184] Also disclosed are compositions comprising a tCAR peptide, a
homing molecule, and a cargo composition, wherein the tCAR peptide
and the cargo composition are covalently coupled or non-covalently
associated with each other, wherein the tCAR peptide and the homing
molecule are covalently coupled or non-covalently associate with
each other. Also disclosed are compositions comprising a tCAR
conjugate, a homing molecule, and a cargo composition, wherein the
tCAR conjugate and the cargo composition are covalently coupled or
non-covalently associated with each other, wherein the tCAR
conjugate and the homing molecule are covalently coupled or
non-covalently associate with each other. Also disclosed are
compositions comprising a tCAR composition, a homing molecule, and
a cargo composition, wherein the tCAR composition and the cargo
composition are covalently coupled or non-covalently associated
with each other, wherein the tCAR composition and the homing
molecule are covalently coupled or non-covalently associate with
each other. Also disclosed are compositions comprising a tCAR
peptide, a homing molecule, and a cargo composition, wherein the
tCAR peptide and the cargo composition are covalently coupled or
non-covalently associated with each other, wherein the tCAR peptide
comprises the homing molecule. Also disclosed are compositions
comprising a tCAR conjugate, a homing molecule, and a cargo
composition, wherein the tCAR conjugate and the cargo composition
are covalently coupled or non-covalently associated with each
other, wherein the tCAR conjugate comprises the homing molecule.
Also disclosed are compositions comprising a tCAR composition, a
homing molecule, and a cargo composition, wherein the tCAR
composition and the cargo composition are covalently coupled or
non-covalently associated with each other, wherein the tCAR
composition comprises the homing molecule. In these compositions,
the homing molecule can be or can comprise a homing peptide. In
these compositions, the composition can comprise one or more cargo
compositions and/or one or more homing molecules, wherein the tCAR
peptide, tCAR conjugate, or tCAR composition and at least one of
the cargo compositions are not covalently coupled or non-covalently
associated with each other, wherein the tCAR peptide, tCAR
conjugate, or tCAR composition and at least one of the homing
molecules are covalently coupled or non-covalently associated with
each other.
[0185] Also disclosed are compositions comprising a protein or
peptide and a co-composition, wherein the protein or peptide
comprises a tCAR peptide and an accessory peptide, wherein the tCAR
peptide and the co-composition are not covalently coupled or
non-covalently associated with each other. Also disclosed are
compositions comprising a protein or peptide and a co-composition,
wherein the protein or peptide comprises an amino acid sequence,
wherein the amino acid sequence comprises a tCAR peptide and an
accessory peptide, wherein the tCAR peptide and the co-composition
are not covalently coupled or non-covalently associated with each
other. In these compositions, the composition can comprise one or
more co-compositions and/or one or more accessory peptides, wherein
the protein or peptide and at least one of the co-compositions are
not covalently coupled or non-covalently associated with each
other, wherein the protein or peptide and at least one of the
accessory peptides are covalently coupled or non-covalently
associated with each other.
[0186] Also disclosed are compositions comprising a protein or
peptide and a co-composition, wherein the protein or peptide
comprises a tCAR peptide and a homing peptide, wherein the tCAR
peptide and the co-composition are not covalently coupled or
non-covalently associated with each other. Also disclosed are
compositions comprising a protein or peptide and a co-composition,
wherein the protein or peptide comprises an amino acid sequence,
wherein the amino acid sequence comprises a tCAR peptide and a
homing peptide, wherein the tCAR peptide and the co-composition are
not covalently coupled or non-covalently associated with each
other. In these compositions, the composition can comprise one or
more co-compositions and/or one or more homing peptides, wherein
the protein or peptide and at least one of the co-compositions are
not covalently coupled or non-covalently associated with each
other, wherein the protein or peptide and at least one of the
homing peptides are covalently coupled or non-covalently associated
with each other.
[0187] Also disclosed are compositions comprising a protein or
peptide and a cargo composition, wherein the protein or peptide
comprises a tCAR peptide and an accessory peptide, wherein the tCAR
peptide and the cargo composition are covalently coupled or
non-covalently associated with each other. Also disclosed are
compositions comprising a protein or peptide and a cargo
composition, wherein the protein or peptide comprises an amino acid
sequence, wherein the amino acid sequence comprises a tCAR peptide
and an accessory peptide, wherein the tCAR peptide and the cargo
composition are covalently coupled or non-covalently associated
with each other. In these compositions, the composition can
comprise one or more cargo compositions and/or one or more
accessory peptides, wherein the protein or peptide and at least one
of the cargo compositions are covalently coupled or non-covalently
associated with each other, wherein the protein or peptide and at
least one of the accessory peptides are covalently coupled or
non-covalently associated with each other.
[0188] Also disclosed are compositions comprising a protein or
peptide and a cargo composition, wherein the protein or peptide
comprises a tCAR peptide and a homing peptide, wherein the tCAR
peptide and the cargo composition are covalently coupled or
non-covalently associated with each other. Also disclosed are
compositions comprising a protein or peptide and a cargo
composition, wherein the protein or peptide comprises an amino acid
sequence, wherein the amino acid sequence comprises a tCAR peptide
and a homing peptide, wherein the tCAR peptide and the cargo
composition are covalently coupled or non-covalently associated
with each other. In these compositions, the composition can
comprise one or more cargo compositions and/or one or more homing
peptides, wherein the protein or peptide and at least one of the
cargo compositions are not covalently coupled or non-covalently
associated with each other, wherein the protein or peptide and at
least one of the homing peptides are covalently coupled or
non-covalently associated with each other.
[0189] As used herein, reference to components (such as a tCAR
peptide and a co-composition) as being "not covalently coupled"
means that the components are not connected via covalent bonds (for
example, that the tCAR peptide and the co-composition are not
connected via covalent bonds). That is, there is no continuous
chain of covalent bonds between, for example, the tCAR peptide and
the co-composition. Conversely, reference to components (such as a
tCAR peptide and a cargo composition) as being "covalently coupled"
means that the components are connected via covalent bonds (for
example, that the tCAR peptide and the cargo composition are
connected via covalent bonds). That is, there is a continuous chain
of covalent bonds between, for example, the tCAR peptide and the
cargo composition. Components can be covalently coupled either
directly or indirectly. Direct covalent coupling refers to the
presence of a covalent bond between atoms of each of the
components. Indirect covalent coupling refers to the absence of a
covalent bond between atoms of each of the components. That is,
some other atom or atoms not belonging to either of the coupled
components intervenes between atoms of the components. Both direct
and indirect covalent coupling involve a continuous chain of
covalent bonds.
[0190] Non-covalent association refers to association of components
via non-covalent bonds and interactions. A non-covalent association
can be either direct or indirect. A direct non-covalent association
refers to a non-covalent bond involving atoms that are each
respectively connected via a chain of covalent bonds to the
components. Thus, in a direct non-covalent association, there is no
other molecule intervening between the associated components. An
indirect non-covalent association refers to any chain of molecules
and bonds linking the components where the components are not
covalently coupled (that is, there is a least one separate molecule
other than the components intervening between the components via
non-covalent bonds).
[0191] Reference to components (such as a tCAR peptide and a
co-composition) as not being "non-covalently associated" means that
there is no direct or indirect non-covalent association between the
components. That is, for example, no atom covalently coupled to a
tCAR peptide is involved in a non-covalent bond with an atom
covalently coupled to a co-composition. Within this meaning, a tCAR
peptide and a co-composition can be together in a composition where
they are indirectly associated via multiple intervening
non-covalent bonds while not being non-covalently associated as
that term is defined herein. For example, a tCAR peptide and a
co-composition can be mixed together in a carrier where they are
not directly non-covalently associated. A tCAR peptide and a
co-composition that are referred to as not indirectly
non-covalently associated cannot be mixed together in a continuous
composition. Reference to components (such as a tCAR peptide and a
co-composition) as not being "directly non-covalently associated"
means that there is no direct non-covalent association between the
components (an indirect non-covalent association may be present).
Reference to components (such as a tCAR peptide and a
co-composition) as not being "indirectly non-covalently associated"
means that there is no direct or indirect non-covalent association
between the components.
[0192] It is understood that components can be non-covalently
associated via multiple chains and paths including both direct and
indirect non-covalent associations. For the purposes of these
definitions, the presence a single direct non-covalent association
makes the association a direct non-covalent association even if
there are also indirect non-covalent associations present.
Similarly, the presence of a covalent connection between components
means the components are covalently coupled even if there are also
non-covalent associations present. It is also understood that
covalently coupled components that happened to lack any
non-covalent association with each other are not considered to fall
under the definition of components that are not non-covalently
associated.
[0193] In some forms, the co-composition does not comprise a
functional internalization element. The co-composition can comprise
a functional internalization element. In some forms, the
co-composition does not comprise a homing molecule. The
co-composition can comprise a homing molecule. In some forms, the
co-composition does not comprise a homing peptide. The
co-composition can comprise a homing peptide. The co-composition
can selectively home to tumors, regenerating tissue, sites of
injury, surgical sites, tumor vasculature, sites of tumor
angiogenesis, sites of inflammation, sites of arthritis, lung
tissue, pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries, or
interstitial space of lungs. In some forms, the co-composition does
not selectively home to tumor vasculature. The co-composition can
selectively home to tumor vasculature. In some forms, the
co-composition does not comprise an accessory molecule. The
co-composition can comprise an accessory molecule. In some forms,
the co-composition does not comprise a accessory peptide. The
co-composition can comprise an accessory peptide. The
co-composition can selectively home to tumors, regenerating tissue,
sites of injury, surgical sites, tumor vasculature, sites of tumor
angiogenesis, sites of inflammation, sites of arthritis, lung
tissue, pulmonary arterial hypertension lung vasculature, pulmonary
arterial hypertension lesions, remodeled pulmonary arteries, or
interstitial space of lungs.
[0194] The tCAR peptide can be associated with one or more
accessory molecules. For example, an accessory molecule can be a
part of an amino acid sequence, protein, peptide, conjugate, or
composition that comprises the tCAR peptide. As another example,
the accessory molecule can be covalently coupled or non-covalently
associated with the tCAR peptide or an amino acid sequence,
protein, peptide, conjugate, or composition that comprises the tCAR
peptide. Accessory molecules can be any molecule, compound,
component, etc. that has a useful function and that can be used in
combination with a tCAR composition, tCAR conjugate, tCAR molecule,
tCAR protein, and/or tCAR peptide. Examples of useful accessory
molecules include homing molecules, targeting molecules, affinity
ligands, cell penetrating molecules, endosomal escape molecules,
subcellular targeting molecules, nuclear targeting molecules.
Different accessory molecules can have similar or different
functions from each other. Accessory molecules having similar
functions, different functions, or both, can be associated a tCAR
composition, tCAR conjugate, tCAR molecule, tCAR protein, and/or
tCAR peptide.
[0195] The accessory molecule can be separate from or overlapping
with the tCAR peptide. For example, some accessory molecules are
amino acid sequences. This can allow the amino acid sequence
consisting of the tCAR peptide to overlap the amino acid sequence
that consists of the accessory amino acid sequence. For example,
iRGD, CAR, LyP-1, iNGR, and RGR peptides each contain both an
accessory sequence and tCAR sequence overlapping with one another
in the peptide. Alternatively the accessory molecule can be a
separate entity that does not overlap with the tCAR peptide. For
example, a HER2 binding peptide, CREKA peptide, NGR peptide, iNGR,
or an RGD peptide that is not a tCAR peptide can consist of amino
acid sequence that does not overlap with a tCAR peptide. In some
forms, the accessory molecule can comprise a sequence in, for
example, a peptide that binds to a specific receptor distinct from
the receptor for the tCAR peptide.
[0196] The tCAR peptide can be associated with one or more
accessory molecules. For example, an accessory molecule can be a
part of an amino acid sequence, protein, peptide, conjugate, or
composition that comprises the tCAR peptide. As another example,
the accessory molecule can be covalently coupled or non-covalently
associated with the tCAR peptide or an amino acid sequence,
protein, peptide, conjugate, or composition that comprises the tCAR
peptide. The tCAR conjugate can be associated with one or more
accessory molecules. For example, an accessory molecule can be a
part of a conjugate or composition that comprises the tCAR
conjugate. As another example, the accessory molecule can be
covalently coupled or non-covalently associated with the tCAR
conjugate or a conjugate or composition that comprises the tCAR
conjugate. The tCAR composition can be associated with one or more
accessory molecules. For example, an accessory molecule can be a
part of a composition that comprises the tCAR composition. As
another example, the accessory molecule can be covalently coupled
or non-covalently associated with the tCAR composition or a
composition that comprises the tCAR composition.
[0197] The amino acid sequence can be associated with one or more
accessory molecules. For example, an accessory molecule can be a
part of an amino acid sequence, protein, peptide, conjugate, or
composition that comprises the amino acid sequence. As another
example, the accessory molecule can be covalently coupled or
non-covalently associated with the amino acid sequence or an amino
acid sequence, protein, peptide, conjugate, or composition that
comprises the amino acid sequence. For example, the amino acid
sequences can comprise a iRGD peptide, a CAR peptide, a LyP-1
peptide, a RGR peptide, a HER2 binding peptide, a CREKA peptide, a
NGR peptide, iNGR, a RGD peptide that is not a tCAR peptide, or a
combination. The amino acid sequence can comprise a CREKA peptide.
The protein or peptide can be associated with one or more accessory
molecules. For example, an accessory molecule can be a part of a
protein, peptide, conjugate, or composition that comprises the
peptide. As another example, the accessory molecule can be
covalently coupled or non-covalently associated with the peptide or
a protein, peptide, conjugate, or composition that comprises the
peptide. For example, an accessory molecule can be a part of a
protein, conjugate, or composition that comprises the protein. As
another example, the accessory molecule can be covalently coupled
or non-covalently associated with the protein or a protein,
conjugate, or composition that comprises the protein. For example,
the protein or peptide can comprise a iRGD peptide, a CAR peptide,
a LyP-1 peptide, a RGR peptide, a HER2 binding peptide, a CREKA
peptide, a NGR peptide, iNGR, a RGD peptide that is not a tCAR
peptide, or a combination. The conjugate can be associated with one
or more accessory molecules. For example, an accessory molecule can
be a part of a conjugate or composition that comprises the
conjugate. As another example, the accessory molecule can be
covalently coupled or non-covalently associated with the conjugate
or a conjugate or composition that comprises the conjugate. For
example, the conjugate can comprise a iRGD peptide, a CAR peptide,
a LyP-1 peptide, a RGR peptide, a HER2 binding peptide, a CREKA
peptide, a NGR peptide, iNGR, a RGD peptide that is not a tCAR
peptide, or a combination. The composition can be associated with
one or more accessory molecules. For example, an accessory molecule
can be a part of a composition that comprises the composition. As
another example, the accessory molecule can be covalently coupled
or non-covalently associated with the composition or a composition
that comprises the composition. For example, the composition can
comprise a iRGD peptide, a CAR peptide, a LyP-1 peptide, a RGR
peptide, a HER2 binding peptide, a CREKA peptide, a NGR peptide,
iNGR, a RGD peptide that is not a tCAR peptide, or a
combination.
[0198] The tCAR peptide can be associated with one or more homing
molecules. For example, a homing molecule can be a part of an amino
acid sequence, protein, peptide, conjugate, or composition that
comprises the tCAR peptide. As another example, the homing molecule
can be covalently coupled or non-covalently associated with the
tCAR peptide or an amino acid sequence, protein, peptide,
conjugate, or composition that comprises the tCAR peptide. The
homing molecule can be separate from or overlapping with the tCAR
peptide. For example, some homing molecules are amino acid
sequences. This can allow the amino acid sequence consisting of the
tCAR peptide to overlap the amino acid sequence that consists of
the homing amino acid sequence. For example, iRGD, CAR, LyP-1,
iNGR, and RGR peptides each contain both a homing sequence and tCAR
sequence overlapping with one another in the peptide. Alternatively
the homing molecule can be a separate entity that does not overlap
with the tCAR peptide. For example, a HER2 binding peptide, CREKA
peptide, NGR peptide, iNGR, or an RGD peptide that is not a tCAR
peptide can consist of amino acid sequence that does not overlap
with a tCAR peptide. In some forms, the homing molecule can
comprise a sequence in, for example, a tCAR peptide that binds to a
specific receptor distinct from the receptor for the tCAR
peptide.
[0199] The tCAR peptide can be associated with one or more homing
molecules. For example, a homing molecule can be a part of an amino
acid sequence, protein, peptide, conjugate, or composition that
comprises the tCAR peptide. As another example, the homing molecule
can be covalently coupled or non-covalently associated with the
tCAR peptide or an amino acid sequence, protein, peptide,
conjugate, or composition that comprises the tCAR peptide. The tCAR
conjugate can be associated with one or more homing molecules. For
example, a homing molecule can be a part of a conjugate or
composition that comprises the tCAR conjugate. As another example,
the homing molecule can be covalently coupled or non-covalently
associated with the tCAR conjugate or a conjugate or composition
that comprises the tCAR conjugate. The tCAR composition can be
associated with one or more homing molecules. For example, a homing
molecule can be a part of a composition that comprises the tCAR
composition. As another example, the homing molecule can be
covalently coupled or non-covalently associated with the tCAR
composition or a composition that comprises the tCAR
composition.
[0200] The amino acid sequence can be associated with one or more
homing molecules. For example, a homing molecule can be a part of
an amino acid sequence, protein, peptide, conjugate, or composition
that comprises the amino acid sequence. As another example, the
homing molecule can be covalently coupled or non-covalently
associated with the amino acid sequence or an amino acid sequence,
protein, peptide, conjugate, or composition that comprises the
amino acid sequence. For example, the amino acid sequences can
comprise a iRGD peptide, a CAR peptide, a LyP-1 peptide, a RGR
peptide, a HER2 binding peptide, a CREKA peptide, a NGR peptide,
iNGR, a RGD peptide that is not a tCAR peptide, or a combination.
The amino acid sequence can comprise a CREKA peptide. The protein
or peptide can be associated with one or more homing molecules. For
example, a homing molecule can be a part of a protein, peptide,
conjugate, or composition that comprises the peptide. As another
example, the homing molecule can be covalently coupled or
non-covalently associated with the peptide or a protein, peptide,
conjugate, or composition that comprises the peptide. For example,
a homing molecule can be a part of a protein, conjugate, or
composition that comprises the protein. As another example, the
homing molecule can be covalently coupled or non-covalently
associated with the protein or a protein, conjugate, or composition
that comprises the protein. For example, the protein or peptide can
comprise a iRGD peptide, a CAR peptide, a LyP-1 peptide, a RGR
peptide, a HER2 binding peptide, a CREKA peptide, a NGR peptide,
iNGR, a RGD peptide that is not a tCAR peptide, or a combination.
The protein or peptide can comprise iRGD. The protein or peptide
can comprise a CAR peptide, a LyP-1 peptide. The protein or peptide
can comprise iNGR. The protein or peptide can comprise RGR peptide.
The protein or peptide can comprise a CREKA peptide. The conjugate
can be associated with one or more homing molecules. For example, a
homing molecule can be a part of a conjugate or composition that
comprises the conjugate. As another example, the homing molecule
can be covalently coupled or non-covalently associated with the
conjugate or a conjugate or composition that comprises the
conjugate. For example, the conjugate can comprise a iRGD peptide,
a CAR peptide, a LyP-1 peptide, a RGR peptide, a HER2 binding
peptide, a CREKA peptide, a NGR peptide, iNGR, a RGD peptide that
is not a tCAR peptide, or a combination. The conjugate can comprise
iRGD. The conjugate can comprise a CAR peptide, a LyP-1 peptide.
The conjugate can comprise iNGR. The conjugate can comprise RGR
peptide. The conjugate can comprise a CREKA peptide. The
composition can be associated with one or more homing molecules.
For example, a homing molecule can be a part of a composition that
comprises the composition. As another example, the homing molecule
can be covalently coupled or non-covalently associated with the
composition or a composition that comprises the composition. For
example, the composition can comprise a iRGD peptide, a CAR
peptide, a LyP-1 peptide, a RGR peptide, a HER2 binding peptide, a
CREKA peptide, a NGR peptide, iNGR, a RGD peptide that is not a
tCAR peptide, or a combination. The composition can comprise iRGD.
The composition can comprise a CAR peptide. The composition can
comprise a LyP-1 peptide. The composition can comprise iNGR. The
composition can comprise RGR peptide. The composition can comprise
a CREKA peptide.
[0201] The amino acid sequence can be selected for internalization
into a cell. The amino acid sequence can be selected for tissue
penetration. The amino acid sequence can be selected for
internalization into a cell and tissue penetration. The protein or
peptide can be selected for internalization into a cell. The
protein or peptide can be selected for tissue penetration. The
protein or peptide can be selected for internalization into a cell
and tissue penetration. The conjugate can be selected for
internalization into a cell. The conjugate can be selected for
tissue penetration. The conjugate can be selected for
internalization into a cell and tissue penetration. The composition
can be selected for internalization into a cell. The composition
can be selected for tissue penetration. The composition can be
selected for internalization into a cell and tissue
penetration.
[0202] The tCAR peptide, tCAR conjugate, tCAR composition, amino
acid sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to tumors, regenerating tissue, sites of injury, surgical
sites, tumor vasculature, sites of tumor angiogenesis, sites of
inflammation, sites of arthritis, lung tissue, pulmonary arterial
hypertension lung vasculature, pulmonary arterial hypertension
lesions, remodeled pulmonary arteries, or interstitial space of
lungs. The tCAR peptide, tCAR conjugate, tCAR composition, amino
acid sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to tumor vasculature. The tCAR peptide, tCAR conjugate, tCAR
composition, amino acid sequence, protein or peptide, conjugate,
composition, co-composition, cargo composition, or a combination
can selectively home to one or more particular types of tumor. The
tCAR peptide, tCAR conjugate, tCAR composition, amino acid
sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to the vasculature of one or more particular types of tumor.
The tCAR peptide, tCAR conjugate, tCAR composition, amino acid
sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to one or more particular stages of a tumor or cancer. The
tCAR peptide, tCAR conjugate, tCAR composition, amino acid
sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to the vasculature of one or more particular stages of a tumor
or cancer. The tCAR peptide, tCAR conjugate, tCAR composition,
amino acid sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to one or more particular stages of one or more particular
types of tumor. The tCAR peptide, tCAR conjugate, tCAR composition,
amino acid sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to the vasculature of one or more different stages of one or
more particular types of tumor.
[0203] The tCAR peptide, tCAR conjugate, tCAR composition, amino
acid sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to lung tissue. The tCAR peptide, tCAR conjugate, tCAR
composition, amino acid sequence, protein or peptide, conjugate,
composition, co-composition, cargo composition, or a combination
can selectively home to lung vasculature. The tCAR peptide, tCAR
conjugate, tCAR composition, amino acid sequence, protein or
peptide, conjugate, composition, co-composition, cargo composition,
or a combination can selectively home to heart tissue. The tCAR
peptide, tCAR conjugate, tCAR composition, amino acid sequence,
protein or peptide, conjugate, composition, co-composition, cargo
composition, or a combination can selectively home to heart
vasculature. The tCAR peptide, tCAR conjugate, tCAR composition,
amino acid sequence, protein or peptide, conjugate, composition,
co-composition, cargo composition, or a combination can selectively
home to brain cells, brain stem cells, brain tissue, and/or brain
vasculature, kidney cells, kidney stem cells, kidney tissue, and/or
kidney vasculature, skin cells, skin stem cells, skin tissue,
and/or skin vasculature, lung cells, lung tissue, and/or lung
vasculature, pancreatic cells, pancreatic tissue, and/or pancreatic
vasculature, intestinal cells, intestinal tissue, and/or intestinal
vasculature, adrenal gland cells, adrenal tissue, and/or adrenal
vasculature, retinal cells, retinal tissue, and/or retinal
vasculature, liver cells, liver tissue, and/or liver vasculature,
prostate cells, prostate tissue, and/or prostate vasculature,
endometriosis cells, endometriosis tissue, and/or endometriosis
vasculature, ovary cells, ovary tissue, and/or ovary vasculature,
tumor cells, tumors, tumor blood vessels, and/or tumor vasculature,
bone cells, bone tissue, and/or bone vasculature, bone marrow
cells, bone marrow tissue, and/or bone marrow vasculature,
cartilage cells, cartilage tissue, and/or cartilage vasculature,
stem cells, embryonic stem cells, pluripotent stem cells, induced
pluripotent stem cells, adult stem cells, hematopoietic stem cells,
neural stem cells, mesenchymal stem cells, mammary stem cells,
endothelial stem cells, olfactory adult stem cells, neural crest
stem cells, cancer stem cells, blood cells, erythrocytes,
platelets, leukocytes, granulocytes, neutrophils, eosinphils,
basophils, lymphoid cells, lymphocytes, monocytes, wound
vasculature, vasculature of injured tissue, vasculature of inflamed
tissue, atherosclerotic plaques, or a combination.
[0204] tCAR compositions, tCAR conjugates, tCAR molecules, tCAR
proteins, and tCAR peptides can be designed and produced in any
suitable manner. For example, the tCAR peptide in the disclosed
tCAR compositions, tCAR conjugates, tCAR molecules, and tCAR
proteins can be designed or produced by selecting an amino acid
sequence for internalization into a cell and/or penetration of
tissue, wherein the amino acid sequence comprises a C-terminal
element, wherein a protein or peptide comprises the selected amino
acid sequence, wherein the selected amino acid sequence is at the
C-terminal end of the protein or peptide.
[0205] The peptide can be an activatable peptide. The activatable
peptide can be a protease-activatable peptide. The
protease-activatable peptide can be activatable by a serine
protease, plasmin, a plasminogen activator, urokinase, a proprotein
convertase, a furin, a carboxypeptidase, carboxypeptidase A, a
glutamate-specific carboxypeptidase, a proline-specific
carboxypeptidase, PSMA, or a combination.
[0206] The tCAR peptide can be comprised in an amino acid sequence.
The amino acid sequence can be comprised in a protein or peptide.
The tCAR peptide can be comprised in a protein or peptide. In some
forms, the protein or peptide can be internalized into a cell,
penetrate tissue, or both when the amino acid sequence is present
in the protein or peptide but not when the amino acid sequence is
not present in the protein or peptide. In some forms, the protein
or peptide can penetrate tissue when the amino acid sequence is
present in the protein or peptide but not when the amino acid
sequence is not present in the protein or peptide. In some forms,
the protein or peptide can be internalized into a cell and
penetrate tissue when the amino acid sequence is present in the
protein or peptide but not when the amino acid sequence is not
present in the protein or peptide.
[0207] In some forms, the amino acid sequence can be internalized
into a cell, penetrate tissue, or both without being associated
with the co-composition. In some forms, the amino acid sequence can
be internalized into a cell, penetrate tissue, or both without
being associated with the cargo composition. In some forms, the
amino acid sequence can penetrate tissue without being associated
with the co-composition. In some forms, the amino acid sequence can
penetrate tissue without being associated with the cargo
composition. In some forms, the amino acid sequence can be
internalized into a cell and penetrate tissue without being
associated with the co-composition. In some forms, the amino acid
sequence can be internalized into a cell and penetrate tissue
without being associated with the cargo composition. In some forms,
the amino acid sequence can be the only functional internalization
element in the protein or peptide.
[0208] In some forms, the internalization, penetration, or both of
the co-composition or cargo composition into or through a cell,
tissue, or both can be enhanced when the amino acid sequence is
present in the protein or peptide but not when the amino acid
sequence is not present in the protein or peptide. In some forms,
the penetration of the co-composition or cargo composition into or
through tissue can be enhanced when the amino acid sequence is
present in the protein or peptide but not when the amino acid
sequence is not present in the protein or peptide. In some forms,
the internalization and penetration of the co-composition or cargo
composition into or through a cell and tissue can be enhanced when
the amino acid sequence is present in the protein or peptide but
not when the amino acid sequence is not present in the protein or
peptide. In some forms, the internalization, penetration, or both
of the co-composition or cargo composition into or through a cell,
tissue, or both can be enhanced when the tCAR peptide is present in
the protein or peptide but not when the amino acid sequence is not
present in the protein or peptide. In some forms, the penetration
of the co-composition or cargo composition into or through tissue
can be enhanced when the tCAR peptide is present in the protein or
peptide but not when the amino acid sequence is not present in the
protein or peptide. In some forms, the internalization and
penetration of the co-composition or cargo composition into or
through a cell and tissue can be enhanced when the tCAR peptide is
present in the protein or peptide but not when the amino acid
sequence is not present in the protein or peptide.
[0209] The amino acid sequence can be associated with one or more
accessory molecules. The protein or peptide can be associated with
one or more accessory molecules. One or more of the accessory
molecules can be independently a homing molecule, a targeting
molecule, an affinity ligand, a cell penetrating peptide, an
endosomal escape molecule, a subcellular targeting molecule, a
nuclear targeting molecule, or a combination. One or more of the
accessory molecules can be homing molecules. One or more of the
homing molecules can be independently an RGD peptide, iRGD, a CAR
peptide, a LyP-1 peptide, NGR peptide, iNGR, RGR peptide, HER2
binding peptide, or a combination.
[0210] The amino acid sequence can be selected for internalization
into a cell. The amino acid sequence can be selected for tissue
penetration. The amino acid sequence can be selected for
internalization into a cell and tissue penetration.
[0211] In some forms, the internalization, penetration, or both of
the co-composition into or through a cell, tissue, or both can be
enhanced when the cell, tissue, or both is exposed to the tCAR
peptide but not when the cell, tissue, or both is not exposed to
the tCAR peptide. In some forms, the penetration of the
co-composition into or through tissue can be enhanced when the
tissue is exposed to the tCAR peptide but not when the tissue is
not exposed to the tCAR peptide. In some forms, the internalization
and penetration of the co-composition into or through a cell and
tissue can be enhanced when the cell and tissue are exposed to the
tCAR peptide but not when the cell and tissue is not exposed to the
tCAR peptide.
[0212] The tCAR peptide can be comprised in a tCAR composition. The
tCAR composition can comprise one or more accessory molecules. The
tCAR composition can comprise one or more cargo compositions. The
tCAR composition can comprise one or more homing molecules. The
tCAR peptide can be comprised in a tCAR conjugate. The tCAR
conjugate can comprise one or more accessory molecules. The tCAR
conjugate can comprise one or more cargo compositions. The tCAR
conjugate can comprise one or more homing molecules.
[0213] The cell, tissue, or both can be exposed to a plurality of
accessory molecules. The cell, tissue, or both can be exposed to a
plurality of homing molecules. The cell, tissue, or both can be
exposed to a plurality of cargo compositions. The cell, tissue, or
both can be exposed to a plurality of tCAR peptides. The cell,
tissue, or both can be exposed to a plurality of
co-compositions.
[0214] As used herein, "selecting an amino acid sequence for
internalization into a cell" refers to selecting, identifying
designing or otherwise categorizing an amino acid sequence with the
specific intention of obtaining entry into a cell of a protein or
peptide that is comprised of the amino acid sequence. Thus, for
example, selecting an amino acid sequence for some purpose or
capability other than obtaining entry into a cell of a protein or
peptide that is comprised of the amino acid sequence and in the
absence of an intention of obtaining entry into a cell of a protein
or peptide that is comprised of the amino acid sequence does not
constitute "selecting an amino acid sequence for internalization
into a cell." Selecting an amino acid sequence for some purpose or
capability as well as for obtaining entry into a cell of a protein
or peptide that is comprised of the amino acid sequence does
constitute "selecting an amino acid sequence for internalization
into a cell." Thus, the presence of additional goals or purposes
does not alter that selection of an amino acid sequence at least
with the specific intention of obtaining entry into a cell of a
protein or peptide that is comprised of the amino acid sequence
constitutes "selecting an amino acid sequence for internalization
into a cell."
[0215] As used herein, "selecting an amino acid sequence for
penetration of tissue" refers to selecting, identifying designing
or otherwise categorizing an amino acid sequence with the specific
intention of obtaining entry into tissue (that is, tissue
penetration) of a protein or peptide that is comprised of the amino
acid sequence. Thus, for example, selecting an amino acid sequence
for some purpose or capability other than obtaining entry into
tissue of a protein or peptide that is comprised of the amino acid
sequence and in the absence of an intention of obtaining entry into
tissue of a protein or peptide that is comprised of the amino acid
sequence does not constitute "selecting an amino acid sequence for
penetration of tissue." Selecting an amino acid sequence for some
purpose or capability as well as for obtaining entry into tissue of
a protein or peptide that is comprised of the amino acid sequence
does constitute "selecting an amino acid sequence for penetration
of tissue." Thus, the presence of additional goals or purposes does
not alter that selection of an amino acid sequence at least with
the specific intention of obtaining entry into tissue of a protein
or peptide that is comprised of the amino acid sequence constitutes
"selecting an amino acid sequence for penetration of tissue."
[0216] As used herein, "selecting an amino acid sequence for
internalization into a cell and/or penetration of tissue" refers to
selecting, identifying designing or otherwise categorizing an amino
acid sequence with the specific intention of obtaining entry into
either or both a cell and tissue of a protein or peptide that is
comprised of the amino acid sequence. Thus, for example, selecting
an amino acid sequence for some purpose or capability other than
obtaining entry into a cell, tissue, or both of a protein or
peptide that is comprised of the amino acid sequence and in the
absence of an intention of obtaining entry into a cell, tissue, or
both of a protein or peptide that is comprised of the amino acid
sequence does not constitute "selecting an amino acid sequence for
internalization into a cell and/or penetration of tissue."
Selecting an amino acid sequence for some purpose or capability as
well as for obtaining entry into either or both a cell and tissue
of a protein or peptide that is comprised of the amino acid
sequence does constitute "selecting an amino acid sequence for
internalization into a cell and/or penetration of tissue." Thus,
the presence of additional goals or purposes does not alter that
selection of an amino acid sequence at least with the specific
intention of obtaining entry into a cell, tissue, or both of a
protein or peptide that is comprised of the amino acid sequence
constitutes "selecting an amino acid sequence for internalization
into a cell and/or penetration of tissue."
[0217] As used herein, unless the context indicates otherwise,
"selecting a co-composition for internalization into a cell" refers
to selecting, identifying designing or otherwise categorizing a
co-composition and a tCAR composition, tCAR conjugate, tCAR
molecule, tCAR protein, or tCAR peptide with the specific intention
of obtaining entry into a cell of both the co-composition and the
tCAR composition, tCAR conjugate, tCAR molecule, tCAR protein, or
tCAR peptide. Thus, for example, selecting a co-composition for
some purpose or capability other than obtaining entry into a cell
in combination with entry of a selected tCAR composition, tCAR
conjugate, tCAR molecule, tCAR protein, or tCAR peptide and in the
absence of an intention of obtaining entry into a cell of both the
co-composition and the tCAR composition, tCAR conjugate, tCAR
molecule, tCAR protein, or tCAR peptide does not constitute
"selecting co-composition for internalization into a cell."
Selecting a co-composition for some purpose or capability as well
as for obtaining entry into a cell of the co-composition does
constitute "selecting co-composition for internalization into a
cell." Thus, the presence of additional goals or purposes does not
alter that selection of a co-composition at least with the specific
intention of obtaining entry into a cell of a co-composition
constitutes "selecting a co-composition for internalization into a
cell."
[0218] As used herein, unless the context indicates otherwise,
"selecting a co-composition for penetration of tissue" refers to
selecting, identifying designing or otherwise categorizing a
co-composition and a tCAR composition, tCAR conjugate, tCAR
molecule, tCAR protein, or tCAR peptide with the specific intention
of obtaining entry into tissue (that is, tissue penetration) of
both the co-composition and the tCAR composition, tCAR conjugate,
tCAR molecule, tCAR protein, or tCAR peptide. Thus, for example,
selecting a co-composition for some purpose or capability other
than obtaining entry into tissue in combination with entry of a
selected tCAR composition, tCAR conjugate, tCAR molecule, tCAR
protein, or tCAR peptide and in the absence of an intention of
obtaining entry into tissue of both the co-composition and the tCAR
composition, tCAR conjugate, tCAR molecule, tCAR protein, or tCAR
peptide does not constitute "selecting co-composition for
penetration of tissue." Selecting a co-composition for some purpose
or capability as well as for obtaining entry into tissue of the
co-composition does constitute "selecting co-composition for
penetration of tissue." Thus, the presence of additional goals or
purposes does not alter that selection of a co-composition at least
with the specific intention of obtaining entry into tissue of a
co-composition constitutes "selecting a co-composition for
penetration of tissue."
[0219] As used herein, unless the context indicates otherwise,
"selecting a co-composition for internalization into a cell and/or
penetration of tissue" refers to selecting, identifying designing
or otherwise categorizing a co-composition and a tCAR composition,
tCAR conjugate, tCAR molecule, tCAR protein, or tCAR peptide with
the specific intention of obtaining entry into either or both a
cell and tissue of both the co-composition and the tCAR
composition, tCAR conjugate, tCAR molecule, tCAR protein, or tCAR
peptide. Thus, for example, selecting a co-composition for some
purpose or capability other than obtaining entry into either or
both a cell and tissue in combination with entry of a selected tCAR
composition, tCAR conjugate, tCAR molecule, tCAR protein, or tCAR
peptide and in the absence of an intention of obtaining entry into
either or both a cell and tissue of both the co-composition and the
tCAR composition, tCAR conjugate, tCAR molecule, tCAR protein, or
tCAR peptide does not constitute "selecting co-composition for
internalization into a cell and/or penetration of tissue."
Selecting a co-composition for some purpose or capability as well
as for obtaining entry into either or both a cell and tissue of the
co-composition does constitute "selecting co-composition for
internalization into a cell and/or penetration of tissue." Thus,
the presence of additional goals or purposes does not alter that
selection of a co-composition at least with the specific intention
of obtaining entry into either or both a cell and tissue of a
co-composition constitutes "selecting a co-composition for
internalization into a cell and/or penetration of tissue."
[0220] As used herein, unless the context indicates otherwise,
"selecting a cargo composition for internalization into a cell"
refers to selecting, identifying designing or otherwise
categorizing a cargo composition and a tCAR composition, tCAR
conjugate, tCAR molecule, tCAR protein, or tCAR peptide with the
specific intention of obtaining entry into a cell of both the cargo
composition and the tCAR composition, tCAR conjugate, tCAR
molecule, tCAR protein, or tCAR peptide. Thus, for example,
selecting a cargo composition for some purpose or capability other
than obtaining entry into a cell in combination with entry of a
selected tCAR composition, tCAR conjugate, tCAR molecule, tCAR
protein, or tCAR peptide and in the absence of an intention of
obtaining entry into a cell of both the cargo composition and the
tCAR composition, tCAR conjugate, tCAR molecule, tCAR protein, or
tCAR peptide does not constitute "selecting cargo composition for
internalization into a cell." Selecting a cargo composition for
some purpose or capability as well as for obtaining entry into a
cell of the cargo composition does constitute "selecting cargo
composition for internalization into a cell." Thus, the presence of
additional goals or purposes does not alter that selection of a
cargo composition at least with the specific intention of obtaining
entry into a cell of a cargo composition constitutes "selecting a
cargo composition for internalization into a cell."
[0221] As used herein, unless the context indicates otherwise,
"selecting a cargo composition for penetration of tissue" refers to
selecting, identifying designing or otherwise categorizing a cargo
composition and a tCAR composition, tCAR conjugate, tCAR molecule,
tCAR protein, or tCAR peptide with the specific intention of
obtaining entry into tissue (that is, tissue penetration) of both
the cargo composition and the tCAR composition, tCAR conjugate,
tCAR molecule, tCAR protein, or tCAR peptide. Thus, for example,
selecting a cargo composition for some purpose or capability other
than obtaining entry into tissue in combination with entry of a
selected tCAR composition, tCAR conjugate, tCAR molecule, tCAR
protein, or tCAR peptide and in the absence of an intention of
obtaining entry into tissue of both the cargo composition and the
tCAR composition, tCAR conjugate, tCAR molecule, tCAR protein, or
tCAR peptide does not constitute "selecting cargo composition for
penetration of tissue." Selecting a cargo composition for some
purpose or capability as well as for obtaining entry into tissue of
the cargo composition does constitute "selecting cargo composition
for penetration of tissue." Thus, the presence of additional goals
or purposes does not alter that selection of a cargo composition at
least with the specific intention of obtaining entry into tissue of
a cargo composition constitutes "selecting a cargo composition for
penetration of tissue."
[0222] As used herein, unless the context indicates otherwise,
"selecting a cargo composition for internalization into a cell
and/or penetration of tissue" refers to selecting, identifying
designing or otherwise categorizing a cargo composition and a tCAR
composition, tCAR conjugate, tCAR molecule, tCAR protein, or tCAR
peptide with the specific intention of obtaining entry into either
or both a cell and tissue of both the cargo composition and the
tCAR composition, tCAR conjugate, tCAR molecule, tCAR protein, or
tCAR peptide. Thus, for example, selecting a cargo composition for
some purpose or capability other than obtaining entry into either
or both a cell and tissue in combination with entry of a selected
tCAR composition, tCAR conjugate, tCAR molecule, tCAR protein, or
tCAR peptide and in the absence of an intention of obtaining entry
into either or both a cell and tissue of both the cargo composition
and the tCAR composition, tCAR conjugate, tCAR molecule, tCAR
protein, or tCAR peptide does not constitute "selecting cargo
composition for internalization into a cell and/or penetration of
tissue." Selecting a cargo composition for some purpose or
capability as well as for obtaining entry into either or both a
cell and tissue of the cargo composition does constitute "selecting
cargo composition for internalization into a cell and/or
penetration of tissue." Thus, the presence of additional goals or
purposes does not alter that selection of a cargo composition at
least with the specific intention of obtaining entry into either or
both a cell and tissue of a cargo composition constitutes
"selecting a cargo composition for internalization into a cell
and/or penetration of tissue."
[0223] As used herein, "causing a compound or composition to be
covalently coupled or non-covalently associated" with something
else refers to any action that results in a compound or composition
that is not covalently coupled or non-covalently associated with
the something else becoming or coming into the state of being
covalently coupled or non-covalently associated with the something
else. As an example, covalently coupling a homing molecule to a
tCAR peptide constitutes "causing a homing molecule to be
covalently coupled or non-covalently associated" with the tCAR
peptide. As another example, a tCAR peptide that starts as a
nonexistent concept and then is synthesized as part of a
composition that includes the thing to which the tCAR peptide is to
be coupled or associated constitutes "causing a tCAR peptide to be
covalently coupled or non-covalently associated" with the thing.
For example, synthesis of a peptide that includes both an amino
acid sequence of interest and an amino acid sequence comprising a
C-terminal element constitutes causing the amino acid sequence of
interest to be covalently coupled or non-covalently associated with
the amino acid sequence comprising a C-terminal element. However,
and in general, synthesis of a protein or peptide that naturally
includes both the amino acid sequence of interest and an amino acid
sequence comprising a C-terminal element can be excluded as a
process of "causing the amino acid sequence of interest to be
covalently coupled or non-covalently associated" with the amino
acid sequence comprising a C-terminal element.
[0224] As used herein, "causing a co-composition to be covalently
coupled or non-covalently associated" with something else refers to
any action that results in a co-composition that is not and the
something else becoming or coming into the state of being and the
something else. More clearly, "causing a co-composition to be
covalently coupled or non-covalently associated" with something
else refers to any action that results in a co-composition and the
something else becoming or coming into the state of being
covalently coupled or non-covalently associated. As an example,
covalently coupling a co-composition to another co-composition
constitutes "causing a co-composition to be covalently coupled or
non-covalently associated" with the other co-composition. As
another example, a co-composition that starts as a nonexistent
concept and then is synthesized as part of a composition that
includes the thing to which the co-composition is to be coupled or
associated constitutes "causing a co-composition to be covalently
coupled or non-covalently associated" with the thing.
[0225] As used herein, "causing a cargo composition to be
covalently coupled or non-covalently associated" with something
else refers to any action that results in a cargo composition that
is not and the something else becoming or coming into the state of
being and the something else. More clearly, "causing a cargo
composition to be covalently coupled or non-covalently associated"
with something else refers to any action that results in a cargo
composition and the something else becoming or coming into the
state of being covalently coupled or non-covalently associated. As
an example, covalently coupling a cargo composition to another
cargo composition constitutes "causing a cargo composition to be
covalently coupled or non-covalently associated" with the other
cargo composition. As another example, a cargo composition that
starts as a nonexistent concept and then is synthesized as part of
a composition that includes the thing to which the cargo
composition is to be coupled or associated constitutes "causing a
cargo composition to be covalently coupled or non-covalently
associated" with the thing.
[0226] tCAR peptides can be composed of, for example, amino acids,
amino acid analogs, peptide analogs, amino acid mimetics, peptide
mimetics, etc. Although structures, design, etc. of tCAR peptides
is described herein in terms of amino acids and peptides composed
of amino acids for convenience, it is understood that analogous
analogs, mimetics, modified forms, etc. of amino acids and peptides
can also be used as tCAR peptides and designed using similar
principles.
[0227] Any component, such as the components disclosed herein, can
overlap, be adjacent to, and/or be upstream, downstream, or both of
a peptide, such as a tCAR peptide. Examples of such components
include accessory molecules, homing molecules, protease cleavage
sites, etc. It is useful to have some components coupled to or
associated with a peptide, such as a tCAR peptide to be downstream
(C-terminal) of the peptide. For example, activatable peptide
having an accessory protein or a homing peptide downstream of the
peptide (and thus downstream from the cleavage site for activation)
will be separated from the peptide when it is activated. As another
example, activatable peptides having an accessory molecule or a
homing molecule downstream of the peptide (and thus downstream from
the cleavage site for activation) will be separated from the
peptide when it is activated. This can have some advantages such as
making the peptide function more efficient or reducing the chance
for extraneous effects of the eliminated component.
[0228] As used herein, "activatable peptide" refers to a peptide,
such as a tCAR peptide, having a molecule, moiety, nanoparticle,
compound or other composition covalently coupled to the peptide,
such as to the terminal carboxyl group of the peptide, where the
molecule, moiety, nanoparticle, compound or other composition can
block internalization and/or tissue penetration of the tCAR
composition, conjugate, molecule, protein, peptide, etc. and where
the molecule, moiety, nanoparticle, compound or other composition
can be removed (to expose the terminal carboxy group of the
peptide, for example). For example, the activatable peptide can be
on the C-terminal end of the protein or peptide, and can prevent
the peptide from being internalized and/or from penetrating tissue.
The molecule, nanoparticle, moiety, compound or other composition
covalently coupled to the peptide can be referred to as the
"blocking group." For example, the blocking group can be coupled to
the terminal carboxyl group of the C-terminal arginine of the tCAR
peptide, to the C-terminal amino acid of the tCAR peptide, or to an
amino acid of the tCAR peptide other than the C-terminal amino
acid. The blocking group can also be coupled, or associated with a
part of a tCAR composition, conjugate, molecule, protein, peptide,
etc. other than the tCAR peptide so long as it can prevent the tCAR
peptide from being internalized and/or from penetrating tissue. A
tCAR composition comprising an activatable peptide, such as an
activatable tCAR peptide, can be referred to as an activatable tCAR
composition. A tCAR molecule comprising an activatable peptide,
such as an activatable tCAR peptide, can be referred to as an
activatable tCAR molecule. A tCAR conjugate comprising an
activatable peptide, such as an activatable tCAR peptide, can be
referred to as an activatable tCAR conjugate. A tCAR protein
comprising an activatable peptide, such as an activatable tCAR
peptide, can be referred to as an activatable tCAR protein. A tCAR
peptide comprising an activatable peptide can be referred to as an
activatable tCAR peptide. The blocking group can comprise or
consist of an amino acid or an amino acid sequence.
[0229] An activatable peptide, such as an activatable tCAR peptide,
can be blocked from internalization into a cell, from tissue
penetration, or both. Generally, an activatable peptide, such as an
activatable tCAR peptide, will be blocked from both internalization
into a cell and penetration of tissue. Such activatable peptides
can be referred to as activatable internalization and penetrating
peptides. However, some activatable peptides could be blocked only
from tissue penetration or only from internalization into a cell.
Such activatable peptides can be referred to as activatable
internalization peptides (for peptides that are blocked only from
internalization into a cell) or as activatable penetrating peptides
(for peptides that are blocked only from penetration of tissue).
Generally, internalization peptides that are activatable will be
activatable internalization peptides. Similarly, penetrating
peptides that are activatable generally will be activatable
penetrating peptides. Internalization and penetrating peptides that
are activatable will be activatable internalization and penetrating
peptides. Removal of the blocking group will allow the peptide to
be internalized into a cell, penetrate tissue, or both.
[0230] The cleavable bond of an activatable peptide, such as an
activatable tCAR peptide, can be cleaved in any suitable way. For
example, the cleavable bond can be cleaved enzymatically or
non-enzymatically. For enzymatic cleavage, the cleaving enzyme can
be supplied or can be present at a site where the peptide is
delivered, homes, travels or accumulates. For example, the enzyme
can be present in proximity to a cell to which the peptide is
delivered, homes, travels, or accumulates. For non-enzymatic
cleavage, the peptide can be brought into contact with a cleaving
agent, can be placed in cleaving conditions, or both. A cleaving
agent is any substance that can mediate or stimulate cleavage of
the cleavable bond. A non-enzymatic cleaving agent is any cleaving
agent except enzymes. Cleaving conditions can be any solution or
environmental conditions that can mediate or stimulate cleavage of
the cleavable bond. For example, some labile bonds can be cleaved
in acid conditions, alkaline conditions, in the presence of a
reactive group, etc. Non-enzymatic cleaving conditions are any
cleaving conditions except the presence of enzymes. Non-agent
cleaving conditions are any cleaving conditions except the presence
of cleaving agents.
[0231] Activatable peptides, such as activatable tCAR peptides, can
be activatable in broad or narrow circumstances. Generally,
activatable peptides are activatable relative to a specific agent
or group of agents that can activate the peptides. Thus, for
example, a particular activatable peptide may only be activatable
by certain proteases. Such a peptide can be referred to as an
activatable peptide but can also be referred to as being
activatable by the particular proteases.
[0232] A "protease-activatable peptide" (or "protease-activated
peptide") refers to an activatable peptide where the blocking group
is coupled to the peptide via a peptide bond and where the peptide
bond can be cleaved by a protease. Cleavage of this peptide bond in
a protease-activatable peptide makes the peptide capable of
internalization into a cell and/or of tissue penetration. In one
example, the blocking group can be coupled to the internalizing
peptide via a cleavable or labile bond. The cleavable bond can be
cleaved by, for example, an enzyme or a chemical compound. Cleavage
or `labilization` of the bond in an activatable peptide makes the
peptide capable of internalization into a cell and/or of tissue
penetration. Such cleavage or `labilization` can be referred to as
activation of the peptide. A protease-activatable peptide is a form
of activatable peptide. The amino acids of an activatable tCAR
peptide, can be selected for specific purposes. For example, the
amino acids following the end of tCAR sequence can be chosen to
form all or a portion of a protease recognition sequence. This
would be useful, for example, to specify or enable cleavage of a
peptide having the tCAR peptide that is activated by cleavage
following the end of the tCAR sequence. Examples of such amino acid
choices are shown in Tables 1 and 2 of U.S. Patent Application
Publication No. 2010-0322862. Protease cleavage sites can be
predicted based on knowledge developed and known to those of skill
in the art. For example, prediction of cleavage can be assessed at
the website cbs.dtu.dk/services/ProP/. A useful class of peptides
can consist of unblocked peptides and activatable peptidess, which
class excludes blocked peptidess that are not activatable.
[0233] Useful proteases include enzymes that cleave on the C
terminal side of basic residues (the C terminal residue of tCAR
peptides is a basic residue) and enzymes that recognize sequence on
the C terminal side of their cleavage site (thus allowing free
choice of the C terminal sequence of the cleavage product).
Examples of useful proteases include, for example, serine proteases
(including, for example, plasmin and plasminogen activators),
urokinase, proprotein convertases (see, for example, Duckert et
al., Prediction of proprotein convertase cleavage sites Protein
engineering Design and Selection 17(1):107-112 (2004)), furins, and
carboxypeptidases, such as carboxypeptidase A (amino acids with
aromatic or branched hydrocarbon side chains), glutamate-specific
carboxypeptidase, proline-specific carboxypeptidase, and PSMA.
Serine proteases are particularly useful for tCAR peptides and tCAR
compositions targeted to cancer cells and tumors. Examples of
enzymes that cleave on the C terminal side of basic residues
include Arg-C protease (which cleaves on the C terminal side of
arginine residues; Keil, Specificity of Proteolysis
(Springer-Verlag, Berlin-Heidelberg-N.Y. (1992)), clostripain
(which cleaves on the C terminal side of arginine residues; Keil,
1992), enterokinase (which cleaves after the sequence
-Asp-Asp-Asp-Asp-Lys-; SEQ ID NO:22), Factor Xa (which cleaves
after the sequence -Gly-Arg-; Fujikawa et al., Activation of bovine
factor X (Stuart factor): conversion of factor Xa alpha to factor
Xa beta, Proc. Natl. Acad. Sci. 72: 3359-3363 (1975)), Lys-C (which
cleaves on the C terminal side of lysine residues; Keil, 1992),
thrombin (which cleaves on the C terminal side of arginine
residues; Keil, 1992), trypsin (which cleaves on the C terminal
side of arginine and lysine residues; Keil, 1992), serine
proteases, proprotein convertases (such as PC1, PC2, PC3, PC4, PC5,
PC6, PC7, PC8, furin, Pace, PACE4, Site 1 protease, S1P, SKI,
NARC-1, PCSK1, PCSK2, PCSK3, PCSK4, PCSK5, PCSK6, PCSK7, PCSK8, and
PCSK9), plasmin, and plasminogen activators. Examples of enzymes
that recognize sequence on the C terminal side of their cleavage
site include Asp-N endopeptidase (which cleaves on the N terminal
side of aspartic acid; Keil, 1992) and carboxypeptidases such as
carboxypeptidase A (which cleaves C-terminal residues except
proline, lysine and arginine).
[0234] Examples of proteases are also described in Hook,
Proteolytic and cellular mechanisms in prohormone and proprotein
processing, RG Landes Company, Austin, Tex., USA (1998); Hooper et
al., Biochem. J. 321: 265-279 (1997); Werb, Cell 91: 439-442
(1997); Wolfsberg et al., J. Cell Biol. 131: 275-278 (1995);
Murakami and Etlinger, Biochem. Biophys. Res. Comm. 146: 1249-1259
(1987); Berg et al., Biochem. J. 307: 313-326 (1995); Smyth and
Trapani, Immunology Today 16: 202-206 (1995); Talanian et al., J.
Biol. Chem. 272: 9677-9682 (1997); and Thornberry et al., J. Biol.
Chem. 272: 17907-17911 (1997).
[0235] As used herein, "activatable CendR element" refers to a
CendR element having a molecule, moiety, nanoparticle, compound or
other composition covalently coupled to the CendR element, such as
to the terminal carboxyl group of the C-terminal element, where the
molecule, moiety, nanoparticle, compound or other composition can
block internalization and/or tissue penetration of the CendR
composition, conjugate, molecule, protein, peptide, etc. and where
the molecule, moiety, nanoparticle, compound or other composition
can be removed (to expose the terminal carboxy group, for example).
Activatable CendR elements are described in U.S. Patent Application
Publication No. 2010-0322862, which is hereby incorporated by
reference in its entirety and, in particular, for its description
of CendR elements and activatable CendR elements.
[0236] Exopeptidases, such as carboxypeptidases, can be used to
activate peptides. For example, carboxypeptidases are useful
proteases for activating peptides. Carboxypeptidases remove the
C-terminal amino acid from proteins and peptides. Carboxypeptidases
can, within the limits of their substrate preferences, remove amino
acids sequentially from a protein or peptide. Thus, for example, a
carboxypeptidase could completely or nearly completely hydrolyze a
protein of peptide. Because various carboxypeptidases have certain
substrate preferences or limitations, and because carboxypeptidases
generally only cleave peptide bonds, the presence of certain amino
acids, modifications, and/or non-peptide bonds can control
carboxypeptidase cleavage of a protein or peptide.
[0237] In the context of tCAR peptides, the structure of and/or
modifications to a protein, peptide or amino acid sequence
comprising a tCAR peptide can be chosen to result in cleavage by a
carboxypeptidase ending at the C-terminal amino acid of the tCAR
peptide. This can be accomplished by, for example, using a bond
between the C-terminal amino acid and the penultimate amino acid in
the tCAR peptide that can be protected from protease cleavage. For
example, the bond can be a non-peptide bond or can include a
modification, such as methylation. As another example, a D-amino
acid can be used as the C-terminal amino acid, the penultimate
amino acid, or both, in a tCAR peptide. As another example, a
D-amino acid can be used as the C-terminal amino acid in a tCAR
peptide. tCAR peptides with limited use of D amino acids retain
internalization and penetration activity. As another example, an
amino acid that serves as a substrate for a carboxypeptidase can be
located C-terminal to the C-terminal amino acid in the tCAR
peptide. For example, for a glutamate-specific carboxypeptidase
such as PSMA, a glutamic acid amino acid can be placed adjacent to
and C-terminal of the C-terminal amino acid in the tCAR peptide and
at the C-terminal end of the protein or peptide containing the tCAR
peptide. Other amino acid-specific (or preferring)
carboxypeptidases can be used in similar ways. In these cases, the
C-terminal amino acid in the tCAR petide should not be a substrate
(or should be a disfavored substrate) for the carboxypeptidase.
[0238] Bonds and modifications to amino acids that can reduce or
eliminate protease cleavage at a bond are known and can be used in
the disclosed tCAR peptides. For example, a variety of chemical
modification techniques and moieties are described in, for example,
U.S. Pat. Nos. 5,554,728, 6,869,932, 6,828,401, 6,673,580,
6,552,170, 6,420,339, U.S. Pat. Pub. 2006/0210526 and Intl. Pat.
App. WO 2006/136586. Some examples of such modifications include
peptide bond surrogates such as those described in Cudic and
Stawikowski, Peptidomimetics: Fmoc Solid-Phase Pseudopeptide
Synthesis, in Methods in Molecular Biology, vol. 294, 223-246
(2008), and chemical modifications, such as maleimide capping,
polyethylene glycol (PEG) attachment, maleidification, acylation,
alkylation, esterification, and amidification, to produce
structural analogs of the peptide. These and other modifications
are further described elsewhere herein.
[0239] The tCAR peptide in a disclosed protein, peptide, amino acid
sequence or tCAR composition generally should be at a free
C-terminal end or on the N-terminal side of the cleavage site in an
activatable tCAR peptide.
[0240] Also disclosed are methods of forming an activatable
peptide, the method comprising causing a blocking group to be
covalently coupled to a peptide, wherein a bond coupling the
blocking group and the peptide is cleavable. Also disclosed are
methods of forming an activatable peptide, the method comprising
causing a blocking group to be covalently coupled to an amino acid
sequence, wherein the amino acid sequence comprises the peptide,
wherein a bond coupling the blocking group and the peptide is
cleavable. Also disclosed are methods of forming an activatable
peptide, the method comprising (a) selecting an amino acid sequence
for internalization into a cell and/or penetration of tissue,
wherein the amino acid sequence comprises a peptide, and (b)
causing a blocking group to be covalently coupled to the peptide,
wherein a bond coupling the blocking group and the peptide is
cleavable. The blocking group covalently coupled to the peptide
reduces or prevents internalization into a cell and/or penetration
of tissue. The blocking group covalently coupled to the peptide can
reduce or prevent internalization into a cell and/or penetration of
tissue compared to the same peptide with no blocking group. For
example, an amino acid sequence comprising tCAR sequence-cleavage
site-homing module can be made and then tested for activatability
(via cleavage of the cleavage site, for example). For example, a
pool of peptides having the amino acid sequence
CARSKNK-XXXXXXXXXXXXXXXXX (SEQ ID NO:8) can be tested for homing
and activatability. That is, such peptides can be identified by
screens using libraries. The activatable peptide can comprise the
selected amino acid sequence and the blocking group. The cell can
be in a subject. The enzyme, cleaving agent, and/or cleaving
conditions present in proximity to the cell of interest can be
identified. The enzyme, cleaving agent, and/or cleaving conditions
present in proximity to the cell of interest can be identified
prior to forming the activatable peptide. The cleavable bond can be
selected based on the enzyme that is present in proximity to the
cell of interest. The cleavable bond can be selected based on the
cleaving agent present at site where the peptide is delivered,
homes, travels or accumulates, such as the cell of interest. The
cleavable bond can be selected based on the cleaving conditions
present at site where the peptide is delivered, homes, travels or
accumulates, such as the cell of interest. The cleavable bond can
be selected prior to forming the activatable peptide. The peptide
can comprise a terminal carboxyl group, wherein the blocking group
is coupled to the terminal carboxyl group.
[0241] "Internalization" refers to passage through a plasma
membrane or other biological barrier. "Penetration" refers to
passage into and through a cell, tissue, or other biological
barrier. Penetration generally involves and includes
internalization. The disclosed tCAR peptides generally promote and
allow both internalization (such as internalization into a cell)
and penetration (such as tissue penetration). Reference to
internalization or to penetration should be understood to refer to
both internalization and penetration unless the context indicates
otherwise (such as separate or distinct discussion and description
of internalization into a cell and tissue penetration
separately--the present paragraph is an example of such).
[0242] By "internalization into a cell" is meant that that tCAR
peptide is capable of penetrating the plasma membrane, thereby
being internalized into the cell. This internalization can occur
with, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%
efficiency for a given tCAR peptide and a given cell. tCAR peptides
generally an promote, mediate, cause, enhance, etc.
internalization; penetration; internalization into and/or through
cells, tissue, or both; penetration into and/or through cells,
tissue, or both; permeabilization of cells and/or tissues; or a
combination. By "permeabilization" is meant promoting, mediating,
causing, enhancing, etc. the ability and/or condition of cells
and/or tissues to allow compositions, conjugates, molecules, etc.
in proximity to the cells and/or tissues to enter and or pass
through the cells and/or tissues. Thus, the disclosed tCAR
proteins, peptides, conjugates, compositions, etc. can be said to
permeabilize the cells and/or tissues. By "permeable" is meant the
ability and/or condition of cells and/or tissues to allow
compositions, conjugates, molecules, etc. in proximity to the cells
and/or tissues to enter and or pass through the cells and/or
tissues.
[0243] As used herein, "tissue penetration" and "penetration of
tissue" refer to passage into or through a tissue beyond or through
the outer or a first layer of cells or through a tissue membrane.
Such passage or penetration through tissue (which can also be
referred to as extravasation and tissue penetration) can be a
function of, for example, cell internalization and passage between
cells in the tissue. Throughout this application, when the term
"tissue penetration" is used, it is understood that such
penetration can also extend to other barriers and suitable
membranes found throughout the body, such as the blood brain
barrier.
[0244] Cells that can internalize a tCAR peptide can be identified
by, for example, (a) exposing a cell to a tCAR peptide; and (b)
determining if the tCAR peptide was internalized. The cell can be
in an assay, for example. Any form or type of tCAR peptide, tCAR
peptide, tCAR protein, tCAR conjugate, or tCAR composition can be
used in these methods.
[0245] Cancer cells, or subjects harboring cancer cells, can be
identified as candidates for tCAR-based therapy by, for example,
(a) exposing the cancer cell to a tCAR peptide; and (b) determining
if the tCAR peptide was internalized by the cancer cell, wherein an
internalized tCAR peptide identifies the cancer cell or the subject
as being a candidate for tCAR-based therapy. The cell can be in an
assay, or can be in a subject, for example. Any form or type of
tCAR peptide, tCAR peptide, tCAR protein, tCAR conjugate, or tCAR
composition can be used in these methods.
[0246] Tumors, or subjects harboring a tumor, can be identified as
a candidate for tCAR-based therapy by, for example, (a) exposing
tissue from the tumor to a tCAR peptide; and (b) determining if the
tCAR peptide passed through the tissue or was internalized by cells
in the tissue, wherein a passed-through or internalized tCAR
peptide identifies the tumor or the subject as being a candidate
for tCAR-based therapy. Any form or type of tCAR peptide, tCAR
peptide, tCAR protein, tCAR conjugate, or tCAR composition can be
used in these methods.
[0247] The co-composition can be, for example, a nanoparticle, or a
molecule, or complex of molecules with therapeutic or diagnostic
applications. Therapeutic co-compositions that can be targeted with
tCAR peptides include but are not limited to a nanoparticle, a
molecule, a complex of molecules, an anti-angiogenic agent, a
pro-angiogenic agent, a cancer chemotherapeutic agent, a cytotoxic
agent, a pro-cell survival agent, a cell differentiating agent, a
neuroprotective agent, an immunomodulatory agent, an
anti-inflammatory agent, an anti-arthritic agent, an anti-viral
agent, or a combination of these. Therapeutic co-compositions that
can be targeted with tCAR peptides include but are not limited to a
therapeutic protein, a therapeutic compound, a therapeutic
composition, an anti-angiogenic agent, a pro-angiogenic agent, a
cancer chemotherapeutic agent, a toxin, a cytotoxic agent, an
anti-inflammatory agent, an anti-arthritic agent, a growth factor,
a cytokine, a chemokine, a compound that modulates one or more
signaling pathways, an antibody, a nucleic acid, a nucleic acid
analog, a cell, a virus, a phage, a viral particle, a phage
particle, a viral capsid, a phage capsid, a virus-like particle, a
liposome, a micelle, a bead, a nanoparticle, a microparticle, a
chemotherapeutic agent, a contrast agent, an imaging agent, a
label, a labeling agent, or a combination. Diagnostic
co-compositions that can be targeted with tCAR peptides include but
are not limited to a nanoparticle, a molecule, a complex of
molecules, a MRI imaging agent, a radioimaging agent, an optical
imaging agent, a molecular tag (such as biotin), a fluorophore, an
epitope tag (that can, for example, be detected using a specific
molecular assay), or a combination of these.
[0248] The cargo composition can be, for example, a nanoparticle,
or a molecule, or complex of molecules with therapeutic or
diagnostic applications. Therapeutic cargo compositions that can be
targeted with tCAR peptides include but are not limited to a
nanoparticle, a molecule, a complex of molecules, an
anti-angiogenic agent, a pro-angiogenic agent, a cancer
chemotherapeutic agent, a cytotoxic agent, a pro-cell survival
agent, a cell differentiating agent, a neuroprotective agent, an
immunomodulatory agent, an anti-inflammatory agent, an
anti-arthritic agent, an anti-viral agent, or a combination of
these. Therapeutic cargo compositions that can be targeted with
tCAR peptides include but are not limited to a therapeutic protein,
a therapeutic compound, a therapeutic composition, an
anti-angiogenic agent, a pro-angiogenic agent, a cancer
chemotherapeutic agent, a toxin, a cytotoxic agent, an
anti-inflammatory agent, an anti-arthritic agent, a growth factor,
a cytokine, a chemokine, a compound that modulates one or more
signaling pathways, an antibody, a nucleic acid, a nucleic acid
analog, a cell, a virus, a phage, a viral particle, a phage
particle, a viral capsid, a phage capsid, a virus-like particle, a
liposome, a micelle, a bead, a nanoparticle, a microparticle, a
chemotherapeutic agent, a contrast agent, an imaging agent, a
label, a labeling agent, or a combination. Diagnostic cargo
compositions that can be targeted with tCAR peptides include but
are not limited to a nanoparticle, a molecule, a complex of
molecules, a MRI imaging agent, a radioimaging agent, an optical
imaging agent, a molecular tag (such as biotin), a fluorophore, an
epitope tag (that can, for example, be detected using a specific
molecular assay), or a combination of these.
[0249] A cell that can internalize a tCAR peptide can be identified
by, for example, (a) exposing a cell to a tCAR peptide, and (b)
determining if the tCAR peptide was internalized. Also disclosed
are methods of identifying a cancer cell as a candidate for
tCAR-based therapy, the method comprising (a) exposing the cancer
cell to a tCAR peptide, and (b) determining if the tCAR peptide was
internalized by the cancer cell, wherein an internalized tCAR
peptide identifies the cancer cell as being a candidate for
tCAR-based therapy. The cell can be in an assay. The tCAR peptide
can be coupled to a protein or peptide. The tCAR peptide can be an
activatable tCAR peptide. The activatable tCAR peptide can be
activated before exposure to the cell. The activatable tCAR peptide
can be a protease-activatable tCAR peptide. The protein or peptide
can be circular. The protein or peptide can be linear. The tCAR
peptide can be at the C-terminal end of the protein or peptide. Any
form or type of tCAR peptide, tCAR peptide, tCAR protein, tCAR
conjugate, or tCAR composition can be used in these methods.
[0250] A tissue that can be penetrated by a tCAR peptide can be
identified by, for example, (a) exposing a tissue to a tCAR
peptide, and (b) determining if the tCAR peptide penetrated the
tissue. Also disclosed are methods of identifying a tumor as a
candidate for tCAR-based therapy, the method comprising (a)
exposing a cell from the tumor to a tCAR peptide, and (b)
determining if the tCAR peptide was internalized by the cell,
wherein an internalized tCAR peptide identifies the tumor as being
a candidate for tCAR-based therapy. A tumor can be identified as a
candidate for tCAR-based therapy by, for example, (a) exposing the
tumor to a tCAR peptide, and (b) determining if the tCAR peptide
penetrated the tumor, wherein a tCAR peptide that penetrated
identifies the tumor as being a candidate for tCAR-based therapy.
The tumor can be in an assay. The tCAR peptide can be coupled to a
protein or peptide. The tCAR peptide can be an activatable tCAR
peptide. The activatable tCAR peptide can be activated before
exposure to the tumor. The activatable tCAR peptide can be a
protease-activatable tCAR peptide. The protein or peptide can be
circular. The protein or peptide can be linear. The tCAR peptide
can be at the C-terminal end of the protein or peptide. Any form or
type of tCAR peptide, tCAR peptide, tCAR protein, tCAR conjugate,
or tCAR composition can be used in these methods.
[0251] The tCAR peptide can have a length of up to 10, 20, 30, 40,
50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In
particular embodiments, a tCAR peptide can have a length of at
least 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or
200 residues. In further embodiments, a tCAR peptide can have a
length of 7 to 200 residues, 7 to 100 residues, 7 to 90 residues, 7
to 80 residues, 7 to 70 residues, 7 to 60 residues, 7 to 50
residues, 7 to 40 residues, 7 to 30 residues, 7 to 20 residues, 7
to 15 residues, 7 to 10 residues, 8 to 200 residues, 8 to 100
residues, 8 to 90 residues, 8 to 80 residues, 8 to 70 residues, 8
to 60 residues, 8 to 50 residues, 8 to 40 residues, 8 to 30
residues, 8 to 20 residues, 8 to 15 residues, 8 to 10 residues, 9
to 200 residues, 9 to 100 residues, 9 to 90 residues, 9 to 80
residues, 9 to 70 residues, 9 to 60 residues, 9 to 50 residues, 9
to 40 residues, 9 to 30 residues, 9 to 20 residues, 9 to 15
residues, 9 to 10 residues, 10 to 200 residues, 10 to 100 residues,
10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60
residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues,
10 to 20 residues, 10 to 15 residues, 15 to 200 residues, 15 to 100
residues, 15 to 90 residues, 15 to 80 residues, 15 to 70 residues,
15 to 60 residues, 15 to 50 residues, 15 to 40 residues, 15 to 30
residues, 15 to 20 residues, 20 to 200 residues, 20 to 100
residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues,
20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30
residues. As used herein, the term "residue" refers to an amino
acid or amino acid analog.
[0252] A protein or peptide containing a tCAR peptide can have a
length of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000
residues. In particular embodiments, the protein or peptide portion
of a tCAR composition can have a length of at least 10, 20, 30, 40,
50, 60, 70, 80, 90, 100 or 200 residues. In further embodiments,
the protein or peptide containing a tCAR peptide can have a length
of 7 to 200 residues, 7 to 100 residues, 7 to 90 residues, 7 to 80
residues, 7 to 70 residues, 7 to 60 residues, 7 to 50 residues, 7
to 40 residues, 7 to 30 residues, 7 to 20 residues, 7 to 15
residues, 7 to 10 residues, 8 to 200 residues, 8 to 100 residues, 8
to 90 residues, 8 to 80 residues, 8 to 70 residues, 8 to 60
residues, 8 to 50 residues, 8 to 40 residues, 8 to 30 residues, 8
to 20 residues, 8 to 15 residues, 8 to 10 residues, 9 to 200
residues, 9 to 100 residues, 9 to 90 residues, 9 to 80 residues, 9
to 70 residues, 9 to 60 residues, 9 to 50 residues, 9 to 40
residues, 9 to 30 residues, 9 to 20 residues, 9 to 15 residues, 9
to 10 residues, 10 to 200 residues, 10 to 100 residues, 10 to 90
residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues,
10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20
residues, 10 to 15 residues, 15 to 200 residues, 15 to 100
residues, 15 to 90 residues, 15 to 80 residues, 15 to 70 residues,
15 to 60 residues, 15 to 50 residues, 15 to 40 residues, 15 to 30
residues, 15 to 20 residues, 20 to 200 residues, 20 to 100
residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues,
20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30
residues.
[0253] The tCAR conjugate can have a length of up to 50, 100, 150,
200, 250, 300, 400, 500, 1000 or 2000 residues. In particular
embodiments, a tCAR conjugate can have a length of at least 10, 20,
30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further
embodiments, a tCAR conjugate can have a length of 10 to 200
residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues,
10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40
residues, 10 to 30 residues, 10 to 20 residues, 10 to 15 residues,
15 to 200 residues, 15 to 100 residues, 15 to 90 residues, 15 to 80
residues, 15 to 70 residues, 15 to 60 residues, 15 to 50 residues,
15 to 40 residues, 15 to 30 residues, 15 to 20 residues, 20 to 200
residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues,
20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40
residues or 20 to 30 residues.
[0254] The protein or peptide portion of a tCAR composition can
have a length of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000
or 2000 residues. In particular embodiments, the protein or peptide
portion of a tCAR composition can have a length of at least 10, 20,
30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further
embodiments, the protein or peptide portion of a tCAR composition
can have a length of 7 to 200 residues, 7 to 100 residues, 7 to 90
residues, 7 to 80 residues, 7 to 70 residues, 7 to 60 residues, 7
to 50 residues, 7 to 40 residues, 7 to 30 residues, 7 to 20
residues, 7 to 15 residues, 7 to 10 residues, 8 to 200 residues, 8
to 100 residues, 8 to 90 residues, 8 to 80 residues, 8 to 70
residues, 8 to 60 residues, 8 to 50 residues, 8 to 40 residues, 8
to 30 residues, 8 to 20 residues, 8 to 15 residues, 8 to 10
residues, 9 to 200 residues, 9 to 100 residues, 9 to 90 residues, 9
to 80 residues, 9 to 70 residues, 9 to 60 residues, 9 to 50
residues, 9 to 40 residues, 9 to 30 residues, 9 to 20 residues, 9
to 15 residues, 9 to 10 residues, 10 to 200 residues, 10 to 100
residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues,
10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30
residues, 10 to 20 residues, 10 to 15 residues, 15 to 200 residues,
15 to 100 residues, 15 to 90 residues, 15 to 80 residues, 15 to 70
residues, 15 to 60 residues, 15 to 50 residues, 15 to 40 residues,
15 to 30 residues, 15 to 20 residues, 20 to 200 residues, 20 to 100
residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues,
20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30
residues.
[0255] The tCAR composition can have a length of up to 50, 100,
150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In particular
embodiments, a tCAR composition can have a length of at least 10,
20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further
embodiments, a tCAR composition can have a length of 10 to 200
residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues,
10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40
residues, 10 to 30 residues, 10 to 20 residues, 10 to 15 residues,
15 to 200 residues, 15 to 100 residues, 15 to 90 residues, 15 to 80
residues, 15 to 70 residues, 15 to 60 residues, 15 to 50 residues,
15 to 40 residues, 15 to 30 residues, 15 to 20 residues, 20 to 200
residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues,
20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40
residues or 20 to 30 residues.
[0256] tCAR (and other) peptides can be stabilized against
proteolysis. For example, the stability and activity of peptides,
such as tumor-homing peptides CREKA (Simberg et al., 2007), by
protecting some of the peptide bonds with N-methylation or
C-methylation. The most important bond to protect in order to
enhance activity is the R-G bond because it would prevent a
cleavage that would inactivate both the integrin-binding and tCAR
activities. Accessory peptides and homing peptides can also or
similarly be stabilized against proteolysis.
[0257] The disclosed tCAR peptides (and other tCAR forms) and
co-compositions can be administered together or separately; in the
same form and manner or in different forms and/or manners; at the
same time or at different times; with the tCAR peptide (or other
tCAR form) administered first or second. Administration can be, for
example, co-administration (at the same time and by the same or
different route/means/form), separate administration (parallel
administration by the same or different route/means/form),
sequential administration (at different times by the same or
different route/means/form), etc. When the co-composition and tCAR
peptide (or other tCAR form) are administered at different times, a
variety of different delays can be used between the
administrations. For example, the tCAR peptide (or other tCAR form)
can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15,
20, 30, 40, 45, 50, 60, 70, 80, 90, 100, 110, or 120 minutes or
more before administering a co-composition. The tCAR peptide (or
other tCAR form) can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 54, 60, 66, or 72 hours or more before
administering a co-composition. The tCAR peptide (or other tCAR
form) can be administered 1, 2, 3, 4, 5, 6, or 7 days or more
before administering a co-composition. The tCAR peptide (or other
tCAR form) can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 15, 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, 110, or 120
minutes or more after administering a co-composition. The tCAR
peptide (or other tCAR form) can be administered 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 54, 60, 66, or 72 hours or more
after administering a co-composition. The tCAR peptide (or other
tCAR form) can be administered 1, 2, 3, 4, 5, 6, or 7 days or more
after administering a co-composition.
[0258] The tCAR peptide (or other tCAR form) can be administered
within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 20, 30, 40,
45, 50, 60, 70, 80, 90, 100, 110, or 120 minutes before
administering a co-composition. The tCAR peptide (or other tCAR
form) can be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 54, 60, 66, or 72 hours before administering a
co-composition. The tCAR peptide (or other tCAR form) can be
administered within 1, 2, 3, 4, 5, 6, or 7 days before
administering a co-composition. The tCAR peptide (or other tCAR
form) can be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 15, 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, 110, or 120
minutes after administering a co-composition. The tCAR peptide (or
other tCAR form) can be administered within 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 54, 60, 66, or 72 hours after administering
a co-composition. The tCAR peptide (or other tCAR form) can be
administered within 1, 2, 3, 4, 5, 6, or 7 days after administering
a co-composition. Administration within the same day or hour is
particularly useful.
[0259] The tCAR composition, tCAR conjugate, tCAR molecule, tCAR
protein, or tCAR peptide and the co-composition can be administered
to the subject simultaneously. By simultaneously is meant during
overlapping or contiguous time periods. The tCAR composition, tCAR
conjugate, tCAR molecule, tCAR protein, or tCAR peptide and the
co-composition can be administered to the subject in a single
composition comprising the tCAR composition, tCAR conjugate, tCAR
molecule, tCAR protein, or tCAR peptide and the co-composition. The
tCAR composition, tCAR conjugate, tCAR molecule, tCAR protein, or
tCAR peptide and the co-composition can be administered to the
subject in separate compositions. The tCAR peptide and the
co-composition can be administered to the subject at different
times. The tCAR peptide and the co-composition can be administered
to the subject in separate compositions. By separate compositions
is meant compositions that are not mixed or in contact with each
other (except as may occur following administration). The tCAR
peptide and the co-composition can be administered to the subject
by separate routes. By separate routes is meant in separate
locations, by different means or mode, or both.
[0260] tCAR peptides can be made in the form of stabilized peptides
and/or formulated as long-circulating forms. For example, a
polyethylene glycol conjugate can be used. tCAR peptides and/or
co-compositions can also be administered over a period of time. For
example, tCAR peptides and/or co-compositions can be delivered with
an osmotic pump. This can extend the permeability of the target
cells and tissues. Modified forms of tCAR peptides can be used. For
example, tCAR peptides can be methylated (which can stabilize the
peptides against proteolysis). Stability against cleavage is
desirable, except for bonds to be cleaved to activate tCAR
peptides. Modifications to tCAR peptides generally should leave
them functional or capable of function after activation.
[0261] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed tCAR
compositions, conjugates, molecules, proteins, peptides, and
elements. For example, there are numerous D amino acids or other
non-natural amino acids which can be used. The opposite
stereoisomers of naturally occurring peptides are disclosed, as
well as the stereo isomers of peptide analogs. These amino acids
can readily be incorporated into polypeptide chains by chemical
synthesis or by charging tRNA molecules with the amino acid of
choice and engineering genetic constructs that utilize, for
example, amber codons, to insert the analog amino acid into a
peptide chain in a site specific way (Thorson et al., Methods in
Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in
Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic
Engineering Reviews 13:197-216 (1995), Cahill et al., TIBS,
14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba
and Hennecke, Bio/technology, 12:678-682 (1994) all of which are
herein incorporated by reference at least for material related to
amino acid analogs).
[0262] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include
CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH--
(cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and
--CHH.sub.2SO-- (These and others can be found in Spatola, A. F. in
Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,
B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);
Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide
Backbone Modifications (general review); Morley, Trends Pharm Sci
(1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res
14:177-185 (1979) (--CH.sub.2NH--, CH.sub.2CH.sub.2--); Spatola et
al. Life Sci 38:1243-1249 (1986) (--CH H.sub.2--S); Hann J. Chem.
Soc Perkin Trans. I 307-314 (1982) (--CH--CH--, cis and trans);
Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH.sub.2--);
Jennings-White et al. Tetrahedron Lett 23:2533 (1982)
(--COCH.sub.2--); Szelke et al. European Appin, EP 45665 CA (1982):
97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay et al. Tetrahedron.
Lett 24:4401-4404 (1983) (--C(OH)CH.sub.2--); and Hruby Life Sci
31:189-199 (1982) (--CH.sub.2--S--); each of which is incorporated
herein by reference. A particularly preferred non-peptide linkage
is --CH.sub.2NH--. It is understood that peptide analogs can have
more than one atom between the bond atoms, such as b-alanine,
g-aminobutyric acid, and the like.
[0263] Amino acid analogs and peptide analogs often have enhanced
or desirable properties, such as, more economical production,
greater chemical stability, enhanced pharmacological properties
(half-life, absorption, potency, efficacy, etc.), altered
specificity (e.g., a broad-spectrum of biological activities),
reduced antigenicity, and others.
[0264] D-amino acids can be used to generate more stable peptides,
because D amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides as
long as activity is preserved. Cysteine residues can be used to
cyclize or attach two or more peptides together. This can be
beneficial to constrain peptides into particular conformations.
(Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated
herein by reference).
[0265] Disclosed are polyfunctional tCAR compositions which, in
addition to the tCAR peptide, contain, for example, an accessory
peptide, an accessory peptide fused to the tCAR peptide, an
accessory molecule covalently coupled to or non-covalently
associated with the tCAR peptide, a homing peptide fused to the
tCAR peptide, a homing molecule covalently coupled to or
non-covalently associated with the tCAR peptide, a cargo
composition fused to the tCAR peptide, and/or a cargo composition
covalently coupled to or non-covalently associated with the tCAR
peptide. Additional compounds having separate functions can be
added to the composition. Such polyfunctional conjugates have at
least two functions conferred by different portions of the
composition and can, for example, display anti-angiogenic activity
or pro-apoptotic activity in addition to selective homing
activity.
[0266] As used herein, the term "peptide" is used broadly to mean
peptides, proteins, fragments of proteins and the like. The term
"peptidomimetic," as used herein, means a peptide-like molecule
that has the activity of the peptide upon which it is structurally
based. Such peptidomimetics include chemically modified peptides,
peptide-like molecules containing non-naturally occurring amino
acids, and peptoids and have an activity such as that from which
the peptidomimetic is derived (see, for example, Goodman and Ro,
Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry
and Drug Discovery" Vol. 1 (ed. M. E. Wolff; John Wiley & Sons
1995), pages 803-861).
[0267] Protein variants and derivatives are well understood by
those of skill in the art and in can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues.
Immunogenic fusion protein derivatives, such as those described in
the examples, are made by fusing a polypeptide sufficiently large
to confer immunogenicity to the target sequence by cross-linking in
vitro or by recombinant cell culture transformed with DNA encoding
the fusion. Deletions are characterized by the removal of one or
more amino acid residues from the protein sequence. Typically, no
more than about from 2 to 6 residues are deleted at any one site
within the protein molecule. These variants ordinarily are prepared
by site specific mutagenesis of nucleotides in the DNA encoding the
protein, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example M13 primer mutagenesis
and PCR mutagenesis. Amino acid substitutions are typically of
single residues, but can occur at a number of different locations
at once; insertions usually will be on the order of about from 1 to
10 amino acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure. Substitutional variants are those in which at least one
residue has been removed and a different residue inserted in its
place. Such substitutions generally are made in accordance with the
following Tables 5 and 6 and are referred to as conservative
substitutions.
TABLE-US-00001 TABLE 5 Amino Acid Abbreviations Amino Acid
Abbreviations Alanine Ala A allosoleucine AIle Arginine Arg R
asparagine Asn N aspartic acid Asp D Cysteine Cys C glutamic acid
Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isolelucine Ile
I Leucine Leu L Lysine Lys K phenylalanine Phe F proline Pro P
pyroglutamic acid pGlu Serine Ser S Threonine Thr T Tyrosine Tyr Y
Tryptophan Trp W Valine Val V
TABLE-US-00002 TABLE 6 Amino Acid Substitutions Original Residue
Exemplary Conservative Substitutions, others are known in the art.
Ala Ser Arg Lys; Gln Asn Gln; His Asp Glu Cys Ser Gln Asn, Lys Glu
Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln Met
Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val
Ile; Leu
[0268] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those in Table 6, i.e., selecting residues that differ more
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site or (c) the bulk
of the side chain. The substitutions which in general are expected
to produce the greatest changes in the protein properties will be
those in which (a) a hydrophilic residue, e.g. seryl or threonyl,
is substituted for (or by) a hydrophobic residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine, in this case, (e) by increasing the
number of sites for sulfation and/or glycosylation.
[0269] For example, the replacement of one amino acid residue with
another that is biologically and/or chemically similar is known to
those skilled in the art as a conservative substitution. For
example, a conservative substitution would be replacing one
hydrophobic residue for another, or one polar residue for another.
The substitutions include combinations such as, for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and
Phe, Tyr. Such conservatively substituted variations of each
explicitly disclosed sequence are included within the mosaic
polypeptides provided herein.
[0270] Substitutional or deletional mutagenesis can be employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation
(Ser or Thr). Deletions of cysteine or other labile residues also
may be desirable. Deletions or substitutions of potential
proteolysis sites, e.g. Arg, is accomplished for example by
deleting one of the basic residues or substituting one by
glutaminyl or histidyl residues.
[0271] Certain post-translational derivatizations are the result of
the action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the o-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco pp
79-86 [1983]), acetylation of the N-terminal amine and, in some
instances, amidation of the C-terminal carboxyl.
[0272] It is understood that one way to define the variants and
derivatives of the disclosed proteins herein is through defining
the variants and derivatives in terms of homology/identity to
specific known sequences. For example, SEQ ID NO:4 sets forth a
particular sequence of tCAR. Specifically disclosed are variants of
these and other peptides herein disclosed which have at least, 70%
or 75% or 80% or 85% or 90% or 95% homology to the stated sequence.
Wherein a sequence is said to have at least about 70% sequence
identity, it is understood to also have at least about 75%, 80%,
85%, 90%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity.
[0273] A design principle for homing peptides has been developed
that combines three functions: tissue-specific homing, spreading
within the target tissue, and internalization into cells in that
tissue. These peptides contain both a tissue-specific homing
sequence and a tissue-penetrating and internalizing motif embodied
in a tCAR peptide.
[0274] The disclosed compounds are useful tools for introducing
materials into the target tissues. They can allow disease-specific
or cell type and tissue-specific targeting of diagnostic and
therapeutic compounds to increase efficacy and decrease side
effects. The principles disclosed herein are applicable to any
cells or tissues for which specific homing peptides can be
obtained.
[0275] Studies have revealed extensive molecular heterogeneity in
the vasculature of different normal tissues. In addition,
pathological lesions, such as tumors, impose their own changes on
the vasculature. This system of molecular markers can be referred
to as `vascular zip codes` (Ruoslahti, 2004). The zip codes enable
docking-based (`synaphic`) targeting to selectively deliver
diagnostics and therapeutics into a specific tissue. This approach
can produce greater efficacy and diminished side effects. The
targeted delivery principle has been established, particularly in
cancer: targeting of radioisotopes to leukemic cells with
antibodies is an established therapy, and several products aimed at
diagnosis and treatment of solid tumors are in clinical trials;
many of them use early generation tumor-homing peptides or their
derivatives. However, one issue in making the synaphic delivery
more generally useful is that efficacy has tended to be low. It has
been realized that it may be more effective to target the delivery
to blood vessels because their inner endothelial lining is readily
available to circulating probes, whereas penetration into tumor
parenchyma has been a problem in the past (Jain, 1990). Thus, while
it has been easy to demonstrate binding of the targeted material to
the target vessels, a substantially higher concentration of the
material in the target tissue has not necessarily been achieved
(e.g. Liu et al., 2007).
[0276] The disclosed peptides can be validated by, for example,
testing in vitro cell binding and internalization, and in vivo
homing. Synthetic peptides can be used to show that the activities
associated with the selected phage are reproduced by the peptide
the phage displays. Techniques for this are well known (e.g. Zhang
et al., 2005; Simberg et al., 2007; Karmali et al., 2008). The
peptides generally can be labeled with a fluorophore to allow
detection in tissues, and both the free peptide and a multimeric
conjugate on nanoparticles (which more closely resembles the
multivalent presentation on phage) can be tested.
[0277] A variety of homing molecules can be used in the disclosed
compositions, conjugates and methods. Such homing molecules
include, without limitation, peptides as disclosed herein. The
disclosed compounds, compositions, conjugates and methods can
include or use the disclosed homing molecules in various forms,
including peptides and peptidomimetics as disclosed. For
convenience of expression, in many places herein the use or
inclusion of peptides will be recited. It is understood that, in
such cases, it is considered that homing molecules in various forms
can also be used or included in the same or similar ways as is
described in terms of peptides, and such use and inclusion is
specifically contemplated and disclosed thereby.
[0278] The term "homing molecule" as used herein, means any
molecule that selectively homes in vivo to specific cells or
specific tissue in preference to normal tissue. Similarly, the term
"homing peptide" or "homing peptidomimetic" means a peptide that
selectively homes in vivo to specific cells or specific tissue in
preference to normal tissue. It is understood that a homing
molecule that selectively homes in vivo to specific cells or
specific tissue or can exhibit preferential homing to r specific
cells or specific tissue.
[0279] By "selectively homes" is meant that, in vivo, the homing
molecule binds preferentially to the target as compared to
non-target. For example, the homing molecule can bind
preferentially to tumors, as compared to non-tumors. Selective
homing to, for example, tumor cells generally is characterized by
at least a two-fold greater localization within tumor cells, as
compared to several tissue types of non-tumor cells. A homing
molecule can be characterized by, for example, 5-fold, 10-fold,
20-fold or more preferential localization to the target as compared
to one or more non-targets. For example, a homing molecule can be
characterized by, for example, 5-fold, 10-fold, 20-fold or more
preferential localization to tumor vasculature as compared to
vasculature of several or many tissue types of non-tumoral tissue,
or as compared to vasculature of most or all non-tumoral tissue. As
another example, a homing molecule can be characterized by, for
example, 5-fold, 10-fold, 20-fold or more preferential localization
to tumors as compared to several or many tissue types of
non-tumoral tissue, or as compared to-most or all non-tumoral
tissue. Thus, it is understood that, in some cases, a homing
molecule homes, in part, to one or more normal organs in addition
to homing to the target tissue. Selective homing can also be
referred to as targeting. The molecules, proteins, cells, tissues,
etc. that are targeted by homing molecules can be referred to as
targeted molecules, proteins, cells, tissues, etc.
[0280] Binding in the context of a homing molecule recognizing
and/or binding to its target can refer to both covalent and
non-covalent binding, for example where a homing molecule can bind,
attach or otherwise couple to its target by covalent and/or
non-covalent binding. Binding can be either high affinity or low
affinity, preferably high affinity. Examples of binding forces that
can be useful include, but are not limited to, covalent bonds,
dipole interactions, electrostatic forces, hydrogen bonds,
hydrophobic interactions, ionic bonds, and/or van der Waals forces.
This binding can occur in addition to that binding which occurs
with the tCAR peptide.
[0281] Surface molecules can be associated with and arranged in the
compositions in a variety of configurations. In some forms, surface
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of homing molecules, a plurality of cargo
molecules, or both. In some forms, surface molecules can be
associated with, conjugated to, and/or covalently coupled to a
plurality of homing molecules, wherein the homing molecules can be
associated with, conjugated to, and/or covalently coupled to a
plurality of cargo molecules. In some forms, surface molecules can
be associated with, conjugated to, and/or covalently coupled to a
plurality of cargo molecules, wherein the cargo molecules can be
associated with, conjugated to, and/or covalently coupled to a
plurality of homing molecules. Combinations of these combinations
can also be used.
[0282] The surface molecules, alternatively referred to as a
surface particles, disclosed herein can be conjugated with homing
molecules and cargo molecules in such a way that the composition is
delivered to a target. The surface molecule can be any substance
that can be used with the homing molecules and cargo molecules, and
is not restricted by size or substance. Examples include, but are
not limited to, nanoparticles (such as iron oxide nanoparticles or
albumin nanoparticles), liposomes, small organic molecules,
microparticles, or microbubbles, such as fluorocarbon microbubbles.
The term surface molecule is used to identify a component of the
disclosed composition but is not intended to be limiting. In
particular, the disclosed surface molecules are not limited to
substances, compounds, compositions, particles or other materials
composed of a single molecule. Rather, the disclosed surface
molecules are any substance(s), compound(s), composition(s),
particle(s) and/or other material(s) that can be conjugated with a
plurality of homing molecules and cargo molecules such that at
least some of the homing molecules and/or cargo molecules are
presented and/or accessible on the surface of the surface molecule.
A variety of examples of suitable surface molecules are described
and disclosed herein.
[0283] The surface molecule can be detectable, or can be a
therapeutic agent such as iRGD, RGD, or Abraxane.TM.. The section
herein which discusses cargo molecules and moieties that can be
detectable or therapeutic also applies to the surface molecule.
[0284] The term "nanoparticle" refers to a nanoscale particle with
a size that is measured in nanometers, for example, a nanoscopic
particle that has at least one dimension of less than about 100 nm.
Examples of nanoparticles include paramagnetic nanoparticles,
superparamagnetic nanoparticles, metal nanoparticles, nanoworms,
fullerene-like materials, inorganic nanotubes, dendrimers (such as
with covalently attached metal chelates), nanofibers, nanohoms,
nano-onions, nanorods, nanoropes and quantum dots. A nanoparticle
can produce a detectable signal, for example, through absorption
and/or emission of photons (including radio frequency and visible
photons) and plasmon resonance.
[0285] Microspheres (or microbubbles) can also be used with the
methods disclosed herein. Microspheres containing chromophores have
been utilized in an extensive variety of applications, including
photonic crystals, biological labeling, and flow visualization in
microfluidic channels. See, for example, Y. Lin, et al., Appl. Phys
Lett. 2002, 81, 3134; D. Wang, et al., Chem. Mater. 2003, 15, 2724;
X. Gao, et al., J. Biomed. Opt. 2002, 7, 532; M. Han, et al.,
Nature Biotechnology. 2001, 19, 631; V. M. Pai, et al., Mag. &
Magnetic Mater. 1999, 194, 262, each of which is incorporated by
reference in its entirety. Both the photostability of the
chromophores and the monodispersity of the microspheres can be
important.
[0286] Nanoparticles, such as, for example, metal nanoparticles,
metal oxide nanoparticles, or semiconductor nanocrystals can be
incorporated into microspheres. The optical, magnetic, and
electronic properties of the nanoparticles can allow them to be
observed while associated with the microspheres and can allow the
microspheres to be identified and spatially monitored. For example,
the high photostability, good fluorescence efficiency and wide
emission tunability of colloidally synthesized semiconductor
nanocrystals can make them an excellent choice of chromophore.
Unlike organic dyes, nanocrystals that emit different colors (i.e.
different wavelengths) can be excited simultaneously with a single
light source. Colloidally synthesized semiconductor nanocrystals
(such as, for example, core-shell CdSe/ZnS and CdS/ZnS
nanocrystals) can be incorporated into microspheres. The
microspheres can be monodisperse silica microspheres.
[0287] The nanoparticle can be a metal nanoparticle, a metal oxide
nanoparticle, or a semiconductor nanocrystal. The metal of the
metal nanoparticle or the metal oxide nanoparticle can include
titanium, zirconium, hafnium, vanadium, niobium, tantalum,
chromium, molybdenum, tungsten, manganese, technetium, rhenium,
iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel,
palladium, platinum, copper, silver, gold, zinc, cadmium, scandium,
yttrium, lanthanum, a lanthanide series or actinide series element
(e.g., cerium, praseodymium, neodymium, promethium, samarium,
europium, gadolinium, terbium, dysprosium, holmium, erbium,
thulium, ytterbium, lutetium, thorium, protactinium, and uranium),
boron, aluminum, gallium, indium, thallium, silicon, germanium,
tin, lead, antimony, bismuth, polonium, magnesium, calcium,
strontium, and barium. In certain embodiments, the metal can be
iron, ruthenium, cobalt, rhodium, nickel, palladium, platinum,
silver, gold, cerium or samarium. The metal oxide can be an oxide
of any of these materials or combination of materials. For example,
the metal can be gold, or the metal oxide can be an iron oxide, a
cobalt oxide, a zinc oxide, a cerium oxide, or a titanium oxide.
Preparation of metal and metal oxide nanoparticles is described,
for example, in U.S. Pat. Nos. 5,897,945 and 6,759,199, each of
which is incorporated by reference in its entirety.
[0288] The nanoparticles can be comprised of cargo molecules and a
carrier protein (such as albumin). Such nanoparticles are useful,
for example, to deliver hydrophobic or poorly soluble compounds.
Nanoparticles of poorly water soluble drugs (such as taxane) have
been disclosed in, for example, U.S. Pat. Nos. 5,916,596;
6,506,405; and 6,537,579 and also in U.S. Pat. Pub. No.
2005/0004002A1.
[0289] In forms, the nanoparticles can have an average or mean
diameter of no greater than about 1000 nanometers (nm), such as no
greater than about any of 900, 800, 700, 600, 500, 400, 300, 200,
and 100 nm. In some forms, the average or mean diameters of the
nanoparticles can be no greater than about 200 nm. In some forms,
the average or mean diameters of the nanoparticles can be no
greater than about 150 nm. In some forms, the average or mean
diameters of the nanoparticles can be no greater than about 100 nm.
In some forms, the average or mean diameter of the nanoparticles
can be about 20 to about 400 nm. In some forms, the average or mean
diameter of the nanoparticles can be about 40 to about 200 nm. In
some embodiments, the nanoparticles are sterile-filterable.
[0290] The nanoparticles can be present in a dry formulation (such
as lyophilized composition) or suspended in a biocompatible medium.
Suitable biocompatible media include, but are not limited to,
water, buffered aqueous media, saline, buffered saline, optionally
buffered solutions of amino acids, optionally buffered solutions of
proteins, optionally buffered solutions of sugars, optionally
buffered solutions of vitamins, optionally buffered solutions of
synthetic polymers, lipid-containing emulsions, and the like.
[0291] Examples of suitable carrier proteins include proteins
normally found in blood or plasma, which include, but are not
limited to, albumin, immunoglobulin including IgA, lipoproteins,
apolipoprotein B, alpha-acid glycoprotein, beta-2-macroglobulin,
thyroglobulin, transferin, fibronectin, factor VII, factor VIII,
factor IX, factor X, and the like. In some embodiments, the carrier
protein is non-blood protein, such as casein, .alpha.-lactalbumin,
and .beta.-lactoglobulin. The carrier proteins may either be
natural in origin or synthetically prepared. In some embodiments,
the pharmaceutically acceptable carrier comprises albumin, such as
human serum albumin. Human serum albumin (HSA) is a highly soluble
globular protein of M.sub.r 65K and consists of 585 amino acids.
HSA is the most abundant protein in the plasma and accounts for
70-80% of the colloid osmotic pressure of human plasma. The amino
acid sequence of HSA contains a total of 17 disulphide bridges, one
free thiol (Cys 34), and a single tryptophan (Trp 214). Intravenous
use of HSA solution has been indicated for the prevention and
treatment of hypovolumic shock (see, e.g., Tullis, JAMA
237:355-360, 460-463 (1977)) and Houser et al., Surgery, Gynecology
and Obstetrics, 150:811-816 (1980)) and in conjunction with
exchange transfusion in the treatment of neonatal
hyperbilirubinemia (see, e.g., Finlayson, Seminars in Thrombosis
and Hemostasis, 6:85-120 (1980)). Other albumins are contemplated,
such as bovine serum albumin. Use of such non-human albumins could
be appropriate, for example, in the context of use of these
compositions in non-human mammals, such as the veterinary
(including domestic pets and agricultural context).
[0292] Carrier proteins (such as albumin) in the composition
generally serve as a carrier for the hydrophobic cargo molecules,
i.e., the carrier protein in the composition makes the cargo
molecules more readily suspendable in an aqueous medium or helps
maintain the suspension as compared to compositions not comprising
a carrier protein. This can avoid the use of toxic solvents (or
surfactants) for solubilizing the cargo molecules, and thereby can
reduce one or more side effects of administration of the cargo
molecules into an individual (such as a human). Thus, in some
embodiments, the composition described herein can be substantially
free (such as free) of surfactants, such as Cremophor (including
Cremophor EL.RTM. (BASF)). In some embodiments, the composition can
be substantially free (such as free) of surfactants. A composition
is "substantially free of Cremophor" or "substantially free of
surfactant" if the amount of Cremophor or surfactant in the
composition is not sufficient to cause one or more side effect(s)
in an individual when the composition is administered to the
individual.
[0293] The amount of carrier protein in the composition described
herein will vary depending on other components in the composition.
In some embodiments, the composition, co-composition, cargo
composition, and/or tCAR composition can comprise a carrier protein
in an amount that is sufficient to stabilize the cargo molecules in
an aqueous suspension, for example, in the form of a stable
colloidal suspension (such as a stable suspension of
nanoparticles). In some embodiments, the carrier protein is in an
amount that reduces the sedimentation rate of the cargo molecules
in an aqueous medium. For particle-containing compositions, the
amount of the carrier protein also depends on the size and density
of nanoparticles of the cargo molecules.
[0294] Methods of making nanoparticle compositions are known in the
art. For example, nanoparticles containing cargo molecules and
carrier protein (such as albumin) can be prepared under conditions
of high shear forces (e.g., sonication, high pressure
homogenization, or the like). These methods are disclosed in, for
example, U.S. Pat. Nos. 5,916,596; 6,506,405; and 6,537,579 and
also in U.S. Pat. Pub. No. 2005/0004002A1.
[0295] Briefly, the hydrophobic carrier molecules can be dissolved
in an organic solvent, and the solution can be added to a human
serum albumin solution. The mixture is subjected to high pressure
homogenization. The organic solvent can then be removed by
evaporation. The dispersion obtained can be further lyophilized.
Suitable organic solvent include, for example, ketones, esters,
ethers, chlorinated solvents, and other solvents known in the art.
For example, the organic solvent can be methylene chloride and
chloroform/ethanol (for example with a ratio of 1:9, 1:8, 1:7, 1:6,
1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or
9:1).
[0296] The nanoparticle can also be, for example, a heat generating
nanoshell. As used herein, "nanoshell" is a nanoparticle having a
discrete dielectric or semi-conducting core section surrounded by
one or more conducting shell layers. U.S. Pat. No. 6,530,944 is
hereby incorporated by reference herein in its entirety for its
teaching of the methods of making and using metal nanoshells.
Targeting molecules can be attached to the disclosed compositions
and/or carriers. For example, the targeting molecules can be
antibodies or fragments thereof, ligands for specific receptors, or
other proteins specifically binding to the surface of the cells to
be targeted.
[0297] As used herein, the term "dendrimer" refers to repeatedly
branched and roughly spherical molecules. A dendrimer is typically
symmetric around a core and usually adopts a spherical
three-dimensional morphology. Dendrimers generally contain three
major portions: a core, an inner shell and an outer shell.
Dendrimers can be synthesized to have different and varying
functionality in each of the major portions in order to control
such variables as solubility, thermal stability and attachment of
compounds suitable for particular applications.
[0298] Dendrimers can be macromolecules having well-defined
hyperbranched structures. Peptide dendrimers are radially branched
macromolecules that contain a peptidyl branching core and/or
peripheral peptide chains, and they can be divided into three
categories. One category consists of "grafted" peptide dendrimers,
having peptides only as surface functionalities. The second
category is peptide dendrimers that composed entirely of amino
acids. The third are dendrimers utilizing amino acids in the
branching core and surface functional groups, but having
non-peptide branching units. Peptide dendrimers can be synthesized
using either divergent or convergent approach, and the availability
of solid-phase combinatorial methods enables large libraries of
peptide dendrimers to be produced and screened for desired
properties.
[0299] Dendrimer variants of SEQ ID NO:4 can be synthesized. For
example, a tCAR dendrimer containing 8 tCAR residues on a
polyamidoamine (PAMAM) core can be constructed. Other cores such as
Poly (ethylene glycol) can also be used to create dendrimer
variants. These dendrimer variants can have dozens, hundreds, or
even thousands of tCAR peptide residues on the surface of the
dendrimer to provide enhanced functional characteristics. These
dendrimers can contain tCAR alone for disease selective homing,
cell penetration and delivery of co-administered bioactive agents
or contain a bioactive agent within the dendrimer for targeted
delivery.
[0300] "Liposome" as the term is used herein refers to a structure
comprising an outer lipid bi- or multi-layer membrane surrounding
an internal aqueous space. Liposomes can be used to package any
biologically active agent for delivery to cells.
[0301] Materials and procedures for forming liposomes are
well-known to those skilled in the art. Upon dispersion in an
appropriate medium, a wide variety of phospholipids swell, hydrate
and form multilamellar concentric bilayer vesicles with layers of
aqueous media separating the lipid bilayers. These systems are
referred to as multilamellar liposomes or multilamellar lipid
vesicles ("MLVs") and have diameters within the range of 10 nm to
100 .mu.m. These MLVs were first described by Bangham, et al., J.
Mol. Biol. 13:238-252 (1965). In general, lipids or lipophilic
substances are dissolved in an organic solvent. When the solvent is
removed, such as under vacuum by rotary evaporation, the lipid
residue forms a film on the wall of the container. An aqueous
solution that typically contains electrolytes or hydrophilic
biologically active materials is then added to the film. Large MLVs
are produced upon agitation. When smaller MLVs are desired, the
larger vesicles are subjected to sonication, sequential filtration
through filters with decreasing pore size or reduced by other forms
of mechanical shearing. There are also techniques by which MLVs can
be reduced both in size and in number of lamellae, for example, by
pressurized extrusion (Barenholz, et al., FEBS Lett. 99:210-214
(1979)).
[0302] Liposomes can also take the form of unilamnellar vesicles,
which are prepared by more extensive sonication of MLVs, and
consist of a single spherical lipid bilayer surrounding an aqueous
solution. Unilamellar vesicles ("ULVs") can be small, having
diameters within the range of 20 to 200 nm, while larger ULVs can
have diameters within the range of 200 nm to 2 .mu.m. There are
several well-known techniques for making unilamellar vesicles. In
Papahadjopoulos, et al., Biochim et Biophys Acta 135:624-238
(1968), sonication of an aqueous dispersion of phospholipids
produces small ULVs having a lipid bilayer surrounding an aqueous
solution. Schneider, U.S. Pat. No. 4,089,801 describes the
formation of liposome precursors by ultrasonication, followed by
the addition of an aqueous medium containing amphiphilic compounds
and centrifugation to form a biomolecular lipid layer system.
[0303] Small ULVs can also be prepared by the ethanol injection
technique described by Batzri, et al., Biochim et Biophys Acta
298:1015-1019 (1973) and the ether injection technique of Deamer,
et al., Biochim et Biophys Acta 443:629-634 (1976). These methods
involve the rapid injection of an organic solution of lipids into a
buffer solution, which results in the rapid formation of
unilamellar liposomes. Another technique for making ULVs is taught
by Weder, et al. in "Liposome Technology", ed. G. Gregoriadis, CRC
Press Inc., Boca Raton, Fla., Vol. I, Chapter 7, pg. 79-107 (1984).
This detergent removal method involves solubilizing the lipids and
additives with detergents by agitation or sonication to produce the
desired vesicles.
[0304] Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes
the preparation of large ULVs by a reverse phase evaporation
technique that involves the formation of a water-in-oil emulsion of
lipids in an organic solvent and the drug to be encapsulated in an
aqueous buffer solution. The organic solvent is removed under
pressure to yield a mixture which, upon agitation or dispersion in
an aqueous media, is converted to large ULVs. Suzuki et al., U.S.
Pat. No. 4,016,100, describes another method of encapsulating
agents in unilamellar vesicles by freezing/thawing an aqueous
phospholipid dispersion of the agent and lipids.
[0305] In addition to the MLVs and ULVs, liposomes can also be
multivesicular. Described in Kim, et al., Biochim et Biophys Acta
728:339-348 (1983), these multivesicular liposomes are spherical
and contain internal granular structures. The outer membrane is a
lipid bilayer and the internal region contains small compartments
separated by bilayer septum. Still yet another type of liposomes
are oligolamellar vesicles ("OLVs"), which have a large center
compartment surrounded by several peripheral lipid layers. These
vesicles, having a diameter of 2-15 .mu.m, are described in Callo,
et al., Cryobiology 22(3):251-267 (1985).
[0306] Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also
describe methods of preparing lipid vesicles. More recently, Hsu,
U.S. Pat. No. 5,653,996 describes a method of preparing liposomes
utilizing aerosolization and Yiournas, et al., U.S. Pat. No.
5,013,497 describes a method for preparing liposomes utilizing a
high velocity-shear mixing chamber. Methods are also described that
use specific starting materials to produce ULVs (Wallach, et al.,
U.S. Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848
and 5,628,936).
[0307] A comprehensive review of all the aforementioned lipid
vesicles and methods for their preparation are described in
"Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca
Raton, Fla., Vol. I, II & III (1984). This and the
aforementioned references describing various lipid vesicles
suitable for use in the disclosed compositions are incorporated
herein by reference.
[0308] "Micelle" as used herein refers to a structure comprising an
outer lipid monolayer. Micelles can be formed in an aqueous medium
when the Critical Micelle Concentration (CMC) is exceeded. Small
micelles in dilute solution at approximately the critical micelle
concentration (CMC) are generally believed to be spherical.
However, under other conditions, they may be in the shape of
distorted spheres, disks, rods, lamellae, and the like. Micelles
formed from relatively low molecular weight amphiphile molecules
can have a high CMC so that the formed micelles dissociate rather
rapidly upon dilution. If this is undesired, amphiphile molecules
with large hydrophobic regions can be used. For example, lipids
with a long fatty acid chain or two fatty acid chains, such as
phospholipids and sphingolipids, or polymers, specifically block
copolymers, can be used.
[0309] Polymeric micelles have been prepared that exhibit CMCs as
low as 10.sup.-6 M (molar). Thus, they tend to be very stable while
at the same time showing the same beneficial characteristics as
amphiphile micelles. Any micelle-forming polymer presently known in
the art or as such may become known in the future may be used in
the disclosed compositions and methods. Examples of micelle-forming
polymers include, without limitation, methoxy poly(ethylene
glycol)-b-poly(.di-elect cons.-caprolactone), conjugates of
poly(ethylene glycol) with phosphatidyl-ethanolamine, poly(ethylene
glycol)-b-polyesters, poly(ethylene glycol)-b-poly(L-aminoacids),
poly(N-vinylpyrrolidone)-bl-poly(orthoesters),
poly(N-vinylpyrrolidone)-b-polyanhydrides and
poly(N-vinylpyrrolidone)-b-poly(alkyl acrylates).
[0310] Micelles can be produced by processes conventional in the
art. Examples of such are described in, for example, Liggins
(Liggins, R. T. and Burt, H. M., "Polyether-polyester diblock
copolymers for the preparation of paclitaxel loaded polymeric
micelle formulations." Adv. Drug Del. Rev. 54: 191-202, (2002));
Zhang, et al. (Zhang, X. et al., "Development of amphiphilic
dibiock copolymers as micellar carriers of taxol." Int. J. Pharm.
132: 195-206, (1996)); and Churchill (Churchill, J. R., and
Hutchinson, F. G., "Biodegradable amphipathic copolymers." U.S.
Pat. No. 4,745,160, (1988)). In one such method,
polyether-polyester block copolymers, which are amphipathic
polymers having hydrophilic (polyether) and hydrophobic (polyester)
segments, are used as micelle forming carriers.
[0311] Another type of micelle can be formed using, for example,
AB-type block copolymers having both hydrophilic and hydrophobic
segments, as described in, for example, Tuzar (Tuzar, Z. and
Kratochvil, P., "Block and graft copolymer micelles in solution.",
Adv. Colloid Interface Sci. 6:201-232, (1976)); and Wilhelm, et al.
(Wilhelm, M. et al., "Poly(styrene-ethylene oxide) block copolymer
micelle formation in water: a fluorescence probe study.",
Macromolecules 24: 1033-1040 (1991)). These polymeric micelles are
able to maintain satisfactory aqueous stability. These micelles, in
the range of approximately <200 nm in size, are effective in
reducing non-selective RES scavenging and show enhanced
permeability and retention.
[0312] Further, U.S. Pat. No. 5,929,177 to Kataoka, et al.
describes a polymeric molecule which is usable as, inter alia, a
drug delivery carrier. The micelle is formed from a block copolymer
having functional groups on both of its ends and which comprises
hydrophilic/hydrophobic segments. The polymer functional groups on
the ends of the block copolymer include amino, carboxyl and
mercapto groups on the .alpha.-terminal and hydroxyl, carboxyl
group, aldehyde group and vinyl group on the .omega.-terminal. The
hydrophilic segment comprises polyethylene oxide, while the
hydrophobic segment is derived from lactide, lactone or
(meth)acrylic acid ester.
[0313] Further, for example,
poly(D,L-lactide)-b-methoxypolyethylene glycol (MePEG:PDLLA)
diblock copolymers can be made using MePEG 1900 and 5000. The
reaction can be allowed to proceed for 3 hr at 160.degree. C.,
using stannous octoate (0.25%) as a catalyst. However, a
temperature as low as 130.degree. C. can be used if the reaction is
allowed to proceed for about 6 hr, or a temperature as high as
190.degree. C. can be used if the reaction is carried out for only
about 2 hr.
[0314] As another example, N-isopropylacrylamide ("IPAAm") (Kohjin,
Tokyo, Japan) and dimethylacrylamide ("DMAAm") (Wako Pure
Chemicals, Tokyo, Japan) can be used to make hydroxyl-terminated
poly(IPAAm-co-DMAAm) in a radical polymerization process, using the
method of Kohori, F. et al. (1998). (Kohori, F. et al.,
"Preparation and characterization of thermally Responsive block
copolymer micelles comprising
poly(N-isopropylacrylamide-b-D,L-lactide)." J. Control. Rel. 55:
87-98, (1998)). The obtained copolymer can be dissolved in cold
water and filtered through two ultrafiltration membranes with a
10,000 and 20,000 molecular weight cut-off. The polymer solution is
first filtered through a 20,000 molecular weight cut-off membrane.
Then the filtrate was filtered again through a 10,000 molecular
weight cut-off membrane. Three molecular weight fractions can be
obtained as a result, a low molecular weight, a middle molecular
weight, and a high molecular weight fraction. A block copolymer can
then be synthesized by a ring opening polymerization of D,L-lactide
from the terminal hydroxyl group of the poly(IPAAm-co-DMAAm) of the
middle molecular weight fraction. The resulting
poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) copolymer can be purified
as described in Kohori, F. et al. (1999). (Kohori, F. et al.,
"Control of adriamycin cytotoxic activity using thermally
responsive polymeric micelles composed of
poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lacide).-
-", Colloids Surfaces B: Biointerfaces 16: 195-205, (1999)).
[0315] Examples of block copolymers from which micelles can be
prepared which can be used to coat a support surface are found in
U.S. Pat. No. 5,925,720, to Kataoka, et al., U.S. Pat. No.
5,412,072 to Sakarai, et al., U.S. Pat. No. 5,410,016 to Kataoka,
et al., U.S. Pat. No. 5,929,177 to Kataoka, et al., U.S. Pat. No.
5,693,751 to Sakurai, et al., U.S. Pat. No. 5,449,513 to Yokoyama,
et al., WO 96/32434, WO 96/33233 and WO 97/0623, the contents of
all of which are incorporated by reference. Modifications thereof
which are prepared by introducing thereon a suitable functional
group (including an ethyleneically unsaturated polymerizable group)
are also examples of block copolymers from which micelles for the
disclosed compositions and methods are preferably prepared.
Preferable block copolymers are those disclosed in the
above-mentioned patents and or international patent publications.
If the block copolymer has a sugar residue on one end of the
hydrophilic polymer segment, as in the block copolymer of WO
96/32434, the sugar residue should preferably be subjected to
Malaprade oxidation so that a corresponding aldehyde group may be
formed.
[0316] Lipids are synthetically or naturally-occurring molecules
which includes fats, waxes, sterols, prenol lipids, fat-soluble
vitamins (such as vitamins A, D, E and K), glycerolipids,
monoglycerides, diglycerides, triglycerides, glycerophospholipids,
sphingolipids, phospholipids, fatty acids monoglycerides,
saccharolipids and others. Lipids can be hydrophobic or amphiphilic
small molecules; the amphiphilic nature of some lipids allows them
to form structures such as monolayers, vesicles, micelles,
liposomes, bi-layers or membranes in an appropriate environment
i.e. aqueous environment. Any of a number of lipids can be used as
amphiphile molecules, including amphipathic, neutral, cationic, and
anionic lipids. Such lipids can be used alone or in combination,
and can also include bilayer stabilizing components such as
polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017, "Polyamide
Oligomers", by Ansell), peptides, proteins, detergents,
lipid-derivatives, such as PEG coupled to phosphatidylethanolamine
and PEG conjugated to ceramides (see, U.S. Pat. No. 5,885,613). In
a preferred embodiment, cloaking agents, which reduce elimination
of liposomes by the host immune system, can also be included, such
as polyamide-oligomer conjugates, e.g., ATTA-lipids, (see, U.S.
patent application Ser. No. 08/996,783, filed Feb. 2, 1998) and
PEG-lipid conjugates (see, U.S. Pat. Nos. 5,820,873, 5,534,499 and
5,885,613).
[0317] Any of a number of neutral lipids can be included, referring
to any of a number of lipid species which exist either in an
uncharged or neutral zwitterionic form at physiological pH,
including diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,
cholesterol, cerebrosides, and diacylglycerols.
[0318] Cationic lipids, carry a net positive charge at
physiological pH, can readily be used as amphiphile molecules. Such
lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy) propyl-N,N--N-triethylammonium chloride
("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP");
3.beta.-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
-ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl
carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine
("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"). Additionally, a number of commercial
preparations of cationic lipids can be used, such as LIPOFECTIN
(including DOTMA and DOPE, available from GIBCO/BRL), LIPOFECTAMINE
(comprising DOSPA and DOPE, available from GIBCO/BRL), and
TRANSFECTAM (comprising DOGS, in ethanol, from Promega Corp.).
[0319] Anionic lipids can be used as amphiphile molecules and
include, but are not limited to, phosphatidylglycerol, cardiolipin,
diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl
phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,
N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and
other anionic modifying groups joined to neutral lipids.
[0320] Amphiphatic lipids can also be suitable amphiphile
molecules. "Amphipathic lipids" refer to any suitable material,
wherein the hydrophobic portion of the lipid material orients into
a hydrophobic phase, while the hydrophilic portion orients toward
the aqueous phase. Such compounds include, but are not limited to,
fatty acids, phospholipids, aminolipids, and sphingolipids.
Representative phospholipids include sphingomyelin,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatdylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or
dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds,
such as sphingolipids, glycosphingolipid families, diacylglycerols,
and .beta.-acyloxyacids, can also be used. Additionally, such
amphipathic lipids can be readily mixed with other lipids, such as
triglycerides and sterols. Zwitterionic lipids are a form of
amphiphatic lipid.
[0321] Sphingolipids are fatty acids conjugated to the aliphatic
amino alcohol sphingosine. The fatty acid can be covalently bond to
sphingosine via an amide bond. Any amino acid as described above
can be covalently bond to sphingosine to form a sphingolipid. A
sphingolipid can be further modified by covalent bonding through
the .alpha.-hydroxyl group. The modification can include alkyl
groups, alkenyl groups, alkynyl groups, aromatic groups,
heteroaromatic groups, cyclyl groups, heterocyclyl groups,
phosphonic acid groups. Non-limiting examples of shingolipids are
N-acylsphingosine, N-Acylsphingomyelin, Forssman antigen.
[0322] Saccharolipids are compounds that contain both fatty acids
and sugars. The fatty acids are covalently bonded to a sugar
backbone. The sugar backbone can contain one or more sugars. The
fatty acids can bond to the sugars via either amide or ester bonds.
The sugar can be any sugar base. The fatty acid can be any fatty
acid as described elsewhere herein. The provided compositions can
comprise either natural or synthetic saccharolipids. Non-limiting
saccharolipids are UDP-3-O-(.beta.-hydroxymyristoyl)-GlcNAc, lipid
IV A, Kdo2-lipid A.
[0323] The disclosed compositions, co-compositions, cargo
compositions, and tCAR compositions can include one or more cargo
molecules. Generally, the disclosed compositions can include a
plurality of cargo molecules. The disclosed compositions can
include a single type of cargo molecule or a plurality of different
types of cargo molecules. Thus, for example, the disclosed
compositions can include a plurality of different types of cargo
molecules where a plurality of one or more of the different types
of cargo molecules can be present.
[0324] Cargo molecules can be any compound, molecule, conjugate,
composition, etc. that is desired to be delivered using the
disclosed compositions. For example, the cargo molecules can be
therapeutic agents, detectable agents, or a combination. For
example, the cargo molecules can be membrane perturbing molecules,
pro-apoptotic molecules, pore-generating molecules, antimicrobial
molecules, mitochondria-affecting molecules, mitochondria-targeted
molecules, or a combination. Examples of some useful cargo
molecules are described below and elsewhere herein.
[0325] Cargo molecules can be associated with and arranged in the
compositions in a variety of configurations. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of homing molecules. In some forms, cargo
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of homing molecules, wherein the homing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. Combinations of these
combinations can also be used.
[0326] Membrane perturbing molecules include molecules that can
disrupt membranes, that can form pores in membranes, that can make
membranes leaky, that can be targeted to or affect intracellular
membranes or organelles, such mitochondria or lysosomes. Some forms
of membrane perturbing molecules can be pro-apoptotic while others
can be non-apoptotic. Some forms of membrane perturbing molecules
can be pro-apoptotic for only some types of cells.
[0327] In some forms, one or more of the homing molecules can
comprise the amino acid sequence CGKRK (SEQ ID NO:1). In some
forms, one or more of the membrane perturbing molecules can
comprise the amino acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID
NO:3) or a conservative variant thereof, (KLAKLAK).sub.2 (SEQ ID
NO:3) or a conservative variant thereof, (KLAKKLA).sub.2 (SEQ ID
NO:5) or a conservative variant thereof, (KAAKKAA).sub.2 (SEQ ID
NO:6) or a conservative variant thereof, (KLGKKLG).sub.3 (SEQ ID
NO:7) or a conservative variant thereof, or a combination. In some
forms, one or more of the membrane perturbing molecules can
comprise the amino acid sequence .sub.D(KLAKLAK).sub.2 (SEQ ID
NO:3), (KLAKLAK).sub.2 (SEQ ID NO:3), (KLAKKLA).sub.2 (SEQ ID
NO:5), (KAAKKAA).sub.2 (SEQ ID NO:6), (KLGKKLG).sub.3 (SEQ ID
NO:7), or a combination. In some forms, one or more of the membrane
perturbing molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3) or a conservative variant
thereof. In some forms, one or more of the membrane perturbing
molecules can comprise the amino acid sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3). Membrane perturbing peptides
of this type are described in Ellerby, Nature Medicine 5, 1032-1038
(1999), which is hereby incorporated by reference for its
description of such peptides.
[0328] A plurality of modified and/or unmodified membrane
perturbing molecules can each be independently selected from, for
example, an amino acid segment comprising a modified or unmodified
form of the amino acid sequence of a homing peptide, an amino acid
segment comprising a modified or unmodified form of the amino acid
sequence .sub.D(KLAKLAK).sub.2 (SEQ ID NO:3), (KLAKLAK).sub.2 (SEQ
ID NO:3), (KLAKKLA).sub.2 (SEQ ID NO:5), (KAAKKAA).sub.2 (SEQ ID
NO:6), (KLGKKLG).sub.3 (SEQ ID NO:7), or a combination. A plurality
of the membrane perturbing molecules can each independently
comprise an amino acid segment comprising a modified or unmodified
form of the amino acid sequence of a homing peptide.
[0329] The composition, co-composition, cargo composition, and/or
tCAR composition can comprise a sufficient number and composition
of membrane perturbing molecules (modified or not) such that the
composition has a membrane perturbing effect on the target. In one
example, sufficiency of the number and composition of modified
and/or unmodified membrane perturbing molecules can be determined
by assessing membrane disruption, apoptosis, and/or therapeutic
effect on the target.
[0330] The composition, co-composition, cargo composition, and/or
tCAR composition can comprise any number of modified and/or
unmodified membrane perturbing molecules. By way of example, the
composition, co-composition, cargo composition, and/or tCAR
composition can comprise at least 1, 5, 10, 15, 20, 25, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,
425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 625,
750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200,
1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750,
3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,
8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000,
40,000, 45,000, 50,000, 75,000, or 100,000, or more modified and/or
unmodified membrane perturbing molecules. The composition can also
comprise any number in between those numbers listed above.
[0331] Membrane perturbing molecules can be associated with and
arranged in the compositions in a variety of configurations. In
some forms, membrane perturbing molecules can be associated with,
conjugated to, and/or covalently coupled to a plurality of surface
molecules. In some forms, membrane perturbing molecules can be
associated with, conjugated to, and/or covalently coupled to a
plurality of homing molecules. In some forms, membrane perturbing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of homing molecules, wherein the homing
molecules can be associated with, conjugated to, and/or covalently
coupled to a plurality of surface molecules. Combinations of these
combinations can also be used.
[0332] The disclosed membrane perturbing molecules can include
modified forms of membrane perturbing molecules. The membrane
perturbing molecules can have any useful modification. For example,
some modifications can stabilize the membrane perturbing molecule.
For example, the disclosed membrane perturbing molecules include
methylated membrane perturbing molecules. Methylated membrane
perturbing molecules are particularly useful when the membrane
perturbing molecule includes a protein, peptide or amino acid
segment. For example, a membrane perturbing molecule can be a
modified membrane perturbing molecule, where, for example, the
modified membrane perturbing molecule includes a modified amino
acid segment or amino acid sequence. For example, a modified
membrane perturbing molecule can be a methylated membrane
perturbing molecule, where, for example, the methylated membrane
perturbing molecule includes a methylated amino acid segment or
amino acid sequence. Other modifications can be used, either alone
or in combination. Where the membrane perturbing molecule is, or
includes, a protein, peptide, amino acid segment and/or amino acid
sequences, the modification can be to the protein, peptide, amino
acid segment, amino acid sequences and/or any amino acids in the
protein, peptide, amino acid segment and/or amino acid sequences.
Amino acid and peptide modifications are known to those of skill in
the art, some of which are described below and elsewhere herein.
Methylation is a particularly useful modification for the disclosed
membrane perturbing molecules. Using modified forms of membrane
perturbing molecules can increase their effectiveness.
[0333] The disclosed compositions, surface molecules, cargo
molecules, peptides, proteins, amino acid sequences, etc. can
comprise one or more internalization elements, tissue penetration
elements, or both. Internalization elements and tissue penetration
elements can be incorporated into or fused with other peptide
components of the composition, such as peptide homing molecules and
peptide cargo molecules. Internalization elements are molecules,
often peptides or amino acid sequences, that allow the
internalization element and components with which it is associated,
to pass through biological membranes. Tissue penetration elements
are molecules, often peptides or amino acid sequences, that allow
the tissue penetration element and components with which it is
associated to passage into and through tissue. Some molecules, such
as tCAR peptides and CendR elements, function as both
internalization elements and tissue penetration elements.
[0334] Internalization elements include, for example,
cell-penetrating peptides (CPPs) and tCAR peptides. Peptides that
are internalized into cells are commonly referred to as
cell-penetrating peptides. There are two main classes of such
peptides; hydrophobic and cationic (Zorko and Langel, 2005). The
cationic peptides, which are commonly used to introduce nucleic
acids, proteins into cells, include the prototypic cell-penetrating
peptides (CPP), Tat, and penetratin (Derossi et al., 1998; Meade
and Dowdy, 2007). A herpes virus protein, VP22, is capable of both
entering and exiting cells and carrying a payload with it (Elliott
and O'Hare, 1997; Brewis et al., 2003).
[0335] Association of the components of the disclosed compositions
can be aided or accomplished via molecules, conjugates and/or
compositions. Where such molecules, conjugates and/or compositions
are other than tCAR peptides, surface molecules, homing molecules,
accessory molecules, co-compositions, cargo compositions, or cargo
molecules (such as membrane perturbing molecules, internalization
elements, tissue penetration elements, and moieties), they can be
referred to herein as linkers. Such linkers can be any molecule,
conjugate, composition, etc. that can be used to associate
components of the disclosed compositions. Generally, linkers can be
used to associate components other than surface molecules to
surface molecules. Useful linkers include materials that are
biocompatible, have low bioactivity, have low antigenicity, etc.
That is, such useful linker materials can serve the
linking/association function without adding unwanted bioreactivity
to the disclosed compositions. Many such materials are known and
used for similar linking and association functions. Polymer
materials are a particularly useful form of linker material. For
example, polyethylene glycols can be used.
[0336] Linkers are useful for achieving useful numbers and
densities of the components (such as homing molecules and membrane
perturbing molecules) on surface molecules. For example, linkers of
fibrous form are useful for increasing the number of components per
surface molecule or per a given area of the surface molecule.
Similarly, linkers having a branching form are useful for
increasing the number of components per surface molecule or per a
given area of the surface molecule. Linkers can also have a
branching fibrous form.
[0337] Sufficiency of the number and composition of homing
molecules in the composition can be determined by assessing homing
to the target and effectively delivery of the cargo molecules in a
non-human animal. The composition, co-composition, cargo
composition, and/or tCAR composition can comprise a sufficient
number and composition of homing molecules (modified or not) such
that the composition homes to the target and effectively delivers
the cargo molecules. In one example, sufficiency of the number and
composition of modified and/or unmodified homing molecules can be
determined by assessing cargo delivery and/or therapeutic effect on
the target. Sufficiency of the number and composition of membrane
perturbing molecules can be determined by assessing membrane
perturbing effect of the composition in a non-human animal. The
composition, co-composition, cargo composition, and/or tCAR
composition can comprise a sufficient number and composition of
membrane perturbing molecules (modified or not) such that the
composition has a membrane perturbing effect on the target. In one
example, sufficiency of the number and composition of modified
and/or unmodified membrane perturbing molecules can be determined
by assessing membrane disruption, apoptosis, and/or therapeutic
effect on the target.
[0338] The composition, co-composition, cargo composition, and/or
tCAR composition can comprise a sufficient density and composition
of homing molecules such that the composition homes to the target
and effectively delivers the cargo molecules. Sufficiency of the
density and composition of homing molecules can be determined by
assessing cargo delivery and/or therapeutic effect on the target in
a non-human animal. The composition, co-composition, cargo
composition, and/or tCAR composition can comprise a sufficient
density and composition of membrane perturbing molecules such that
the composition has a membrane perturbing effect on the target.
Sufficiency of the density and composition of membrane perturbing
molecules can be determined by assessing membrane disruption,
apoptosis, and/or therapeutic effect on the target in a non-human
animal.
[0339] The density of homing molecules and/or membrane perturbing
molecules on a surface molecule can be described in any suitable
manner. For example, the density can be expressed as the number of
homing molecules and/or membrane perturbing molecules per, for
example, a given area, surface area, volume, unit, subunit, arm,
etc. of the surface molecule. The density can also be relative to,
for example, the area, surface area, volume, unit, subunit, arm,
etc. of the entire surface molecule or to the area, surface area,
volume, unit, subunit, arm, etc. of a portion of the surface
molecule. For example, a sufficient density of homing molecule
and/or membrane perturbing molecule can be present in a portion of
the surface molecule. The presence of this dense portion can cause
clotting and amplify the accumulation of the composition. Thus, a
composition having a sufficient density of homing molecules and/or
membrane perturbing molecules can have a threshold density (or
above) for the entire surface molecule or for just one or more
portions of the surface molecule. Unless otherwise stated,
densities refer to average density over the designated portion of
the surface molecule. For example, a density of 1 homing molecule
per square nM of the surface molecule refers to an average density
of the homing molecules over the entire surface molecule. As
another example, a density of 1 homing molecule per square nM of a
portion of the surface molecule refers to an average density of the
homing molecules over just that portion of the surface
molecule.
[0340] The density can be measured or calculated in any suitable
manner. For example, the number or amount of homing molecules
and/or membrane perturbing molecules present on a surface molecule
or group of surface molecules can be measured by, for example,
detecting the level or intensity of signal produced by labeled
homing molecules and/or membrane perturbing molecules and
calculating the density based on the structural characteristics of
the surface molecule.
[0341] The density or threshold density of homing molecules and/or
membrane perturbing molecules can be, for example, at least 0.001,
0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 homing
molecules and/or membrane perturbing molecules per square nM of the
entire or a portion of the surface molecule. The composition can
also comprise any density in between those densities listed
above.
[0342] The density or threshold density of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules per square .mu.M of the entire or a
portion of the surface molecule. The composition can also comprise
any density in between those densities listed above.
[0343] The density or threshold density of homing molecules and/or
membrane perturbing molecules can be, for example, at least 0.001,
0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 homing
molecules and/or membrane perturbing molecules per cubic nM of the
entire or a portion of the surface molecule. The composition can
also comprise any density in between those densities listed
above.
[0344] The density or threshold density of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules per cubic .mu.M of the entire or a
portion of the surface molecule. The composition can also comprise
any density in between those densities listed above.
[0345] The number of homing molecules and/or membrane perturbing
molecules on a surface molecule can be described in any suitable
manner. For example, the number can be expressed as the number of
homing molecules and/or membrane perturbing molecules per, for
example, a given area, surface area, volume, unit, subunit, arm,
etc. of the surface molecule. The number can also be relative to,
for example, the area, surface area, volume, unit, subunit, arm,
etc. of the entire surface molecule or to the area, surface area,
volume, unit, subunit, arm, etc. of a portion of the surface
molecule. For example, a sufficient number of homing molecule
and/or membrane perturbing molecule can be present in a portion of
the surface molecule. The presence of this dense portion can cause
clotting and amplify the accumulation of the composition. Thus, a
composition having a sufficient number of homing molecules and/or
membrane perturbing molecules can have a threshold number (or
above) for the entire surface molecule or for just one or more
portions of the surface molecule.
[0346] The number can be measured or calculated in any suitable
manner. For example, the number or amount of homing molecules
and/or membrane perturbing molecules present on a surface molecule
or group of surface molecules can be measured by, for example,
detecting the level or intensity of signal produced by labeled
homing molecules and/or membrane perturbing molecules and
calculating the number based on the structural characteristics of
the surface molecule.
[0347] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules on the surface molecule. The
composition can also comprise any number in between those numbers
listed above.
[0348] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 0.001,
0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 homing
molecules and/or membrane perturbing molecules per square nM of the
entire or a portion of the surface molecule. The composition can
also comprise any number in between those numbers listed above.
[0349] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules per square .mu.M of the entire or a
portion of the surface molecule. The composition can also comprise
any number in between those numbers listed above.
[0350] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 0.001,
0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 homing
molecules and/or membrane perturbing molecules per cubic nM of the
entire or a portion of the surface molecule. The composition can
also comprise any number in between those numbers listed above.
[0351] The number or threshold number of homing molecules and/or
membrane perturbing molecules can be, for example, at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, 4200, 4400, 4600, 4800, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 900, 9500, 10,000 homing molecules and/or
membrane perturbing molecules per cubic .mu.M of the entire or a
portion of the surface molecule. The composition can also comprise
any number in between those numbers listed above.
[0352] In some forms, the compositions not only home to tumors, but
also amplify their own homing. Homing molecules can be used that
are clot-binding compounds that recognize clotted plasma proteins
and selectively homes to tumors, where it binds to vessel walls and
tumor stroma. Surface molecules coupled with the clot-binding
compounds can accumulate in tumor vessels or at wound sites, where
they induce additional local clotting, thereby producing new
binding sites for more particles. The system mimics platelets,
which also circulate freely but accumulate at a diseased site and
amplify their own accumulation at that site. The clotting-based
amplification greatly enhances cargo delivery and tumor
imaging.
[0353] Disclosed are linkers for associating components of the
disclosed compositions. Such linkers can be any molecule,
conjugate, composition, etc. that can be used to associate
components of the disclosed compositions. Generally, linkers can be
used to associate components other than surface molecules to
surface molecules. Useful linkers include materials that are
biocompatible, have low bioactivity, have low antigenicity, etc.
That is, such useful linker materials can serve the
linking/association function without adding unwanted bioreactivity
to the disclosed compositions. Many such materials are know and
used for similar linking and association functions. Polymer
materials are a particularly useful form of linker material. For
example, polyethylene glycols can be used.
[0354] Linkers are useful for achieving useful numbers and
densities of the components (such as homing molecules and membrane
perturbing molecules) on surface molecules. For example, linkers of
fibrous form are useful for increasing the number of components per
surface molecule or per a given area of the surface molecule.
Similarly, linkers having a branching form are useful for
increasing the number of components per surface molecule or per a
given area of the surface molecule. Linkers can also have a
branching fibrous form.
[0355] Linkers of different lengths can be used to bind the
disclosed components to surface molecules and to each other. A
flexible linker can function well even if relatively short, while a
stiffer linker may can be longer to allow effective exposure and
density. The length of a linker can refer to the number of atoms in
a continuous covalent chain between the attachment points on the
components being linked or to the length (in nanometers, for
example) of a continuous covalent chain between the attachment
points on the components being linked. Unless the context clearly
indicates otherwise, the length refers to the shortest continuous
covalent chain between the attachment points on the components
being linked not accounting for side chains, branches, or loops.
Due to flexibility of the linker, all of the linkers may not have
same distance from the surface molecule. Thus linkers with
different chain lengths can make the resulting composition more
effective (by increasing density, for example). Branched linkers
bearing multiple components also allow attachment of more than one
component at a given site of the surface molecule. Useful lengths
for linkers include at least, up to, about, exactly, or between 10,
15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150,
160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000,
7,000, 8,000, 9,000, and 10,000 atoms. Useful lengths for linkers
include at least, up to, about, exactly, or between 10, 15, 20, 25,
30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,
8,000, 9,000, and 10,000 nanometers. Any range of these lengths and
all lengths between the listed lengths are specifically
contemplated.
[0356] Hydrophilic or water-solubility linkers can increase the
mobility of the attached components. Examples of water-soluble,
biocompatible polymers which can serve as linkers include, but are
not limited to polymers such polyethylene glycol (PEG),
polyethylene oxide (PEO), polyvinyl alcohol, polyhydroxyethyl
methacrylate, polyacrylamide, and natural polymers such as
hyaluronic acid, chondroitin sulfate, carboxymethylcellulose, and
starch. Useful forms of branched tethers include star PEO and comb
PEO. Star PEO can be formed of many PEO "arms" emanating from a
common core.
[0357] Polyethylene glycols (PEGs) are simple, neutral polyethers
which have been given much attention in biotechnical and biomedical
applications (Milton Harris, J. (ed) "Poly(ethylene glycol)
chemistry, biotechnical and biomedical applications" Plenum Press,
New York, 1992). PEGs are soluble in most solvents, including
water, and are highly hydrated in aqueous environments, with two or
three water molecules bound to each ethylene glycol segment; this
hydration phenomenon has the effect of preventing adsorption either
of other polymers or of proteins onto PEG-modified surfaces.
Furthermore, PEGs may readily be modified and bound to other
molecules with only little effect on their chemistry. Their
advantageous solubility and biological properties are apparent from
the many possible uses of PEGs and copolymers thereof, including
block copolymers such as PEG-polyurethanes and PEG-polypropylenes.
Appropriate molecular weights for PEG linkers used in the disclosed
compositions can be from about 120 daltons to about 20 kilodaltons.
For example, PEGs can be at least, up to, about, exactly, or
between 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1000, 1200, 1400, 1500, 1600, 1800, 2000, 2500, 3000, 3500,
4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,
9500, 10,000, 20,000, 30,000, 40,000, and 50,000 daltons. Any range
of these masses and all masses between the listed masses are
specifically contemplated. PEGs are usually available as mixtures
of somewhat heterogeneous masses with a stated average mass
(PEG-5000, for example).
[0358] The disclosed compositions can be produced using any
suitable techniques. Many techniques, reactive groups, chemistries,
etc. for linking components of the types disclosed herein are known
and can be used with the disclosed components and compositions.
[0359] Protein crosslinkers that can be used to crosslink other
molecules, elements, moieties, etc. to the disclosed compositions,
surface molecules, homing molecules, membrane perturbing molecules,
internalization elements, tissue penetration elements, cargo
compositions, tCAR peptides, compositions, peptides, amino acid
sequences, etc. are known in the art and are defined based on
utility and structure and include DSS (Disuccinimidylsuberate), DSP
(Dithiobis(succinimidylpropionate)), DTSSP (3,3'-Dithiobis
(sulfosuccinimidylpropionate)), SULFO BSOCOES
(Bis[2-(sulfosuccinimdooxycarbonyloxy)ethyl]sulfone), BSOCOES
(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST
(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO
EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene
glycolbis(sulfosuccinimidylsuccinate)), DPDPB
(1,2-Di[3'-(2'-pyridyldithio)propionamido]butane), BSSS
(Bis(sulfosuccinimdyl)suberate), SMPB
(Succinimdyl-4-(p-maleimidophenyl)butyrate), SULFO SMPB
(Sulfosuccinimdyl-4-(p-maleimidophenyl)butyrate), MBS
(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS
(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB
(N-Succinimidyl(4-iodoacetyl)aminobenzoate), SULFO SIAB
(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC
(Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate),
SULFO SMCC
(Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy
late), NHS LC SPDP
(Succinimidyl-6-[3-(2-pyridyldithio)propionamido)hexanoate), SULFO
NHS LC SPDP
(Sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido)hexanoate),
SPDP (N-Succinimdyl-3-(2-pyridyldithio)propionate), NHS
BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHS IODOACETATE
(N-Hydroxysuccinimidyliodoacetate), MPBH
(4-(N-Maleimidophenyl)butyric acid hydrazide hydrochloride), MCCH
(4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid hydrazide
hydrochloride), MBH (m-Maleimidobenzoic acid
hydrazidehydrochloride), SULFO
EMCS(N-(epsilon-Maleimidocaproyloxy)sulfosuccinimide),
EMCS(N-(epsilon-Maleimidocaproyloxy)succinimide), PMPI
(N-(p-Maleimidophenyl)isocyanate), KMUH
(N-(kappa-Maleimidoundecanoic acid)hydrazide), LC SMCC
(Succinimidyl-4-(N-maleimidomethyp-cyclohexane-1-carboxy(6-amidocaproate)-
), SULFO GMBS (N-(gamma-Maleimidobutryloxy)sulfosuccinimide ester),
SMPH (Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO
KMUS (N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS
(N-(gamma-Maleimidobutyrloxy)succinimide), DMP
(Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate
hydrochloride), MHBH (Wood's Reagent; Methyl-p-hydroxybenzimidate
hydrochloride, 98%), DMA (Dimethyladipimidate hydrochloride).
[0360] Components of the disclosed compositions, such as surface
molecules, homing molecules, membrane perturbing molecules,
internalization elements, tissue penetration elements, etc., can
also be coupled using, for example, maleimide coupling. By way of
illustration, components can be coupled to lipids by coupling to,
for example,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene
glycol).sub.20000; DSPE-PEG.sub.2000-maleimide] (Avanti Polar
Lipids) by making use of a free cysteine sulfhydryl group on the
component. The reaction can be performed, for example, in aqueous
solution at room temperature for 4 hours. This coupling chemistry
can be used to couple components of co-compositions and cargo
compositions.
[0361] Components of the disclosed compositions, such as surface
molecules, homing molecules, membrane perturbing molecules,
internalization elements, tissue penetration elements, etc., can
also be coupled using, for example, amino group-functionalized
dextran chemistry. Particles, such as, for example, nanoparticles,
nanoworms, and micelles, can be coated with amino group
functionalized dextran. Attachment of PEG to aminated particles
increases the circulation time, presumably by reducing the binding
of plasma proteins involved in opsonization (Moghimi et al., Pharm.
Rev. 53, 283-318 (2001)). The particles can have surface
modifications, for example, for reticuloendothelial system
avoidance (PEG) and homing (homing molecules), endosome escape
(pH-sensitive peptide; for example, Pirollo et al., Cancer Res. 67,
2938-43 (2007)), a detectable agent, a therapeutic compound, or a
combination. To accommodate all these functions on one particle,
optimization studies can be conducted to determine what proportion
of the available linking sites at the surface of the particles any
one of these elements should occupy to give the best combination of
targeting and payload delivery. The cell internalization and/or
tissue penetration of such compositions can be mediated by the
disclosed tCAR peptides, amino acid sequences, proteins, molecules,
conjugates, and compositions.
[0362] The provided peptides and polypeptides can have additional
N-terminal, C-terminal, or intermediate amino acid sequences, e.g.,
amino acid linkers or tags. The term "amino acid linker" refers to
an amino acid sequences or insertions that can be used to connect
or separate two distinct peptides, polypeptides, or polypeptide
fragments, where the linker does not otherwise contribute to the
essential function of the composition. The term "amino acid tag"
refers to a distinct amino acid sequence that can be used to detect
or purify the provided polypeptide, wherein the tag does not
otherwise contribute to the essential function of the composition.
The provided peptides and polypeptides can further have deleted
N-terminal, C-terminal or intermediate amino acids that do not
contribute to the essential activity of the peptides and
polypeptides.
[0363] Components can be directly or indirectly covalently bound to
surface molecules or each other by any functional group (e.g.,
amine, carbonyl, carboxyl, aldehyde, alcohol). For example, one or
more amine, alcohol or thiol groups on the components can be
reacted directly with isothiocyanate, acyl azide,
N-hydroxysuccinimide ester, aldehyde, epoxide, anhydride, lactone,
or other functional groups incorporated onto the surface molecules
or other components. Schiff bases formed between the amine groups
on the components and aldehyde groups on the surface molecule or
other components can be reduced with agents such as sodium
cyanoborohydride to form hydrolytically stable amine links
(Ferreira et al., J. Molecular Catalysis B: Enzymatic 2003, 21,
189-199). Components can be coupled to surface molecules and other
components by, for example, the use of a heterobifunctional
silane-linker reagent, or by other reactions that activate
functional groups on either the surface molecule or the
components.
[0364] Useful modes for linking components to surface molecules and
to other components include heterobifunctional linkers or spacers.
Such linkers can have both terminal amine and thiol reactive
functional groups for reacting amines on components with sulfhydryl
groups, thereby coupling the components in an oriented way. These
linkers can contain a variable number of atoms. Examples of such
linkers include, but are not limited to, N-Succinimidyl
3-(2-pyridyldithio)propionate (SPDP, 3- and 7-atom spacer),
long-chain-SPDP (12-atom spacer),
(Succinimidyloxycarbonyl-a-methyl-2-(2-pyridyldithio)toluene)
(SMPT, 8-atom spacer),
Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC,
11-atom spacer) and
Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
(sulfo-SMCC, 11-atom spacer), m-Maleimidobenzoyl-N
hydroxysuccinimide ester (MBS, 9-atom spacer),
N-(g-maleimidobutyryloxy)succinimide ester (GMBS, 8-atom spacer),
N-(g-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS, 8-atom
spacer), Succinimidyl 6-((iodoacetyl)amino)hexanoate (SIAX, 9-atom
spacer), Succinimidyl
6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)hexanoate (SIAXX, 16-atom
spacer), and p-nitrophenyl iodoacetate (NPIA, 2-atom spacer). One
ordinarily skilled in the art also will recognize that a number of
other coupling agents or links, with different number of atoms, may
be used.
[0365] Hydrophilic spacer atoms can be incorporated into linkers to
increase the distance between the reactive functional groups. For
example, polyethylene glycol (PEG) can be incorporated into
sulfo-GMBS. Hydrophilic molecules such as PEG have also been shown
to decrease non-specific binding (NSB) and increase hydrophilicity
of surfaces when covalently coupled. PEG can also be used as the
primary linker material.
[0366] Free amine groups of components can also be attached to
surface molecules or other components containing reactive amine
groups via homobifunctional linkers. Linkers such as
dithiobis(succinimidylpropionate) (DSP, 8-atom spacer),
disuccinimidyl suberate (DSS, 8-atom spacer), glutaraldehyde
(4-atom spacer), Bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone
(BSOCOES, 9-atom spacer), all requiring high pH, can be used for
this purpose. Examples of homobifunctional sulfhydryl-reactive
linkers include, but are not limited to,
1,4-Di-[3'-2'-pyridyldithio)propion-amido]butane (DPDPB, 16-atom
spacer) and Bismaleimidohexane (BMH, 14-atom spacer). For example,
these homobifunctional linkers are first reacted with a thiolated
surface in aqueous solution (for example PBS, pH 7.4), and then in
a second step, the thiolated antibody or protein is joined by the
link. Homo- and heteromultifunctional linkers can also be used.
[0367] Direct binding of components to thiol, amine, or carboxylic
acid functional groups on surface molecules and other components be
used to produce compositions which exhibit viral binding (due to
increased density of components, for example), resulting in
enhanced sensitivity.
[0368] As an example, when necessary to achieve high peptide
coupling density, additional amino groups can be added to the
surface molecules (such as commercially obtained SPIO) as follows:
First, to crosslink the particles before the amination step, 3 ml
of the colloid (.about.10 mgFe/ml in double-distilled water) was
added to 5 ml of 5M NaOH and 2 ml of epichlorohydrin (Sigma, St.
Louis, Mo.). The mixture was agitated for 24 hours at room
temperature to promote interaction between the organic phase
(epichlorohydrin) and aqueous phase (dextran-coated particle
colloid). In order to remove excess epichlorohydrin, the reacted
mixture was dialyzed against double-distilled water for 24 hours
using a dialysis cassette (10,000 Da cutoff, Pierce, Rockford
Ill.). Amino groups were added to the surface of the particles as
follows: 0.02 ml of concentrated ammonium hydroxide (30%) was added
to 1 ml of colloid (.about.10 mg Fe/ml). The mixture was agitated
at room temperature for 24 hours. The reacted mixture was dialyzed
against double-distilled water for 24 hours. To further rinse the
particles, the colloid was trapped on a MACS.RTM.Midi magnetic
separation column (Miltenyi Biotec, Auburn Calif.), rinsed with PBS
three times, and eluted from the column with 1 ml PBS.
[0369] To conjugate CGKRK peptide (and other peptides) to SPIO, the
particles were re-suspended at a concentration of 1 mg Fe/ml, and
heterobifunctional linker N-[a-maleimidoacetoxy]succinimide ester
(AMAS; Pierce) was added (2.5 mg linker per 2 mg Fe) under
vortexing. After incubation at room temperature for 40 min, the
particles were washed 3 times with 10 ml PBS on a MACS column. The
peptide with free terminal cysteine was then added (100 .mu.g
peptide per 2 mg Fe). After incubation overnight at 4.degree. C.
the particles were washed again and re-suspended in PBS at a
concentration of 0.35 mg/ml of Fe). To quantify the number of
peptide molecules conjugated to the particles, a known amount of
stock or AMAS-activated particles was incubated with varying
amounts of the peptide. After completion of the incubation the
particles were pelleted at 100.000G using Beckman TLA 100.3
ultracentrifuge rotor (30 min) and the amount of the unbound
peptide was quantified by fluorescence. To cleave the conjugated
peptide from the particles, the particles were incubated at
37.degree. C. overnight at pH 10. The concentration of free peptide
in the supernatant was determined by reading fluorescence and by
using the calibration curve obtained for the same peptide. The
fluorescence intensity of known amounts of particles was plotted as
a function of peptide conjugation density, and the slope equation
was used to determine conjugation density in different batches.
[0370] Many homing molecules and homing peptides home to the
vasculature of the target tissue. However, for the sake of
convenience homing is referred to in some places herein as homing
to the tissue associated with the vasculature to which the homing
molecule or homing peptide may actually home. Thus, for example, a
homing peptide that homes to tumor vasculature can be referred to
herein as homing to tumor tissue or to tumor cells. By including or
associating a homing molecule or homing peptide with, for example,
a protein, peptide, amino acid sequence, co-composition, cargo
composition, or tCAR peptide the protein, peptide, amino acid
sequence, co-composition, cargo composition, or tCAR peptide can be
targeted or can home to the target of the homing molecule or homing
peptide. In this way, the protein, peptide, amino acid sequence,
co-composition, cargo composition, or tCAR peptide can be said to
home to the target of the homing molecule or homing peptide. For
convenience and unless otherwise indicated, reference to homing of
a protein, peptide, amino acid sequence, co-composition, cargo
composition, tCAR peptide, etc. is intended to indicate that the
protein, peptide, amino acid sequence, co-composition, cargo
composition, tCAR peptide, etc. includes or is associated with an
appropriate homing molecule or homing peptide.
[0371] The homing molecule can selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. The
homing molecule can selectively home to tumor vasculature. The
homing molecule can selectively home to one or more particular
types of tumor. The homing molecule can selectively home to the
vasculature of one or more particular types of tumor. The homing
molecule can selectively home to one or more particular stages of a
tumor or cancer. The homing molecule can selectively home to the
vasculature of one or more particular stages of a tumor or cancer.
The homing molecule can selectively home to one or more particular
stages of one or more particular types of tumor. The homing
molecule can selectively home to the vasculature of one or more
different stages of one or more particular types of tumor.
[0372] The composition can selectively home to tumors, regenerating
tissue, sites of injury, surgical sites, tumor vasculature, sites
of tumor angiogenesis, sites of inflammation, sites of arthritis,
lung tissue, pulmonary arterial hypertension lung vasculature,
pulmonary arterial hypertension lesions, remodeled pulmonary
arteries, or interstitial space of lungs. The composition can
selectively home to tumor vasculature. The composition can
selectively home to one or more particular types of tumor. The
composition can selectively home to the vasculature of one or more
particular types of tumor. The composition can selectively home to
one or more particular stages of a tumor or cancer. The composition
can selectively home to the vasculature of one or more particular
stages of a tumor or cancer. The composition can selectively home
to one or more particular stages of one or more particular types of
tumor. The composition can selectively home to the vasculature of
one or more different stages of one or more particular types of
tumor.
[0373] The co-composition can selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. The
co-composition can selectively home to tumor vasculature. The
co-composition can selectively home to one or more particular types
of tumor. The co-composition can selectively home to the
vasculature of one or more particular types of tumor. The
co-composition can selectively home to one or more particular
stages of a tumor or cancer. The co-composition can selectively
home to the vasculature of one or more particular stages of a tumor
or cancer. The co-composition can selectively home to one or more
particular stages of one or more particular types of tumor. The
co-composition can selectively home to the vasculature of one or
more different stages of one or more particular types of tumor.
[0374] The tCAR composition can selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. The
tCAR composition can selectively home to tumor vasculature. The
tCAR composition can selectively home to one or more particular
types of tumor. The tCAR composition can selectively home to the
vasculature of one or more particular types of tumor. The tCAR
composition can selectively home to one or more particular stages
of a tumor or cancer. The tCAR composition can selectively home to
the vasculature of one or more particular stages of a tumor or
cancer. The tCAR composition can selectively home to one or more
particular stages of one or more particular types of tumor. The
tCAR composition can selectively home to the vasculature of one or
more different stages of one or more particular types of tumor.
[0375] The tCAR peptide can selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. The
tCAR peptide can selectively home to tumor vasculature. The tCAR
peptide can selectively home to one or more particular types of
tumor. The tCAR peptide can selectively home to the vasculature of
one or more particular types of tumor. The tCAR peptide can
selectively home to one or more particular stages of a tumor or
cancer. The tCAR peptide can selectively home to the vasculature of
one or more particular stages of a tumor or cancer. The tCAR
peptide can selectively home to one or more particular stages of
one or more particular types of tumor. The tCAR peptide can
selectively home to the vasculature of one or more different stages
of one or more particular types of tumor.
[0376] The cargo composition can selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. The
cargo composition can selectively home to tumor vasculature. The
cargo composition can selectively home to one or more particular
types of tumor. The cargo composition can selectively home to the
vasculature of one or more particular types of tumor. The cargo
composition can selectively home to one or more particular stages
of a tumor or cancer. The cargo composition can selectively home to
the vasculature of one or more particular stages of a tumor or
cancer. The cargo composition can selectively home to one or more
particular stages of one or more particular types of tumor. The
cargo composition can selectively home to the vasculature of one or
more different stages of one or more particular types of tumor.
[0377] The cargo molecule can selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. The
cargo molecule can selectively home to tumor vasculature. The cargo
molecule can selectively home to one or more particular types of
tumor. The cargo molecule can selectively home to the vasculature
of one or more particular types of tumor. The cargo molecule can
selectively home to one or more particular stages of a tumor or
cancer. The cargo molecule can selectively home to the vasculature
of one or more particular stages of a tumor or cancer. The cargo
molecule can selectively home to one or more particular stages of
one or more particular types of tumor. The cargo molecule can
selectively home to the vasculature of one or more different stages
of one or more particular types of tumor.
[0378] The surface molecule can selectively home to tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. The
surface molecule can selectively home to tumor vasculature. The
surface molecule can selectively home to one or more particular
types of tumor. The surface molecule can selectively home to the
vasculature of one or more particular types of tumor. The surface
molecule can selectively home to one or more particular stages of a
tumor or cancer. The surface molecule can selectively home to the
vasculature of one or more particular stages of a tumor or cancer.
The surface molecule can selectively home to one or more particular
stages of one or more particular types of tumor. The surface
molecule can selectively home to the vasculature of one or more
different stages of one or more particular types of tumor.
[0379] The membrane perturbing molecule can selectively home to
tumors, regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, pulmonary arterial hypertension
lung vasculature, pulmonary arterial hypertension lesions,
remodeled pulmonary arteries, or interstitial space of lungs. The
membrane perturbing molecule can selectively home to tumor
vasculature. The membrane perturbing molecule can selectively home
to one or more particular types of tumor. The membrane perturbing
molecule can selectively home to the vasculature of one or more
particular types of tumor. The membrane perturbing molecule can
selectively home to one or more particular stages of a tumor or
cancer. The membrane perturbing molecule can selectively home to
the vasculature of one or more particular stages of a tumor or
cancer. The membrane perturbing molecule can selectively home to
one or more particular stages of one or more particular types of
tumor. The membrane perturbing molecule can selectively home to the
vasculature of one or more different stages of one or more
particular types of tumor.
[0380] The disclosed amino acid sequences, co-compositions, cargo
compositions, proteins or peptides can, for example, home to brain
cells, brain stem cells, brain tissue, and/or brain vasculature,
kidney cells, kidney stem cells, kidney tissue, and/or kidney
vasculature, skin cells, skin stem cells, skin tissue, and/or skin
vasculature, lung cells, lung tissue, and/or lung vasculature,
pancreatic cells, pancreatic tissue, and/or pancreatic vasculature,
intestinal cells, intestinal tissue, and/or intestinal vasculature,
adrenal gland cells, adrenal tissue, and/or adrenal vasculature,
retinal cells, retinal tissue, and/or retinal vasculature, liver
cells, liver tissue, and/or liver vasculature, prostate cells,
prostate tissue, and/or prostate vasculature, endometriosis cells,
endometriosis tissue, and/or endometriosis vasculature, ovary
cells, ovary tissue, and/or ovary vasculature, tumor cells, tumors,
tumor blood vessels, and/or tumor vasculature, bone cells, bone
tissue, and/or bone vasculature, bone marrow cells, bone marrow
tissue, and/or bone marrow vasculature, cartilage cells, cartilage
tissue, and/or cartilage vasculature, stem cells, embryonic stem
cells, pluripotent stem cells, induced pluripotent stem cells,
adult stem cells, hematopoietic stem cells, neural stem cells,
mesenchymal stem cells, mammary stem cells, endothelial stem cells,
olfactory adult stem cells, neural crest stem cells, cancer stem
cells, blood cells, erythrocytes, platelets, leukocytes,
granulocytes, neutrophils, eosinphils, basophils, lymphoid cells,
lymphocytes, monocytes, wound vasculature, vasculature of injured
tissue, vasculature of inflamed tissue, atherosclerotic plaques, or
a combination.
[0381] Examples of homing molecules and homing peptides are known.
Examples include: Brain homing peptides such as: CNSRLHLRC (SEQ ID
NO:114), CENWWGDVC (SEQ ID NO:115), WRCVLREGPAGGCAWFNRHRL (SEQ ID
NO:116), CLSSRLDAC (SEQ ID NO:117), CVLRGGRC (SEQ ID NO:118),
CNSRLQLRC (SEQ ID NO:119), CGVRLGC (SEQ ID NO:120), CKDWGRIC (SEQ
ID NO:121), CLDWGRIC (SEQ ID NO:122), CTRITESC (SEQ ID NO:123),
CETLPAC (SEQ ID NO:124), CRTGTLFC (SEQ ID NO:125), CGRSLDAC (SEQ ID
NO:126), CRHWFDVVC (SEQ ID NO:127), CANAQSHC (SEQ ID NO:128),
CGNPSYRC (SEQ ID NO:129), YPCGGEAVAGVSSVRTMCSE (SEQ ID NO:130),
LNCDYQGTNPATSVSVPCTV (SEQ ID NO:131); kidney homing peptides such
as: CLPVASC (SEQ ID NO:132), CGAREMC (SEQ ID NO:133), CKGRSSAC (SEQ
ID NO:134), CWARAQGC (SEQ ID NO:135), CLGRSSVC (SEQ ID NO:136),
CTSPGGSC (SEQ ID NO:137), CMGRWRLC (SEQ ID NO:138), CVGECGGC (SEQ
ID NO:139), CVAWLNC (SEQ ID NO:140), CRRFQDC (SEQ ID NO:141),
CLMGVHC (SEQ ID NO:142), CKLLSGVC (SEQ ID NO:143), CFVGHDLC (SEQ ID
NO:144), CRCLNVC (SEQ ID NO:145), CKLMGEC (SEQ ID NO:146); skin
homing peptides such as: CARSKNKDC (SEQ ID NO:147), CRKDKC (SEQ ID
NO:2), CVALCREACGEGC (SEQ ID NO:149), CSSGCSKNCLEMC (SEQ ID
NO:150), CIGEVEVC (SEQ ID NO:151), CKWSRLHSC (SEQ ID NO:152),
CWRGDRKIC (SEQ ID NO:153), CERVVGSSC (SEQ ID NO:154), CLAKENVVC
(SEQ ID NO:155); lung homing peptides such as: CGFECVRQCPERC (SEQ
ID NO:156), CGFELETC (SEQ ID NO:157), CTLRDRNC (SEQ ID NO:158),
CIGEVEVC (SEQ ID NO:151), CGKRYRNC (SEQ ID NO:161), CLRPYLNC (SEQ
ID NO:162), CTVNEAYKTRMC (SEQ ID NO:163), CRLRSYGTLSLC (SEQ ID
NO:164), CRPWHNQAHTEC (SEQ ID NO:165); pancreas homing peptides
such as: SWCEPGWCR (SEQ ID NO:166), CKAAKNK (SEQ ID NO:167),
CKGAKAR (SEQ ID NO:168), VGVGEWSV (SEQ ID NO:92); intestine homing
peptides such as: YSGKWGW (SEQ ID NO:93); uterus homing peptides
such as: GLSGGRS (SEQ ID NO:94); adrenal gland homing peptides such
as: LMLPRAD (SEQ ID NO:95), LPRYLLS (SEQ ID NO:96); retina homing
peptides such as: CSCFRDVCC (SEQ ID NO:97), CRDVVSVIC (SEQ ID
NO:98); gut homing peptides such as: YSGKWGK (SEQ ID NO:99),
GISALVLS (SEQ ID NO:100), SRRQPLS (SEQ ID NO:101), MSPQLAT (SEQ ID
NO:102), MRRDEQR (SEQ ID NO:103), QVRRVPE (SEQ ID NO:104), VRRGSPQ
(SEQ ID NO:105), GGRGSWE (SEQ ID NO:106), FRVRGSP (SEQ ID NO:25),
RVRGPER (SEQ ID NO:26); liver homing peptides such as: VKSVCRT (SEQ
ID NO:27), WRQNMPL (SEQ ID NO:28), SRRFVGG (SEQ ID NO:29), ALERRSL
(SEQ ID NO:30), ARRGWTL (SEQ ID NO:31); prostate homing peptides
such as: SMSIARL (SEQ ID NO:32), VSFLEYR (SEQ ID NO:33), RGRWLAL
(SEQ ID NO:34); ovary homing peptides such as: EVRSRLS (SEQ ID
NO:35), VRARLMS (SEQ ID NO:36), RVGLVAR (SEQ ID NO:37), RVRLVNL
(SEQ ID NO:38); Clot binding homing peptide such as: CREKA (SEQ ID
NO:12), CLOT1, and CLOT2; heart homing peptides such as: CRPPR (SEQ
ID NO:39), CGRKSKTVC (SEQ ID NO:40), CARPAR (SEQ ID NO:41), CPKRPR
(SEQ ID NO:42), CKRAVR (SEQ ID NO:43), CRNSWKPNC (SEQ ID NO:44),
RGSSS (SEQ ID NO:19), CRSTRANPC (SEQ ID NO:16), CPKTRRVPC (SEQ ID
NO:17), CSGMARTKC (SEQ ID NO:45), GGGVFWQ (SEQ ID NO:61), HGRVRPH
(SEQ ID NO:107), VVLVTSS (SEQ ID NO:148), CLHRGNSC (SEQ ID NO:159),
CRSWNKADNRSC (SEQ ID NO:160), CGRKSKTVC (SEQ ID NO:199), CKRAVR
(SEQ ID NO:202), CRNSWKPNC (SEQ ID NO:203), CPKTRRVPC (SEQ ID
NO:17), CSGMARTKC (SEQ ID NO:45), CARPAR (SEQ ID NO:107), CPKRPR
(SEQ ID NO:201); tumor blood vessel homing peptide such as: CNGRC
(SEQ ID NO:68) and other peptides with the NGR motif (U.S. Pat.
Nos. 6,177,542 and 6,576,239; U.S. Patent Application Publication
No. 20090257951); RGD peptides, and RGR peptides. Other homing
peptides include CSRPRRSEC (SEQ ID NO:108), CSRPRRSVC (SEQ ID
NO:109) and CSRPRRSWC (SEQ ID NO:110) (Hoffman et al., Cancer Cell,
vol. 4 (2003)), F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK; (SEQ ID
NO:111)), PQRRSARLSA (SEQ ID NO:112), PKRRSARLSA (SEQ ID NO:113)
(U.S. Pat. No. 7,544,767), and CGRECPRLCQSSC (SEQ ID NO:62), which
home to tumors.
[0382] Homing molecules can also be defined by their targets. For
example, numerous antigens and proteins are known that can be
useful for targeting. Any molecule that can bind, selectively bind,
home, selectively, target, selectively target, etc. such target
molecules can be used as a homing molecule. For example,
antibodies, nucleic acid aptamers, and compounds that can bind to
target molecules can be used as homing molecules. Examples of
useful target molecules for homing molecules include .alpha.v
integrins, .alpha.v.beta.3 integrin, .alpha.v.beta.5 integrin,
.alpha.5.beta.1 integrin, aminopeptidase N, tumor endothelial
markers (TEMs), endosialin, p32, gC1q receptor, annexin-1,
nucleolin, fibronectin ED-B, fibrin-fibronectin complexes,
interleukin-11 receptor .alpha., and protease-cleaved collagen IV.
These and other examples are described and referred to in Ruoslahti
et al., J. Cell Biology, 2010 (doi: 10.1083/jbc.200910104), which
is hereby incorporated by reference in its entirety and
specifically for its description of and references to target
molecules.
[0383] The composition, tCAR composition, co-composition, or cargo
composition can comprise any number of homing molecules. By way of
example, the composition, tCAR composition, co-composition, or
cargo composition can comprise at least 1, 5, 10, 15, 20, 25, 50,
75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,
400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700,
625, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100,
1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500,
2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500,
8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000,
35,000, 40,000, 45,000, 50,000, 75,000, or 100,000, or more homing
molecules. The composition, tCAR composition, co-composition, or
cargo composition can also comprise any number in between those
numbers listed above.
[0384] It is understood that, although many homing and targeting
motifs and sequences are shown with cysteine residues at one or
both ends, such cysteine residues are generally not required for
homing function. Generally, such cysteines are present due to the
methods by which the homing and targeting sequences were
identified. Such terminal cysteines can be used to, for example,
circularize peptides, such as those disclosed herein. For these
reasons, it is also understood that cysteine residues can be added
to the ends of any of the disclosed peptides.
[0385] Useful NGR peptides include peptide such as
X.sub.2CNGRCX.sub.2 (SEQ ID NO:89), CX.sub.2(C/X)NGR(C/X)X.sub.2C
(SEQ ID NO:90), and CNGRCX.sub.6 (SEQ ID NO:91) (where "X" is any
amino acid), which can be linear or circular. Examples of NGR
peptides include CNGRCVSGCAGRC (SEQ ID NO:63), NGRAHA (SEQ ID
NO:24), CVLNGRMEC (SEQ ID NO:67), CNGRC (SEQ ID NO:68), ALNGREESP
(SEQ ID NO:66), CVLNGRME (SEQ ID NO:87), CKVCNGRCCG (SEQ ID NO:88),
CEMCNGRCMG (SEQ ID NO:69), CPLCNGRCAL (SEQ ID NO:70), CPTCNGRCVR
(SEQ ID NO:71), CGVCNGRCGL (SEQ ID NO:72), CEQCNGRCGQ (SEQ ID
NO:73), CRNCNGRCEG (SEQ ID NO:74), CVLCNGRCWS (SEQ ID NO:75),
CVTCNGRCRV (SEQ ID NO:76), CTECNGRCQL (SEQ ID NO:77), CRTCNGRCLE
(SEQ ID NO:78), CETCNGRCVG (SEQ ID NO:79), CAVCNGRCGF (SEQ ID
NO:80), CRDLNGRKVM (SEQ ID NO:81), CSCCNGRCGD (SEQ ID NO:82),
CWGCNGRCRM (SEQ ID NO:83), CPLCNGRCAR (SEQ ID NO:84), CKSCNGRCLA
(SEQ ID NO:85), CVPCNGRCHE (SEQ ID NO:86), CQSCNGRCVR (SEQ ID
NO:47), CRTCNGRCQV (SEQ ID NO:48), CVQCNGRCAL (SEQ ID NO:49),
CRCCNGRCSP (SEQ ID NO:50), CASNNGRVVL (SEQ ID NO:51), CGRCNGRCLL
(SEQ ID NO:52), CWLCNGRCGR (SEQ ID NO:53), CSKCNGRCGH (SEQ ID
NO:54), CVWCNGRCGL (SEQ ID NO:55), CIRCNGRCSV (SEQ ID NO:56),
CGECNGRCVE (SEQ ID NO:57), CEGVNGRRLR (SEQ ID NO:58), CLSCNGRCPS
(SEQ ID NO:59), CEVCNGRCAL (SEQ ID NO:60).
[0386] Useful peptides for tumor targeting include, for example,
iRGD, CAR, LyP-1, iNGR, and RGR peptides. The prototypic
tumor-homing CendR peptide, iRGD, which was used in generating the
results described herein. CAR has tumor-penetrating properties.
This peptide has a unique target within tumors; it preferentially
accumulates in the hypoxic/low nutrient areas of tumors (Laakkonen
et al., 2002; 2004; Karmali et al., 2009). CRGRRST (RGR; Joyce et
al., 2003) is a peptide that has been successfully used in
targeting a cytokine antibody combination into tumors (Hamzah et
al., 2008). This peptide is linear, which simplifies the synthesis.
NGR peptides home to angiogenic vasculature, including angiogenic
vasculature associated with tumors, and .alpha..sub.v integrin and
.alpha..sub.5.beta..sub.1 integrin (U.S. Pat. Nos. 6,576,239 and
6,177,542 and U.S. Patent Application Publication No. 20090257951).
RGR is at least to some extent tumor type-specific (Joyce et al.,
2003), but the tumor types recognized by the two peptides seem to
be partially different, which may be an advantage in testing
combinations with the pan-tumor iRGD.
[0387] RGD peptides are peptides that contain the RGD (Arg-Gly-Asp)
motif and that home to angiogenesis and tumor vasculature. NGR
peptides are peptides that contain the NGR (Asn-Gly-Arg) motif and
that home to angiogenesis and tumor vasculature. Examples of NGR
peptides include CNGRCVSGCAGRC (SEQ ID NO:63), NGRAHA (SEQ ID
NO:24), CVLNGRMEC (SEQ ID NO:67), and CNGRC (SEQ ID NO:68). GSL
peptides are peptides that contain the GSL (Gly-Ser-Leu) motif and
that home to tumor vasculature. Examples of a GSL peptide include
CGSLVRC (SEQ ID NO:65) and CLSGSLSC (SEQ ID NO:64).
[0388] Internalizing RGD (iRGD) refers to peptides that combine an
RGD motif and a CendR element. For example, cyclic RGD peptide
having the sequence CRGDK/RGPD/EC (SEQ ID NO:11) is exceptionally
effective in orchestrating extravasation and spreading of linked
payloads within tumor tissue, and subsequently internalizing within
tumor cells. The iRGD peptide incorporates two functional elements:
the RGD motif that gives tumor specificity (Pierschbacher and
Ruoslahti, E. Cell attachment activity of fibronectin can be
duplicated by small synthetic fragments of the molecule. Nature
309, 30-33 (1984); Ruoslahti (2003); Eliceiri and Cheresh (2001);
Ruoslahti (2002); Arap et al. (1998); Cumis et al. (2004); Sipkins
et al. (1998); Murphy et al. (2008)), and a CendR motif that
mediates penetration. iRGD readily adheres to cultured cells
expressing .alpha.v integrins, and is internalized far more
effectively than other RGD peptides. Internalization was dependent
on expression of neuropilin-1, the receptor for the CendR motif.
iRGD coupled to a payload of fluorescein, phage, or artificial
nanoparticles, accumulated around tumor vessels in vivo, spread
through the tumor interstitium, and became internalized within
tumor cells in various tumor models. Systemic administration of
iRGD micelles labeled with a near infrared dye produced a strong
and specific tumor signal in whole body imaging of mice. The CendR
element in iRGD is an activatable CendR element that is activated,
likely by cleavage after the Lys/Arg, to allow the peptide to
mediate internalization.
[0389] Internalizing NGR (iNGR) refers to peptides that combine a
NGR motif and a CendR element. For example, NGR peptide having the
sequence K/RNGR (SEQ ID NO:46) can be effective in orchestrating
extravasation and spreading of linked payloads within tumor tissue,
and subsequently internalizing within tumor cells. The iNGR peptide
incorporates two functional elements: the NGR motif that gives
tumor specificity, and a CendR motif that mediates penetration.
Another example of an iNGR peptide is NGRAHA (SEQ ID NO:24). The
CendR element in the iNGR peptide NGRAHA (SEQ ID NO:24) is an
activatable CendR element that is activated, likely by cleavage
after the Arg, to allow the peptide to mediate internalization.
[0390] Accessory molecules can be any molecule, compound,
component, etc. that has a useful function and that can be used in
combination with a tCAR composition, tCAR conjugate, tCAR molecule,
tCAR protein, tCAR peptide, composition, co-composition, and/or
cargo composition. Examples of useful accessory molecules include
homing molecules, targeting molecules, affinity ligands, cell
penetrating molecules, endosomal escape molecules, subcellular
targeting molecules, nuclear targeting molecules. Different
accessory molecules can have similar or different functions from
each other.
[0391] Molecules that target, home, or have affinity for certain
molecules, structures, cells, tissues, etc. are particularly useful
as accessory molecules. In addition to the homing peptides
described elsewhere herein, there are numerous molecules and
compounds known that have affinity for particular target molecules,
structures, cells, tissues, etc. and can aid in accumulating and/or
directing the disclosed components and compositions to desired
targets. For convenience, such affinity effects can be referred to
as homing. Descriptions of homing and homing effects elsewhere
herein can be applied to these molecules.
[0392] An affinity ligand is a molecule that interacts specifically
with a particular molecule, moiety, cell tissue, etc. The molecule,
moiety, cell tissue, etc. that interacts specifically with an
affinity ligand is referred to herein as a target or target
molecule, moiety, cell tissue, etc. It is to be understood that the
term target molecule refers to both separate molecules and to
portions of such molecules, such as an epitope of a protein, that
interacts specifically with an affinity ligand. Antibodies, either
member of a receptor/ligand pair, synthetic polyamides (Dervan and
Burli, Sequence-specific DNA recognition by polyamides. Curr Opin
Chem Biol, 3(6):688-93 (1999); Wemmer and Dervan, Targeting the
minor groove of DNA. Curr Opin Struct Biol, 7(3):355-61 (1997)),
and other molecules with specific binding affinities are examples
of affinity ligands.
[0393] An affinity ligand that interacts specifically with a
particular target molecule is said to be specific for that target
molecule. For example, where the affinity ligand is an antibody
that binds to a particular antigen, the affinity ligand is said to
be specific for that antigen. The antigen is the target molecule.
The affinity ligand can also be referred to as being specific for a
particular target molecule. Examples of useful affinity ligands are
antibodies, ligands, binding proteins, receptor proteins, haptens,
aptamers, carbohydrates, lectins, folic acid, synthetic polyamides,
and oligonucleotides. Useful binding proteins include DNA binding
proteins. Useful DNA binding proteins include zinc finger motifs,
leucine zipper motifs, and helix-turn-helix motifs. These motifs
can be combined in the same affinity ligand.
[0394] Antibodies are useful as the affinity ligands. Antibodies
can be obtained commercially or produced using well established
methods. For example, Johnstone and Thorpe, Immunochemistry In
Practice (Blackwell Scientific Publications, Oxford, England, 1987)
on pages 30-85, describe general methods useful for producing both
polyclonal and monoclonal antibodies. The entire book describes
many general techniques and principles for the use of antibodies in
assay systems. Numerous antibodies and other affinity ligands are
known that bind to particular proteins, carbohydrates,
glycoproteins, molecules, cells, tissues, etc. Such antibodies can
be used in the disclosed components and compositions.
[0395] Examples of cell penetrating peptides are described in, for
example, U.S. Patent Application Publication Nos. 20100061942,
20100061932, 20100048487, 20100022466, 20100016215, 20090280058,
20090186802, 20080234183, 20060014712, 20050260756, and
20030077289, which are hereby incorporated by reference in their
entirety and specifically for their description of cell penetrating
peptides and motifs. Examples of endosomal escape molecules are
described in, for example, U.S. Patent Application Publication Nos.
20090325866, 20090317802, 20080305119, 20070292920, 20060147997,
20050038239, 20040219169, 20030148263, 20030082143, 20020132990,
and 20020068272, which are hereby incorporated by reference in
their entirety and specifically for their description of endosomal
escape molecules and motifs. Examples of subcellular targeting
molecules are described in, for example, U.S. Patent Application
Publication Nos. 2009031733, 20090258926, 20090176660, 20080311136,
20070287680, 20070157328, 20070111270, 20070111251, 20060257942,
20060154340, 20060014712, 20050281805, 20050233356, 20040005309,
20030082176, and 20010021500, which are hereby incorporated by
reference in their entirety and specifically for their description
of subcellular targeting molecules and motifs. Examples of nuclear
targeting molecules are described in, for example, U.S. Patent
Application Publication Nos. 10100143454, 20100099627, 20090305329,
20090176710, 20090087899, 20070231862, 20070212332, 20060242725,
20060233807, 20060147922, 20060070133, 20060051315, 20050147993,
20050071088, 20030166601, 20030125283, 20030083261, 20030003100,
20020068272, and 20020055174, which are hereby incorporated by
reference in their entirety and specifically for their description
of nuclear targeting molecules and motifs.
[0396] As disclosed herein, the term "co-composition" refers to any
composition of matter that can be used with the tCAR peptide.
Similarly, the term "cargo composition" refers to any composition
of matter that can be used with the tCAR peptide. Generally, for
example, a co-composition or cargo composition can be any
composition to be internalized and/or to penetrate into cells
and/or tissues. For example, a co-composition or cargo composition
can be a molecule, a conjugate, an association of molecules, a
composition, a mixture. Examples of co-compositions and cargo
compositions include, but are not limited to, cancer
chemotherapeutic agents, cytotoxic agents, anti-inflammatory
agents, anti-arthritic agents, polypeptides, nucleic acid
molecules, small molecules, nanoparticles, microparticles,
fluorophores, fluorescein, rhodamine, a radionuclide, Lutetium-177
(.sup.177Lu), Rhenium-188 (.sup.188Re), Gallium-68 (.sup.68Ga),
Yttrium-90 (.sup.90Y), Technetium-99m (.sup.99mTc), Holmium-166
(.sup.166Ho), Iodine-131 (.sup.131I), Indium-111 (.sup.111In),
Flourine-18 (.sup.18F), Carbon-11 (.sup.11C), Nitrogen-13
(.sup.13N), Oxygen-15 (.sup.15O), Bromine-75 (.sup.75Br),
Bromine-76 (.sup.76Br), Iodine-124 (.sup.124I), Thalium-201
(.sup.201Tl), Technetium-99 (.sup.99Tc), Iodine-123 (.sup.123I), an
anti-angiogenic agents, pro-angiogenic agents, or a combination
thereof.
[0397] The disclosed tCAR components can be used with any
therapeutic agents since they represent a general mode and platform
for aiding in delivery of therapeutic agents to cells and tissues.
Thus, any therapeutic agent can be used in or with the disclosed
compositions. Comprehensive lists of therapeutic agents and drugs
can be found in a number of places, such as the Orange Book and
other lists maintained by the U.S. Food and Drug Administration
(information available at websites
fda.gov/Drugs/InformationOnDrugs/ucm129662.htm and
fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/default.htm) and
similar lists maintained by other countries, and at
clinicaltrials.gov/ (for drugs and therapeutic agents undergoing
clinical trials).
[0398] Co-compositions and cargo compositions can be moieties. As
used herein, the term "moiety" is used broadly to mean a physical,
chemical, or biological material that generally imparts a
biologically useful function to a linked co-composition or a linked
cargo composition. A moiety can be any natural or normatural
material including, without limitation, a biological material, such
as a cell, phage or other virus; an organic chemical such as a
small molecule; a nanoparticle, a radionuclide; a nucleic acid
molecule or oligonucleotide; a polypeptide; or a peptide. For
example, moieties that affect the target, such as moieties with
therapeutic effect, or that facilitate detection, visualization or
imaging of the target, such as fluorescent molecule or
radionuclides.
[0399] Components of the disclosed co-compositions and cargo
compositions can be combined, linked and/or coupled in any suitable
manner. For example, moieties and other molecules can be associated
covalently or non-covalently, directly or indirectly, with or
without a linker moiety.
[0400] In some embodiments, a co-composition or cargo composition
can comprise a cancer chemotherapeutic agent. As used herein, a
"cancer chemotherapeutic agent" is a chemical agent that inhibits
the proliferation, growth, life-span or metastatic activity of
cancer cells. Such a cancer chemotherapeutic agent can be, without
limitation, a taxane such as docetaxel; an anthracyclin such as
doxorubicin; an alkylating agent; a vinca alkaloid; an
anti-metabolite; a platinum agent such as cisplatin or carboplatin;
a steroid such as methotrexate; an antibiotic such as adriamycin; a
isofamide; or a selective estrogen receptor modulator; an antibody
such as trastuzumab; paclitaxel such as Abraxane; Doxil.
[0401] A co-composition or cargo composition can comprise a
therapeutic agent. Useful therapeutic agents can be, for example, a
cytotoxic agent, which, as used herein, can be any molecule that
directly or indirectly promotes cell death. Useful cytotoxic agents
include, without limitation, small molecules, polypeptides,
peptides, peptidomimetics, nucleic acid-molecules, cells and
viruses. As non-limiting examples, useful cytotoxic agents include
cytotoxic small molecules such as doxorubicin, docetaxel or
trastuzumab; antimicrobial peptides such as those described further
below; pro-apoptotic polypeptides such as caspases and toxins, for
example, caspase-8; diphtheria toxin A chain, Pseudomonas exotoxin
A, cholera toxin, ligand fusion toxins such as DAB389EGF, ricinus
communis toxin (ricin); and cytotoxic cells such as cytotoxic T
cells. See, for example, Martin et al., Cancer Res. 60:3218-3224
(2000); Kreitman and Pastan, Blood 90:252-259 (1997); Allam et al.,
Cancer Res. 57:2615-2618 (1997); and Osborne and Coronado-Heinsohn,
Cancer J. Sci. Am. 2:175 (1996). One skilled in the art understands
that these and additional cytotoxic agents described herein or
known in the art can be useful in the disclosed compositions and
methods.
[0402] In some forms, a therapeutic agent can be a therapeutic
polypeptide. As used herein, a therapeutic polypeptide can be any
polypeptide with a biologically useful function. Useful therapeutic
polypeptides encompass, without limitation, cytokines, antibodies,
cytotoxic polypeptides; pro-apoptotic polypeptides; and
anti-angiogenic polypeptides. As non-limiting examples, useful
therapeutic polypeptides can be a cytokine such as tumor necrosis
factor-.alpha. (TNF-.alpha.), tumor necrosis factor-.beta.
(TNF-.beta.), granulocyte macrophage colony stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF),
interferon-.alpha.. (IFN-.alpha.); interferon-.gamma.
(IFN-.gamma.), interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6),
interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12
(IL-12), lymphotactin (LTN) or dendritic cell chemokine 1 (DC-CK1);
an anti-HER2 antibody or fragment thereof; a cytotoxic polypeptide
including a toxin or caspase, for example, diphtheria toxin A
chain, Pseudomonas exotoxin A, cholera toxin, a ligand fusion toxin
such as DAB389EGF or ricin; or an anti-angiogenic polypeptide such
as angiostatin, endostatin, thrombospondin, platelet factor 4;
anastellin; or one of those described further herein or known in
the art. It is understood that these and other polypeptides with
biological activity can be a "therapeutic polypeptide."
[0403] A therapeutic agent useful in the disclosed co-compositions
and cargo compositions can be an anti-angiogenic agent. As used
herein, the term "anti-angiogenic agent" means a molecule that
reduces or prevents angiogenesis, which is the growth and
development of blood vessels. The co-compositions and cargo
compositions can be used to treat or diagnose any disease,
condition, or disorder associated with angiogenesis. For example,
macular degeneration and diabetic vascular complications can be
diagnosed and/or treated. A variety of anti-angiogenic agents can
be prepared by routine methods. Such anti-angiogenic agents
include, without limitation, small molecules; proteins such as
dominant negative forms of angiogenic factors, transcription
factors and antibodies; peptides; and nucleic acid molecules
including ribozymes, antisense oligonucleotides, and nucleic acid
molecules encoding, for example, dominant negative forms of
angiogenic factors and receptors, transcription factors, and
antibodies and antigen-binding fragments thereof. See, for example,
Hagedorn and Bikfalvi, Crit. Rev. Oncol. Hematol. 34:89-110 (2000),
and Kirsch et al., J. Neurooncol. 50:149-163 (2000).
[0404] Some other examples of useful therapeutic agents include
nitrogen mustards, nitrosoureas, ethyleneimine, alkane sulfonates,
tetrazine, platinum compounds, pyrimidine analogs, purine analogs,
antimetabolites, folate analogs, anthracyclines, taxanes, vinca
alkaloids, topoisomerase inhibitors and hormonal agents. Exemplary
chemotherapy drugs are Actinomycin-D, Alkeran, Ara-C, Anastrozole,
Asparaginase, BiCNU, Bicalutamide, Bleomycin, Busulfan,
Capecitabine, Carboplatin, Carboplatinum, Carmustine, CCNU,
Chlorambucil, Chlomaphazine, Cholophosphamide, Cisplatin,
Cladribine, CPT-11, Cyclophosphamide, Cytarabine, Cytosine
arabinoside, Cytoxan, Dacarbazine, Dactinomycin, Daunorubicin,
Dexrazoxane, Docetaxel, Doxorubicin, DTIC, Epirubic in,
Estramustine, Ethyleneimine, Etoposide, Floxuridine, Fludarabine,
Fluorouracil, Flutamide, Fotemustine, Gemcitabine, Herceptin,
Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan,
Lomustine, Mechlorethamine, mechlorethamine oxide hydrochloride,
Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane,
Mitoxantrone, Novembiehin, Oxaliplatin, Paclitaxel, Pamidronate,
Pentostatin, Phenesterine, Plicamycin, Prednimustine, Procarbazine,
Rituximab, Steroids, Streptozocin, STI-571, Streptozocin,
Tamoxifen, Temozolomide, Teniposide, Tetrazine, Thioguanine,
Thiotepa, Tomudex, Topotecan, Treosulphan, Trimetrexate,
Trofosfamide, Vinblastine, Vincristine, Vindesine, Vinorelbine,
VP-16, and Xeloda. Alkylating agents such as Thiotepa and; alkyl
sulfonates such as Busulfan, Improsulfan and Piposulfan; aziridines
such as Benzodopa, Carboquone, Meturedopa, and Uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitroureas
such as Cannustine, Chlorozotocin, Fotemustine, Lomustine,
Nimustine, and Ranimustine; antibiotics such as Aclacinomysins,
Actinomycin, Authramycin, Azaserine, Bleomycins, Cactinomycin,
Calicheamicin, Carabicin, Caminomycin, Carzinophilin,
Chromoinycins, Dactinomycin, Daunorubicin, Detorubicin,
6-diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Esorubicin,
Idambicin, Marcellomycin, Mitomycins, mycophenolic acid,
Nogalamycin, Olivomycins, Peplomycin, Potfiromycin, Puromycin,
Quelamycin, Rodorubicin, Streptonigrin, Streptozocin, Tubercidin,
Ubenimex, Zinostatin, and Zorubicin; anti-metabolites such as
Methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as Denopterin, Methotrexate, Pteropterin, and Trimetrexate; purine
analogs such as Fludarabine, 6-mercaptopurine, Thiamiprine, and
Thioguanine; pyrimidine analogs such as Ancitabine, Azacitidine,
6-azauridine, Carmofur, Cytarabine, Dideoxyuridine, Doxifluridine,
Enocitabine, Floxuridine, and 5-FU; androgens such as Calusterone,
Dromostanolone Propionate, Epitiostanol, Rnepitiostane, and
Testolactone; anti-adrenals such as aminoglutethimide, Mitotane,
and Trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine;
Demecolcine; Diaziquone; Elformithine; elliptinium acetate;
Etoglucid; gallium nitrate; hydroxyurea; Lentinan; Lonidamine;
Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin;
Phenamet; Pirarubicin; podophyllinic acid; 2-ethylhydrazide;
Procarbazine; PSK.RTM.; Razoxane; Sizofrran; Spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
Urethan; Vindesine; Dacarbazine; Mannomustine; Mitobronitol;
Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C");
cyclophosphamide; thiotEPa; taxoids, e.g., Paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and Doxetaxel
(TAXOTERE.RTM., Rhone-Poulenc Rorer, Antony, France); Gemcitabine;
6-thioguanine; Mercaptopurine; Methotrexate; platinum analogs such
as Cisplatin and Carboplatin; Vinblastine; platinum; etoposide
(VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine;
Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin;
Aminopterin; Xeloda; Ibandronate; CPT-11; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
Esperamicins; Capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included are
anti-hormonal agents that act to regulate or inhibit hormone action
on tumors such as anti-estrogens including for example Tamoxifen,
Raloxifene, aromatase inhibiting 4(5)-imidazoles, 4
Hydroxytamoxifen, Trioxifene, Keoxifene, Onapristone, And
Toremifene (Fareston); and anti-androgens such as Flutamide,
Nilutamide, Bicalutamide, Leuprolide, and Goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. Useful co-compositions and cargo compositions include,
for example, doxorubicin, Herceptin, and liposomal doxorubicin.
[0405] The co-composition or cargo composition can also comprise a
boron containing compound. Boron containing compounds have received
increasing attention as therapeutic agents over the past few years
as technology in organic synthesis has expanded to include this
atom (Boron Therapeutics on the horizon, Groziak, M. P.; American
Journal of Therapeutics (2001) 8, 321-328). The most notable boron
containing therapeutic is the boronic acid bortezomib which was
recently launched for the treatment of multiple myeloma. This
breakthrough demonstrates the feasibility of using boron containing
compounds as pharmaceutical agents. Boron containing compounds have
been shown to have various biological activities including
herbicides (Organic boron compounds as herbicides. Barnsley, G. E.;
Eaton, J. K.; Airs, R. S.; (1957), DE 1016978 19571003), boron
neutron capture therapy (Molecular Design and Synthesis of B-10
Carriers for Neutron Capture Therapy. Yamamoto, Y.; Pure Appl.
Chem., (1991) 63, 423-426), serine protease inhibition (Borinic
acid inhibitors as probes of the factors involved in binding at the
active sites of subtilisin Carlsberg and .alpha.-chymotrypsin.
Simpelkamp, J.; Jones, J. B.; Bioorganic & Medicinal Chemistry
Letters, (1992), 2(11), 1391-4; Design, Synthesis and Biological
Evaluation of Selective Boron-containing Thrombin Inhibitors.
Weinand, A.; Ehrhardt, C.; Metternich, R.; Tapparelli, C.;
Bioorganic and Medicinal Chemistry, (1999), 7, 1295-1307),
acetylcholinesterase inhibition (New, specific and reversible
bifunctional alkylborinic acid inhibitor of acetylcholinesterase.
Koehler, K. A.; Hess, G. P.; Biochemistry (1974), 13, 5345-50) and
as antibacterial agents (Boron-Containing Antibacterial Agents:
Effects on Growth and Morphology of Bacteria Under Various Culture
Conditions. Bailey, P. J.; Cousins, G.; Snow, G. A.; and White, A.
J.; Antimicrobial Agents and Chemotherapy, (1980), 17, 549-553).
The boron containing compounds with antibacterial activity can be
sub-divided into two main classes, the diazaborinines, which have
been known since the 1960's, and dithienylborinic acid complexes.
This latter class has been expanded to include many different
diarylborinic acid complexes with potent antibacterial activity
(Preparation of diarylborinic acid esters as DNA methyl transferase
inhibitors. Benkovic, S. J.; Shapiro, L.; Baker, S. J.; Wahnon, D.
C.; Wall, M.; Shier, V. K.; Scott, C. P.; Baboval, J.; PCT Int.
Appl. (2002), WO 2002044184).
[0406] The compositions disclosed herein can also be used to treat
wounds or tissue injuries. Moieties, cargo compositions, and
co-compositions useful for this purpose can include molecules
belonging to several basic groups including anti-inflammatory
agents which prevent inflammation, restenosis preventing drugs
which prevent tissue growth, anti-thrombogenic drugs which inhibit
or control formation of thrombus or thrombolytics, and bioactive
agents which regulate tissue growth and enhance healing of the
tissue.
[0407] Examples of active agents include but are not limited to
steroids, fibronectin, anti-clotting drugs, anti-platelet function
drugs, drugs which prevent smooth muscle cell growth on inner
surface wall of vessel, heparin, heparin fragments, aspirin,
coumadin, tissue plasminogen activator (TPA), urokinase, hirudin,
streptokinase, antiproliferatives (methotrexate, cisplatin,
fluorouracil, Adriamycin), antioxidants (ascorbic acid, beta
carotene, vitamin E), antimetabolites, thromboxane inhibitors,
non-steroidal and steroidal anti-inflammatory drugs, beta and
calcium channel blockers, genetic materials including DNA and RNA
fragments, complete expression genes, antibodies, lymphokines,
growth factors, prostaglandins, leukotrienes, laminin, elastin,
collagen, and integrins.
[0408] Useful therapeutic agents also can be antimicrobial agents
and antimicrobial peptides. This can be particularly useful for
targeting a wound or other infected sites. Thus, also disclosed are
compositions in which a homing molecule that selectively homes to
tumor stroma, wounds, or plasma clots and interacts with
fibrin-fibronectin is linked to an antimicrobial peptide, where the
composition is selectively internalized and exhibits a high
toxicity to the targeted area, and where the antimicrobial peptide
has low mammalian cell toxicity when not linked to the homing
molecule. As used herein, the term "antimicrobial peptide" means a
naturally occurring or synthetic peptide having antimicrobial
activity, which is the ability to kill or slow the growth of one or
more microbes and which has low mammalian cell toxicity when not
linked to a homing molecule. An antimicrobial peptide can, for
example, kill or slow the growth of one or more strains of bacteria
including a Gram-positive or Gram-negative bacteria, or a fungi or
protozoa. Thus, an antimicrobial peptide can have, for example,
bacteriostatic or bacteriocidal activity against, for example, one
or more strains of Escherichia coli, Pseudomonas aeruginosa or
Staphylococcus aureus. An antimicrobial peptide can have biological
activity due to, for example, the ability to form ion channels
through membrane bilayers as a consequence of self-aggregation.
[0409] Antimicrobial peptide can be highly basic and can have a
linear or cyclic structure. As discussed further below, an
antimicrobial peptide can have an amphipathic .alpha.-helical
structure (see U.S. Pat. No. 5,789,542; Javadpour et al., J. Med.
Chem. 39:3107-3113 (1996); and Blondelle and Houghten, Biochem. 31:
12688-12694 (1992)). An antimicrobial peptide also can be, for
example, a .beta.-strand/sheet-forming peptide as described in
Mancheno et al., J. Peptide Res. 51:142-148 (1998).
[0410] An antimicrobial peptide can be a naturally occurring or
synthetic peptide. Naturally occurring antimicrobial peptides have
been isolated from biological sources such as bacteria, insects,
amphibians, and mammals and are thought to represent inducible
defense proteins that can protect the host organism from bacterial
infection. Naturally occurring antimicrobial peptides include the
gramicidins, magainins, mellitins, defensins and cecropins (see,
for example, Maloy and Kari, Biopolymers 37:105-122 (1995);
Alvarez-Bravo et al., Biochem. J. 302:535-538 (1994); Bessalle et
al., FEBS 274:-151-155 (1990.); and Blondelle and Houghten in
Bristol (Ed.), Annual Reports in Medicinal Chemistry pages 159-168
Academic Press, San Diego). An antimicrobial peptide also can be an
analog of a natural peptide, especially one that retains or
enhances amphipathicity.
[0411] An antimicrobial peptide incorporated into a composition can
have low mammalian cell toxicity when not linked to a tumor homing
molecule. Mammalian cell toxicity readily can be assessed using
routine assays. As an example, mammalian cell toxicity can be
assayed by lysis of human erythrocytes in vitro as described in
Javadpour et al., 1996. An antimicrobial peptide having low
mammalian cell toxicity is not lytic to human erythrocytes or
requires concentrations of greater than 100 .mu.M for lytic
activity, preferably concentrations greater than 200, 300, 500 or
1000
[0412] In some embodiments, disclosed are compositions in which the
antimicrobial peptide portion promotes disruption of mitochondrial
membranes when internalized by eukaryotic cells. In particular,
such an antimicrobial peptide preferentially disrupts mitochondrial
membranes as compared to eukaryotic membranes. Mitochondrial
membranes, like bacterial membranes but in contrast to eukaryotic
plasma membranes, have a high content of negatively charged
phospholipids. An antimicrobial peptide can be assayed for activity
in disrupting mitochondrial membranes using, for example, an assay
for mitochondrial swelling or another assay well known in the art.
.sub.D(KLAKLAK).sub.2, (SEQ ID NO:3) for example, is an
antimicrobial peptide which induces marked mitochondrial swelling
at a concentration of 10 .mu.M, significantly less than the
concentration required to kill eukaryotic cells.
[0413] An antimicrobial peptide that induces significant
mitochondrial swelling at, for example, 50 .mu.M, 40 .mu.M, 30
.mu.M, 20 .mu.M, 10 .mu.M, or less, is considered a peptide that
promotes disruption of mitochondrial membranes.
[0414] An antimicrobial peptide can include, for example, the
sequence (KLAKLAK).sub.2 (SEQ ID NO:3), (KLAKKLA).sub.2 (SEQ ID
NO:5), (KAAKKAA).sub.2 (SEQ ID NO:6), or (KLGKKLG).sub.3 (SEQ ID
NO:7), and, in one embodiment, includes the sequence
.sub.D(KLAKLAK).sub.2 (SEQ ID NO:3).
[0415] Antimicrobial peptides can have random coil conformations in
dilute aqueous solutions, yet high levels of helicity can be
induced by helix-promoting solvents and amphipathic media such as
micelles, synthetic bilayers or cell membranes. .alpha.-Helical
structures are well known in the art, with an ideal .alpha.-helix
characterized by having 3.6 residues per turn and a translation of
1.5 .ANG. per residue (5.4 .ANG. per turn; see Creighton, Proteins:
Structures and Molecular Properties W. H Freeman, New York (1984)).
In an amphipathic .alpha.-helical structure, polar and non-polar
amino acid residues are aligned into an amphipathic helix, which is
an .alpha.-helix in which the hydrophobic amino acid residues are
predominantly on one face, with hydrophilic residues predominantly
on the opposite face when the peptide is viewed along the helical
axis.
[0416] Antimicrobial peptides of widely varying sequence have been
isolated, sharing an amphipathic .alpha.-helical structure as a
common feature (Saberwal et al., Biochim. Biophys. Acta
1197:109-131 (1994)). Analogs of native peptides with amino acid
substitutions predicted to enhance amphipathicity and helicity
typically have increased antimicrobial activity. In general,
analogs with increased antimicrobial activity also have increased
cytotoxicity against mammalian cells (Maloy et al., Biopolymers
37:105-122 (1995)).
[0417] As used herein in reference to an antimicrobial peptide, the
term "amphipathic .alpha.-helical structure" means an .alpha.-helix
with a hydrophilic face containing several polar residues at
physiological pH and a hydrophobic face containing nonpolar
residues. A polar residue can be, for example, a lysine or arginine
residue, while a nonpolar residue can be, for example, a leucine or
alanine residue. An antimicrobial peptide having an amphipathic
.alpha.-helical structure generally has an equivalent number of
polar and nonpolar residues within the amphipathic domain and a
sufficient number of basic residues to give the peptide an overall
positive charge at neutral pH (Saberwal et al., Biochim. Biophys.
Acta 1197:109-131 (1994)). One skilled in the art understands that
helix-promoting amino acids such as leucine and alanine can be
advantageously included in an antimicrobial peptide (see, for
example, Creighton, supra, 1984). Synthetic, antimicrobial peptides
having an amphipathic .alpha.-helical structure are known in the
art, for example, as described in U.S. Pat. No. 5,789,542 to
McLaughlin and Becker.
[0418] It is understood by one skilled in the art of medicinal
oncology that these and other agents are useful therapeutic agents,
which can be used separately or together in the disclosed
compositions and methods. Thus, it is understood that a composition
can contain one or more of such therapeutic agents and that
additional components can be included as part of the composition,
if desired. As a non-limiting example, it can be desirable in some
cases to utilize an oligopeptide spacer between the homing molecule
and the therapeutic agent (Fitzpatrick and Garnett, Anticancer Drug
Des. 10:1-9 (1995)).
[0419] Other useful agents include thrombolytics, aspirin,
anticoagulants, painkillers and tranquilizers, beta-blockers,
ace-inhibitors, nitrates, rhythm-stabilizing drugs, and diuretics.
The disclosed compositions can use any of these or similar
agents.
[0420] In some embodiments, a composition can contain a cancer
chemotherapeutic agent. As used herein, a "cancer chemotherapeutic
agent" is a chemical agent that inhibits the proliferation, growth,
life-span or metastatic activity of cancer cells. Such a cancer
chemotherapeutic agent can be, without limitation, a taxane such as
docetaxel; an anthracyclin such as doxorubicin; an alkylating
agent; a vinca alkaloid; an anti-metabolite; a platinum agent such
as cisplatin or carboplatin; a steroid such as methotrexate; an
antibiotic such as adriamycin; a isofamide; or a selective estrogen
receptor modulator; an antibody such as trastuzumab.
[0421] Taxanes are chemotherapeutic agents useful in the
compositions. Useful taxanes include, without limitation, docetaxel
(Taxotere; Aventis Pharmaceuticals, Inc.; Parsippany, N.J.) and
paclitaxel (Taxol; Bristol-Myers Squibb; Princeton, N.J.). See, for
example, Chan et al., J. Clin. Oncol. 17:2341-2354 (1999), and
Paridaens et al., J. Clin. Oncol. 18:724 (2000).
[0422] A cancer chemotherapeutic agent useful in a composition also
can be an anthracyclin such as doxorubicin, idarubicin or
daunorubicin. Doxorubicin is a commonly used cancer
chemotherapeutic agent and can be useful, for example, for treating
breast cancer (Stewart and Ratain, In: "Cancer: Principles and
practice of oncology" 5th ed., chap. 19 (eds. DeVita, Jr., et al.;
J. P. Lippincott 1997); Harris et al., In "Cancer: Principles and
practice of oncology," supra, 1997). In addition, doxorubicin has
anti-angiogenic activity (Folkman, Nature Biotechnology 15:510
(1997); Steiner, In "Angiogenesis: Key principles-Science,
technology and medicine," pp. 449-454 (eds. Steiner et al.;
Birkhauser Verlag, 1992)), which can contribute to its
effectiveness in treating cancer.
[0423] An alkylating agent such as melphalan or chlorambucil also
can be a cancer chemotherapeutic agent useful in a composition.
Similarly, a vinca alkaloid such as vindesine, vinblastine or
vinorelbine; or an antimetabolite such as 5-fluorouracil,
5-fluorouridine or a derivative thereof can be a cancer
chemotherapeutic agent useful in a composition.
[0424] A platinum agent also can be a cancer chemotherapeutic agent
useful in the compositions. Such a platinum agent can be, for
example, cisplatin or carboplatin as described, for example, in
Crown, Seminars in Oncol. 28:28-37 (2001). Other cancer
chemotherapeutic agents useful in a composition include, without
limitation, methotrexate, mitomycin-C, adriamycin, ifosfamide and
ansamycins.
[0425] A cancer chemotherapeutic agent for treatment of breast
cancer and other hormonally-dependent cancers also can be an agent
that antagonizes the effect of estrogen, such as a selective
estrogen receptor modulator or an anti-estrogen. The selective
estrogen receptor modulator, tamoxifen, is a cancer
chemotherapeutic agent that can be used in a composition for
treatment of breast cancer (Fisher et al., J. Natl. Cancer Instit.
90:1371-1388 (1998)).
[0426] A therapeutic agent useful in a composition can be an
antibody such as a humanized monoclonal antibody. As an example,
the anti-epidermal growth factor receptor 2 (HER2) antibody,
trastuzumab (Herceptin; Genentech, South San Francisco, Calif.) is
a therapeutic agent useful in a composition for treating HER2/neu
overexpressing breast cancers (White et al., Annu. Rev. Med.
52:125-141 (2001)).
[0427] Useful therapeutic agents also can be a cytotoxic agent,
which, as used herein, can be any molecule that directly or
indirectly promotes cell death. Useful cytotoxic agents include,
without limitation, small molecules, polypeptides, peptides,
peptidomimetics, nucleic acid-molecules, cells and viruses. As
non-limiting examples, useful cytotoxic agents include cytotoxic
small molecules such as doxorubicin, docetaxel or trastuzumab;
antimicrobial peptides such as those described further below;
pro-apoptotic polypeptides such as caspases and toxins, for
example, caspase-8; diphtheria toxin A chain, Pseudomonas exotoxin
A, cholera toxin, ligand fusion toxins such as DAB389EGF, ricinus
communis toxin (ricin); and cytotoxic cells such as cytotoxic T
cells. See, for example, Martin et al., Cancer Res. 60:3218-3224
(2000); Kreitman and Pastan, Blood 90:252-259 (1997); Allam et al.,
Cancer Res. 57:2615-2618 (1997); and Osborne and Coronado-Heinsohn,
Cancer J. Sci. Am. 2:175 (1996). One skilled in the art understands
that these and additional cytotoxic agents described herein or
known in the art can be useful in the disclosed compositions and
methods.
[0428] In one embodiment, a therapeutic agent can be a therapeutic
polypeptide. As used herein, a therapeutic polypeptide can be any
polypeptide with a biologically useful function. Useful therapeutic
polypeptides encompass, without limitation, cytokines, antibodies,
cytotoxic polypeptides; pro-apoptotic polypeptides; and
anti-angiogenic polypeptides. As non-limiting examples, useful
therapeutic polypeptides can be a cytokine such as tumor necrosis
factor-.alpha. (TNF-.alpha.), tumor necrosis factor-.beta.
(TNF-.beta.), granulocyte macrophage colony stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF),
interferon-.alpha.. (IFN-.alpha.); interferon-.gamma.
(IFN-.gamma.), interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-3 (IL-3), interleukin-4 interleukin-6 (IL-6),
interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12
(IL-12), lymphotactin (LTN) or dendritic cell chemokine 1 (DC-CK1);
an anti-HER2 antibody or fragment thereof; a cytotoxic polypeptide
including a toxin or caspase, for example, diphtheria toxin A
chain, Pseudomonas exotoxin A, cholera toxin, a ligand fusion toxin
such as DAB389EGF or ricin; or an anti-angiogenic polypeptide such
as angiostatin, endostatin, thrombospondin, platelet factor 4;
anastellin; or one of those described further herein or known in
the art (see below). It is understood that these and other
polypeptides with biological activity can be a "therapeutic
polypeptide."
[0429] A therapeutic agent useful in a composition also can be an
anti-angiogenic agent. As used herein, the term "anti-angiogenic
agent" means a molecule that reduces or prevents angiogenesis,
which is the growth and development of blood vessels. The
compositions can be used to treat or diagnose any disease,
condition, or disorder associated with angiogenesis. For example,
macular degeneration and diabetic vascular complications can be
diagnosed and/or treated. A variety of anti-angiogenic agents can
be prepared by routine methods. Such anti-angiogenic agents
include, without limitation, small molecules; proteins such as
dominant negative forms of angiogenic factors, transcription
factors and antibodies; peptides; and nucleic acid molecules
including ribozymes, antisense oligonucleotides, and nucleic acid
molecules encoding, for example, dominant negative forms of
angiogenic factors and receptors, transcription factors, and
antibodies and antigen-binding fragments thereof. See, for example,
Hagedorn and Bikfalvi, Crit. Rev. Oncol. Hematol. 34:89-110 (2000),
and Kirsch et al., J. Neurooncol. 50:149-163 (2000).
[0430] Vascular endothelial growth factor (VEGF) has been shown to
be important for angiogenesis in many types of cancer, including
breast cancer angiogenesis in vivo (Borgstrom et al., Anticancer
Res. 19:4213-4214 (1999)). The biological effects of VEGF include
stimulation of endothelial cell proliferation, survival, migration
and tube formation, and regulation of vascular permeability. An
anti-angiogenic agent can be, for example, an inhibitor or
neutralizing antibody that reduces the expression or signaling of
VEGF or another angiogenic factor, for example, an anti-VEGF
neutralizing monoclonal antibody (Borgstrom et al., supra, 1999).
An anti-angiogenic agent also can inhibit another angiogenic factor
such as a member of the fibroblast growth factor family such as
FGF-1 (acidic), FGF-2 (basic), FGF-4 or FGF-5 (Slavin et al., Cell
Biol. Int. 19:431-444 (1995); Folkman and Shing, J. Biol. Chem.
267:10931-10934 (1992)) or an angiogenic factor such as
angiopoietin-1, a factor that signals through the endothelial
cell-specific Tie2 receptor tyrosine kinase (Davis et al., Cell
87:1161-1169 (1996); and Suri et al., Cell 87:1171-1180 (1996)), or
the receptor of one of these angiogenic factors. It is understood
that a variety of mechanisms can act to inhibit activity of an
angiogenic factor including, without limitation, direct inhibition
of receptor binding, indirect inhibition by reducing secretion of
the angiogenic factor into the extracellular space, or inhibition
of expression, function or signaling of the angiogenic factor.
[0431] A variety of other molecules also can function as
anti-angiogenic agents including, without limitation, angiostatin;
a kringle peptide of angiostatin; endostatin; anastellin,
heparin-binding fragments of fibronectin; modified forms of
antithrombin; collagenase inhibitors; basement membrane turnover
inhibitors; angiostatic steroids; platelet factor 4 and fragments
and peptides thereof; thrombospondin and fragments and peptides
thereof; and doxorubicin (O'Reilly et al., Cell 79:315-328 (1994));
O'Reilly et al., Cell 88:277-285 (1997); Homandberg et al., Am. J.
Path. 120:327-332 (1985); Homandberg et-al., Biochim. Biophys. Acta
874:61-71 (1986); and O'Reilly et al., Science 285:1926-1928
(1999)). Commercially available anti-angiogenic agents include, for
example, angiostatin, endostatin, metastatin and 2ME2 (EntreMed;
Rockville, Md.); anti-VEGF antibodies such as Avastin (Genentech;
South San Francisco, Calif.); and VEGFR-2 inhibitors such as
SU5416, a small molecule inhibitor of VEGFR-2 (SUGEN; South San
Francisco, Calif.) and SU6668 (SUGEN), a small molecule inhibitor
of VEGFR-2, platelet derived growth factor and fibroblast growth
factor I receptor. It is understood that these and other
anti-angiogenic agents can be prepared by routine methods and are
encompassed by the term "anti-angiogenic agent" as used herein.
[0432] The disclosed compositions and methods can be used to
diagnose and deliver targeted therapies for pulmonary diseases such
as pulmonary hypertension, interstitial lung disease, acute lung
injury (ALI), acute respiratory distress syndrome (ARDS), sepsis,
septic shock, sarcoidosis of the lung, pulmonary manifestations of
connective tissue diseases, including systemic lupus erythematosus,
rheumatoid arthritis, scleroderma, and polymyositis,
dermatomyositis, bronchiectasis, asbestosis, berylliosis,
silicosis, Histiocytosis X, pneumotitis, smoker's lung,
bronchiolitis obliterans, the prevention of lung scarring due to
tuberculosis and pulmonary fibrosis, other fibrotic diseases such
as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs;
inflammatory bowel disease, crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, atherosclerosis, ischemic
heart disease, vasculitis, neoplastic/metastatic/oncological
diseases (including cancer), pneumoconiosis, autoimmune diseases,
angiogenic diseases, wound healing, infections, trauma injuries and
systemic connective tissue diseases including systemic lupus
erythematosus, rheumatoid arthritis, scleroderma, polymyositis, and
dermatomyositis.
[0433] For example, if the disease is pulmonary hypertension and
the desired goal is targeted pulmonary arterial vasodilation, an
effective dose of tCAR peptide can be co-administered with a
minimal dose of systemic vasodilator to achieve targeted pulmonary
vasodilation and a significant decrease in pulmonary pressure with
minimal systemic hypotension.
[0434] Similarly, tCAR peptide can be co-administered with other
medications to increase therapeutic bioavailability, boost
therapeutic efficacy, and minimize side effects. tCAR may be
administered in a linear or cyclical form, or in any conformation
deemed physiologically appropriate as a means of conveying
treatment.
[0435] In addition to targeted vasodilation, we can also deliver
targeted anti-coagulation. For example, in a disease like acute
lung injury, which is often marked by pulmonary intra-alveolar
coagulation, targeted anti-coagulation can be delivered to the
affected pulmonary area by co-administering an effective dose of
tCAR with an anti-coagulant such as tissue factor pathway inhibitor
(TFPI) or site-inactivated factor VIIa (Welty-Wolf et al., 2001) in
a minimal dose to achieve targeted pulmonary anticoagulation with
minimal changes in clotting ability over the areas of the body not
undergoing thrombosis. Selective pulmonary anti-coagulation can
also be utilized to treat other pulmonary diseases marked by
pulmonary thrombosis such as pulmonary hypertension, lung
transplant rejection and others.
[0436] In a disease like chronic obstructive pulmonary disease,
which is often marked by shortness of breath, CAR peptide can be
co-administered to boost the effective concentration and potency of
drugs to relax airway smooth muscles such as long lasting B-2
agonists such as salmeterol or formoterol (Richter, et al., 2002).
Many pulmonary diseases are often marked by a decrease in
glutathione (GSH), a powerful antioxidant (Morris and Bernard,
1994). CAR peptide can be co-administered with N-Acetylcysteine
(NAC), a glutathione precursor, in diseases like pulmonary
fibrosis, PAH, ALI, and other pulmonary disorders to boost GSH
production and scavenge reactive oxidants often found in pulmonary
diseases. GSH may also serve to dampen the inflammatory immune
response by binding to triggering receptor expressed on myeloid
cells 1 (TREM1) and diminishing monocyte/macrophage- and
neutrophil-mediated inflammatory responses. Co-administration of
tCAR with NAC can serve to lessen the severe inflammatory immune
response that often characterizes severe pulmonary and fibrotic
diseases like ALI, pulmonary hypertension, autoimmune diseases and
many other conditions.
[0437] The levels of antioxidants such as Superoxide Dismutase
(SOD) (Rosenfeld, et al., 1996), or synthetic superoxide dismutase
mimetics like EUK-8 (Gonzalez et al., 1996) can be increased
through co-administration of tCAR.
[0438] Treatments for pulmonary diseases like pulmonary fibrosis,
PAH and ALI can also be improved by co-administering tCAR with
TGF-.beta. inhibitors like decorin. Decorin, which has been
previously enhanced through direct conjugation with CAR (Jarvinen
and Ruoslahti, 2010), can also be co-administered with tCAR to
achieve the benefits of targeting without direct conjugation
between the CAR and decorin molecules.
[0439] In pulmonary hypertension, pulmonary fibrosis and other
pulmonary diseases, the benefits of endothelin (ET-1) receptor
antagonists (Kuklin et al., 2004), prostacyclin derivatives
(Olschewski et al., 1999), phosphodiesterase type 5 inhibitors
(Kanthapillai et al., 2004) and oncological agents such as imatinib
(Ghofrani et al., 2005) (Aono et al., 2005) can be increased for
patients through the co-administration of tCAR.
[0440] Other pulmonary and fibrotic disease treatments such as
Ketoconazole which inhibits thromboxane and leukotriene synthesis
(Sinuff et al., 1999) can be improved in its efficacy while
minimizing side effects through co-administration with tCAR.
[0441] Newer therapeutic approaches such as small interfering RNA
(siRNA), and microRNA (miRNA) therapies (Wurdinger and Costa, 2007)
can also be improved and side effects minimized through the
selective targeting of diseased tissue through the
co-administration of tCAR.
[0442] The co-composition or cargo composition can also have one or
more isotopes. Such isotopes can be useful, for example, as a
therapeutic agent, as a detectable agent, or both. Examples of
useful isopes include Lutetium-177 (.sup.177Lu), Rhenium-188
(.sup.188Re), Gallium-68 (.sup.68Ga), Yttrium-90 (.sup.90Y),
Technetium-99m (.sup.99mTc), Holmium-166 (.sup.166Ho), Iodine-131
(.sup.131I), Indium-111 (.sup.111In), Flourine-18 (.sup.18F),
Carbon-11 (.sup.11C), Nitrogen-13 (.sup.13N), Oxygen-15 (.sup.15O),
Bromine-75 (.sup.75Br), Bromine-76 (.sup.76Br), Iodine-124
(.sup.124I), Thalium-201 (.sup.201Tl), Technetium-99 (.sup.99Tc),
and Iodine-123 (.sup.123I).
[0443] The co-composition or cargo composition can also comprise a
detectable agent. A variety of detectable agents are useful in the
disclosed methods. As used herein, the term "detectable agent"
refers to any molecule which can be detected. Useful detectable
agents include moieties that can be administered in vivo and
subsequently detected. Detectable agents useful in the disclosed
compositions and imaging methods include yet are not limited to
radiolabels and fluorescent molecules. The detectable agent can be,
for example, any moiety that facilitates detection, either directly
or indirectly, preferably by a non-invasive and/or in vivo
visualization technique. For example, a detectable agent can be
detectable by any known imaging techniques, including, for example,
a radiological technique. Detectable agents can include, for
example, a contrast agent. The contrast agent can be, for example,
Feridex. In some embodiments, for instance, the detectable agent
comprises a tantalum compound. In some embodiments, the detectable
agent comprises iodine, such as radioactive iodine. In some
embodiments, for instance, the detectable agent comprises an
organic iodo acid, such as iodo carboxylic acid, triiodophenol,
iodoform, and/or tetraiodoethylene. In some embodiments, the
detectable agent comprises a non-radioactive detectable agent,
e.g., a non-radioactive isotope. For example, iron oxide and Gd can
be used as a non-radioactive detectable agent in certain
embodiments. Detectable agents can also include radioactive
isotopes, enzymes, fluorophores, and quantum dots (Qdot.RTM.). For
example, the detection moiety can be an enzyme, biotin, metal, or
epitope tag. Other known or newly discovered detectable markers are
contemplated for use with the provided compositions. In some
embodiments, for instance, the detectable agent comprises a barium
compound, e.g., barium sulfate.
[0444] The detectable agent can be (or the co-composition or cargo
composition can include) one or more imaging agents. Examples of
imaging agents include radiologic contrast agent, such as
diatrizoic acid sodium salt dihydrate, iodine, and barium sulfate,
a fluorescing imaging agent, such as Lissamine Rhodamine PE, a
fluorescent or non-fluorescent stain or dye, for example, that can
impart a visible color or that reflects a characteristic spectrum
of electromagnetic radiation at visible or other wavelengths, for
example, infrared or ultraviolet, such as Rhodamine, a
radioisotope, a positron-emitting isotope, such as .sup.18F or
.sup.124I (although the short half-life of a positron-emitting
isotope may impose some limitations), a metal, a ferromagnetic
compound, a paramagnetic compound, such as gadolinium, a
superparamagnetic compound, such as iron oxide, and a diamagnetic
compound, such as barium sulfate. Imaging agents can be selected to
optimize the usefulness of an image produced by a chosen imaging
technology. For example, the imaging agent can be selected to
enhance the contrast between a feature of interest, such as a
gastrointestinal polyp, and normal gastrointestinal tissue. Imaging
can be accomplished using any suitable imaging techniques such as
X-Ray, computed tomography (CT), MRI, Positron Emission Tomography
(PET) or SPECT. In some forms, the co-composition or cargo
composition can be coupled to a nuclear medicine imaging agent such
as Indium-III or Technetium-99, to PET imaging agents, or to MRI
imaging agents such as nanoparticles.
[0445] Examples of imaging techniques include magnetic resonance
imaging (MRI), computerized tomography (CT), single photon emission
computerized tomography (SPECT), and positron emission tomography
(PET). Imaging agents generally can be classified as either being
diagnostic or therapeutic in their application. Because of
radiation's damaging effect on tissues, it is useful to target the
biodistribution of radiopharmaceuticals as accurately as possible.
PET can use imaging agents labeled with, for example, the
positron-emitters such as .sup.18F, .sup.11C, .sup.13N and
.sup.15O, .sup.75Br, .sup.76Br and .sup.124I. SPECT can use imaging
agents labeled with, for example, the single-photon-emitters such
as .sup.201Tl, .sup.99Tc, .sup.123I, and .sup.131I.
[0446] Glucose-based and amino acid-based compounds can be used as
imaging agents. Amino acid-based compounds are more useful in
analyzing tumor cells, due to their faster uptake and incorporation
into protein synthesis. Of the amino acid-based compounds,
.sup.11C- and .sup.18F-containing compounds have been used with
success. .sup.11C-containing radiolabeled amino acids suitable for
imaging include, for example, L-[1-.sup.11C]leucine (Keen et al. J.
Cereb. Blood Flow Metab. 1989 (9):429-45), L-[1-.sup.11C]tyrosine
(Wiesel et al. J. Nucl. Med. 1991 (32):2041-49),
L-[methyl-.sup.11C]methionine (Comar et al. Eur. J. Nucl. Med. 1976
(1):11-14) and L-[1-.sup.11C]methionine (Bolster et al. Appl.
Radiat. Isot. 1986 (37):1069-70).
[0447] PET involves the detection of gamma rays in the form of
annihilation photons from short-lived positron emitting radioactive
isotopes including, but not limited to, .sup.18F with a half-life
of approximately 110 minutes, .sup.11C with a half-life of
approximately 20 minutes, .sup.13N with a half-life of
approximately 10 minutes and .sup.15O with a half-life of
approximately 2 minutes, using the coincidence method. For PET
imaging studies, compounds such as [.sup.11C]meta-hydroxyephedrine
(HED) and 2-[.sup.18F]fluoro-2-deoxy-D-glucose (FDG) can be used.
SPECT can use longer-lived isotopes including, but not limited to,
.sup.99mTc with a half-life of approximately 6 hours and .sup.201Tl
with a half-life of approximately 74 hours. Radio-iodinated
meta-iodobenzylguanidine (MIBG) is a radiotracing agent that can be
used in nuclear medicine imaging studies.
[0448] The disclosed tCAR compositions and co-compositions and
cargo compositions can be administered in vivo in a
pharmaceutically acceptable carrier. By "pharmaceutically
acceptable" is meant a material that is not biologically or
otherwise undesirable, i.e., the material can be administered to a
subject, along with the nucleic acid or vector, without causing any
undesirable biological effects or interacting in a deleterious
manner with any of the other components of the pharmaceutical
composition in which it is contained. The carrier would naturally
be selected to minimize any degradation of the active ingredient
and to minimize any adverse side effects in the subject, as would
be well known to one of skill in the art. The materials can be in
solution, suspension (for example, incorporated into
microparticles, liposomes, or cells).
[0449] The tCAR compositions and co-compositions and cargo
compositions can be used therapeutically in combination with a
pharmaceutically acceptable carrier. Suitable carriers and their
formulations are described in Remington: The Science and Practice
of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,
Easton, Pa. 1995. Typically, an appropriate amount of a
pharmaceutically-acceptable salt is used in the formulation to
render the formulation isotonic. Examples of the
pharmaceutically-acceptable carrier include, but are not limited
to, saline, Ringer's solution and dextrose solution. The pH of the
solution is preferably from about 5 to about 8, and more preferably
from about 7 to about 7.5. Further carriers include sustained
release preparations such as semipermeable matrices of solid
hydrophobic polymers containing the antibody, which matrices are in
the form of shaped articles, e.g., films, liposomes or
microparticles. It will be apparent to those persons skilled in the
art that certain carriers can be more preferable depending upon,
for instance, the route of administration and concentration of
composition being administered.
[0450] The preparation can be administered to a subject or organism
per se, or in a pharmaceutical composition where it is mixed with
suitable carriers or excipients.
[0451] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to a
subject or organism.
[0452] Herein the term "active ingredient" refers to the
preparation accountable for the biological effect. For example tCAR
peptides, tCAR compositions, tCAR conjugates, tCAR molecules, tCAR
proteins, compositions, co-compositions, and cargo compositions
that have a biological effect can be considered active
ingredients.
[0453] As used herein, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which can be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to a subject or organism and does not
abrogate the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0454] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0455] Techniques for formulation and administration of drugs may
be found in Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0456] Any suitable route of administration can be used for the
disclosed compositions. Routes of administration can, for example,
include topical, enteral, local, systemic, or parenteral. For
example, administration can be intratumoral, peritumoral,
epicutaneous, inhalational, enema, conjunctival, eye drops, ear
drops, alveolar, nasal, intranasal, vaginal, intravaginal,
transvaginal, enteral, oral, intraoral, transoral, intestinal,
rectal, intrarectal, transrectal, injection, infusion, intravenous,
intraarterial, intramuscular, intracerebral, intraventricular,
intracerebroventricular, intracardiac, subcutaneous, intraosseous,
intradermal, intrathecal, intraperitoneal, intravesical,
intracavernosal, intramedullar, intraocular, intracranial,
transdermal, transmucosal, transnasal, inhalational,
intracisternal, epidural, peridural, intravitreal, etc.
[0457] For homing to cells and tissue, particularly suitable routes
of administration include parenteral, either local or systemic. For
example, particularly suitable routes of administration for homing
to cells and tissues include intravenous, injection, infusion,
intraarterial, intramuscular, intratumoral, peritumoral,
intracerebral, intraventricular, intracerebroventricular,
intracardiac, subcutaneous, intraosseous, intradermal, intrathecal,
intraperitoneal, intravesical, intramedullar, intraocular,
intracranial, intracisternal, epidural, peridural, and
intravitreal. The disclosed compositions can be used in and with
any other procedure. For example, the disclosed compositions can be
administered as part of HIPEC therapy. In HIPEC a heated sterile
solution containing a composition of interest is continuously
circulated throughout the peritoneal cavity.
[0458] Pharmaceutical compositions can be manufactured by processes
well known in the art, e.g., by means of conventional mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes.
[0459] Pharmaceutical compositions for use in the disclosed methods
thus can be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active ingredients
into preparations which, can be used pharmaceutically. Proper
formulation is dependent upon the route of administration
chosen.
[0460] For injection, the active ingredients can be formulated in
aqueous solutions, preferably in physiologically compatible buffers
such as Hank's solution, Ringer's solution, or physiological salt
buffer. For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0461] The preparations described herein can be formulated for
parenteral administration, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multidose containers with
optionally, an added preservative. The compositions can be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0462] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients can be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions can contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension can also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0463] Alternatively, the active ingredient can be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0464] The disclosed compositions can be provided in any suitable
formulation. For example, solid, liquid, solution, gel, slow
release, timed release, etc.
[0465] Pharmaceutical compositions for use in the disclosed methods
include compositions wherein the active ingredients are contained
in an amount effective to achieve the intended purpose. More
specifically, a therapeutically effective amount means an amount of
active ingredients effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subject being
treated.
[0466] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0467] For any preparation used in the disclosed methods, the
therapeutically effective amount or dose can be estimated initially
from in vitro and cell culture assays. For example, a dose can be
formulated in animal models to achieve a desired circulating
antibody concentration or titer. Such information can be used to
more accurately determine useful doses in humans.
[0468] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl et al in The Pharmacological Basis of Therapeutics, Ch.
1 p. 1. (1975)).
[0469] Dosage amount and interval can be adjusted individually to
provide plasma of antibodies which are sufficient to prevent or
reduce viral entry (minimal effective concentration, MEC). The MEC
will vary for each preparation, but can be estimated from in vitro
data. Dosages necessary to achieve the MEC will depend on
individual characteristics and route of administration. Binding
assays can be used to determine plasma concentrations.
[0470] Dosage intervals can also be determined using the MEC value.
Preparations should be administered using a regimen, which
maintains plasma levels, target site measurments, or other suitable
measure above the MEC for 10-90% of the time, preferable between
30-90% and most preferably 50-90%.
[0471] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected, diminution of the
disease state is achieved, or other therapeutic effect is
achieved.
[0472] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0473] The co-composition or cargo composition can be a
microparticle or a nanoparticle, such as a nanosphere, nanoshell,
nanoworm, heat generating nanoshell, and the like. As used herein,
"nanoshell" is a nanoparticle having a discrete dielectric or
semi-conducting core section surrounded by one or more conducting
shell layers. U.S. Pat. No. 6,530,944 is hereby incorporated by
reference herein in its entirety for its teaching of the methods of
making and using metal nanoshells. Nanoshells can be formed with,
for example, a core of a dielectric or inert material such as
silicon, coated with a material such as a highly conductive metal
which can be excited using radiation such as near infrared light
(approximately 800 to 1300 nm). Upon excitation, the nanoshells
emit heat. The resulting hyperthermia can kill the surrounding
cell(s) or tissue. The combined diameter of the shell and core of
the nanoshells ranges from the tens to the hundreds of nanometers.
Near infrared light is advantageous for its ability to penetrate
tissue. Other types of radiation can also be used, depending on the
selection of the nanoparticle coating and targeted cells. Examples
include x-rays, magnetic fields, electric fields, and ultrasound.
The particles can also be used to enhance imaging, especially using
infrared diffuse photon imaging methods. Targeting molecules can be
antibodies or fragments thereof, ligands for specific receptors, or
other proteins specifically binding to the surface of the cells to
be targeted.
[0474] Fatty acids (i.e., lipids) that can be conjugated to the
disclosed tCAR compositions and co-compositions and cargo
compositions include those that allow the efficient incorporation
of the peptide into liposomes. Generally, the fatty acid is a polar
lipid. Thus, the fatty acid can be a phospholipid. The provided
compositions can comprise either natural or synthetic phospholipid.
The phospholipids can be selected from phospholipids containing
saturated or unsaturated mono or disubstituted fatty acids and
combinations thereof. These phospholipids can be, for example,
dioleoylphosphatidylcholine, dioleoylphosphatidylserine,
dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol,
dioleoylphosphatidic acid, palm itoyloleoylphosphatidylcholine,
palmitoyloleoylphosphatidylserine,
palmitoyloleoylphosphatidylethanolamine,
palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic
acid, palmitelaidoyloleoylphosphatidylcholine,
palmitelaidoyloleoylphosphatidylserine,
palmitelaidoyloleoylphosphatidylethanolamine,
palmitelaidoyloleoylphosphatidylglycerol,
palmitelaidoyloleoylphosphatidic acid,
myristoleoyloleoylphosphatidylcholine,
myristoleoyloleoylphosphatidylserine,
myristoleoyloleoylphosphatidylethanoamine,
myristoleoyloleoylphosphatidylglycerol,
myristoleoyloleoylphosphatidic acid,
dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine,
dilinoleoylphosphatidylethanolamine,
dilinoleoylphosphatidylglycerol, dilinoleoylphosphatidic acid,
palmiticlinoleoylphosphatidylcholine,
palmiticlinoleoylphosphatidylserine,
palmiticlinoleoylphosphatidylethanolamine,
palmiticlinoleoylphosphatidylglycerol,
palmiticlinoleoylphosphatidic acid. These phospholipids may also be
the monoacylated derivatives of phosphatidylcholine
(lysophophatidylidylcholine), phosphatidylserine
(lysophosphatidylserine), phosphatidylethanolamine
(lysophosphatidylethanolamine), phophatidylglycerol
(lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidic
acid). The monoacyl chain in these lysophosphatidyl derivatives may
be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or
myristoleoyl. The phospholipids can also be synthetic. Synthetic
phospholipids are readily available commercially from various
sources, such as AVANTI Polar Lipids (Albaster, Ala.); Sigma
Chemical Company (St. Louis, Mo.). These synthetic compounds may be
varied and may have variations in their fatty acid side chains not
found in naturally occurring phospholipids. The fatty acid can have
unsaturated fatty acid side chains with C14, C16, C18 or C20 chains
length in either or both the PS or PC. Synthetic phospholipids can
have dioleoyl (18:1)-PS; palmitoyl (16:0)-oleoyl (18:1)-PS,
dimyristoyl (14:0)-PS; dipalmitoleoyl (16:1)-PC, dipalmitoyl
(16:0)-PC, dioleoyl (18:1)-PC, palmitoyl (16:0)-oleoyl (18:1)-PC,
and myristoyl (14:0)-oleoyl (18:1)-PC as constituents. Thus, as an
example, the provided compositions can comprise palmitoyl 16:0.
[0475] The other molecules, elements, moieties, etc. can be
covalently linked to or non-covalently associated with, for
example, the disclosed co-compositions, cargo compositions, tCAR
composition, protein, peptide, or amino acid sequence. Such
molecules, elements, moieties, etc. can be linked, for example, to
the amino terminal end of the disclosed protein, peptide, amino
acid sequence, or tCAR peptide; to an internal amino acid of the
disclosed protein, peptide, amino acid sequence, or tCAR peptide;
to the carboxy terminal end of the disclosed protein, peptide, or
amino acid sequence; to the protein, peptide, amino acid sequence
on the N terminal side of the tCAR peptide; via a linker to the
disclosed protein, peptide, amino acid sequence, or tCAR peptide;
or a combination. The disclosed tCAR compositions can further
comprise a linker connecting such molecules, elements, moieties,
etc. and disclosed tCAR composition, protein, peptide, amino acid
sequence, or tCAR peptide. The disclosed tCAR composition, protein,
peptide, amino acid sequence, or tCAR peptide can also be
conjugated to a coating molecule such as bovine serum albumin (BSA;
see Tkachenko et al., (2003) J Am Chem Soc, 125, 4700-4701) that
can be used to coat nanoparticles, nanoworms, nanoshells, and the
like with the protein, peptide, amino acid sequence, or tCAR
peptide.
[0476] Protein crosslinkers that can be used to crosslink other
molecules, elements, moieties, etc. to the disclosed
co-compositions, cargo compositions, tCAR compositions, proteins,
peptides, amino acid sequences, etc. are known in the art and are
defined based on utility and structure and include DSS
(Disuccinimidylsuberate), DSP (Dithiobis(succinimidylpropionate)),
DTSSP (3,3'-Dithiobis (sulfosuccinimidylpropionate)), SULFO BSOCOES
(Bis[2-(sulfosuccinimdooxycarbonyloxy)ethyl]sulfone), BSOCOES
(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST
(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO
EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene
glycolbis(sulfosuccinimidylsuccinate)), DPDPB
(1,2-Di[3'-(2'-pyridyldithio)propionamido]butane), BSSS
(Bis(sulfosuccinimdyl)suberate), SMPB
(Succinimdyl-4-(p-maleimidophenyl)butyrate), SULFO SMPB
(Sulfosuccinimdyl-4-(p-maleimidophenyl)butyrate), MBS
(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS
(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB
(N-Succinimidyl(4-iodoacetyl)aminobenzoate), SULFO SIAB
(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC
(Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate),
SULFO SMCC
(Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate),
NHS LC SPDP
(Succinimidyl-6-[3-(2-pyridyldithio)propionamido)hexanoate), SULFO
NHS LC SPDP
(Sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido)hexanoate),
SPDP (N-Succinimdyl-3-(2-pyridyldithio)propionate), NHS
BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHS IODOACETATE
(N-Hydroxysuccinimidyliodoacetate), MPBH
(4-(N-Maleimidophenyl)butyric acid hydrazide hydrochloride), MCCH
(4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid hydrazide
hydrochloride), MBH (m-Maleimidobenzoic acid
hydrazidehydrochloride), SULFO
EMCS(N-(epsilon-Maleimidocaproyloxy)sulfosuccinimide),
EMCS(N-(epsilon-Maleimidocaproyloxy)succinimide), PMPI
(N-(p-Maleimidophenyl)isocyanate), KMUH
(N-(kappa-Maleimidoundecanoic acid) hydrazide), LC SMCC
(Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate-
)), SULFO GMBS (N-(gamma-Maleimidobutryloxy)sulfosuccinimide
ester), SMPH
(Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS
(N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS
(N-(gamma-Maleimidobutyrloxy)succinimide), DMP
(Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate
hydrochloride), MHBH (Wood's Reagent; Methyl-p-hydroxybenzimidate
hydrochloride, 98%), DMA (Dimethyladipimidate hydrochloride).
[0477] Components of co-compositions or cargo composition can also
be coupled using, for example, maleimide coupling. By way of
illustration, components can be coupled to lipids by coupling to,
for example,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene
glycol).sub.2000; DSPE-PEG.sub.2000-maleimide] (Avanti Polar
Lipids) by making use of a free cysteine sulfhydryl group on the
component. The reaction can be performed, for example, in aqueous
solution at room temperature for 4 hours. This coupling chemistry
can be used to couple components of co-compositions and cargo
compositions.
[0478] The disclosed compounds, components, and compositions can
also be coupled using, for example, amino group-functionalized
dextran chemistry. Particles, such as, for example, nanoparticles,
nanoworms, and micelles, can be coated with amino group
functionalized dextran. Attachment of PEG to aminated particles
increases the circulation time, presumably by reducing the binding
of plasma proteins involved in opsonization (Moghimi et al., 2001).
The particles can have surface modifications, for example, for
reticuloendothelial system avoidance (PEG) and homing (homing
molecules), endosome escape (pH-sensitive peptide; for example,
Pirello et al., 2007), a detectable agent, a therapeutic compound,
or a combination. To accommodate all these functions on one
particle, optimization studies can be conducted to determine what
proportion of the available linking sites at the surface of the
particles any one of these elements should occupy to give the best
combination of targeting and payload delivery. The cell
internalization and/or tissue penetration of such co-compositions
and cargo compositions can be mediated by the disclosed tCAR
peptides, amino acid sequences, proteins, molecules, conjugates,
and compositions.
[0479] The tCAR peptides, amino acid sequences, proteins,
molecules, conjugates, and compositions themselves can be coupled
to other components as disclosed herein using any known technique
or the techniques described herein (although generally not, as
described elsewhere herein, to the disclosed co-compositions). A
maleimide function can also be used as a coupling group. These
chemistries can be used to couple tCAR peptides, amino acid
sequences, proteins, molecules, conjugates, and compositions to
each other and to other components.
[0480] tCAR peptides, amino acid sequences, and proteins can also
be coupled to other components using, for example, maleimide
coupling. By way of illustration, tCAR peptides, amino acid
sequences, and proteins can be coupled to lipids by coupling to,
for example,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene
glycol).sub.2000; DSPE-PEG.sub.2000-maleimide] (Avanti Polar
Lipids) by making use of a free cysteine sulfhydryl group on the
tCAR peptides, amino acid sequence, or protein. The reaction can be
performed, for example, in aqueous solution at room temperature for
4 hours. This coupling chemistry can be used to couple the
disclosed tCAR peptides, amino acid sequences, and proteins to many
other components, molecules and compositions.
[0481] By "treatment" is meant the medical management of a patient
with the intent to cure, ameliorate, stabilize, or prevent a
disease, pathological condition, or disorder. This term includes
active treatment, that is, treatment directed specifically toward
the improvement of a disease, pathological condition, or disorder,
and also includes causal treatment, that is, treatment directed
toward removal of the cause of the associated disease, pathological
condition, or disorder. In addition, this term includes palliative
treatment, that is, treatment designed for the relief of symptoms
rather than the curing of the disease, pathological condition, or
disorder; preventative treatment, that is, treatment directed to
minimizing or partially or completely inhibiting the development of
the associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0482] As used herein, "subject" includes, but is not limited to,
animals, plants, bacteria, viruses, parasites and any other
organism or entity that has nucleic acid. The subject may be a
vertebrate, more specifically a mammal (e.g., a human, horse, pig,
rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig
or rodent), a fish, a bird or a reptile or an amphibian. In
particular, pets and livestock can be a subject. The subject can be
an invertebrate, such as a worm or an arthropod (e.g., insects and
crustaceans). The term does not denote a particular age or sex.
Thus, adult and newborn subjects, as well as fetuses, whether male
or female, are intended to be covered. A patient refers to a
subject afflicted with a disease or disorder. The term "patient"
includes human and veterinary subjects. In the context of
endometriosis and endometriosis cells, it is understood that a
subject is a subject that has or can have endometriosis and/or
endometriosis cells.
[0483] Tissue-penetrating tCAR peptides can be used to augment
tissue imaging and treatment with drugs. The effect of tCAR
peptides on imaging can be tested. For example, optical imaging
with, for example, near infrared fluorphores using a Kodak IN VIVO
Fx imager and Li-Cor Odyssey imager (e.g. Simberg et al., 2007;
Sugahara et al., 2009), and MRI imaging can be used. For MRI
imaging, the co-composition or cargo composition can be an MRI
contrast agent such as Feridex iron oxide nanoparticles and
gadolinium compounds. These compounds can be injected into
tumor-bearing mice, for example, with and without a tumor-homing
tCAR peptide or a combination of peptides, followed by imaging. The
results can be use to determine effectiveness of treatments and to
assess different treatment protocols for using tCAR peptides with
therapeutics as the co-composition or cargo composition.
[0484] Combinations of different tCAR peptides and different
co-compositions and/or cargo compositions can be tested for optimal
accumulation and distribution of the co-composition or cargo
composition in the target cells and tissue by, for example, varying
the dose of the drug and using the dose of the peptide that gives
the maximal effect. The disclosed results show that tCAR-drug
combinations can reduce the amount of drug needed and therefore,
the side effects, while producing the same anti-tumor effect. tCAR
peptides can also produce effects not achievable by using the
co-composition or cargo composition alone. For example, use of tCAR
peptides can allow higher concentrations of the co-composition or
cargo composition in cells and tissues that is otherwise possible.
In such cases, the effectiveness of the co-composition or cargo
composition can be beyond that obtainable with conventional
therapy.
[0485] As defined herein, a C-terminal element (CendR element)
refers to either an arginine, a lysine, or a lysine-glycine (for a
type 1 CendR element), or a histidine or an amino acid sequence
having the sequence X.sub.1X.sub.2X.sub.3X.sub.4, where X.sub.1 can
be R, K or H, where X.sub.4 can be R, K, H, or KG, and where
X.sub.2 and X.sub.3 can each be, independently, any amino acid (for
a type 2 CendR element).
[0486] Type 1 CendR elements are a C-terminal arginine, a
C-terminal lysine, or a C-terminal lysine-glycine pair, where
glycine is at the furthest C-terminal position. In other words, in
the case where a lysine is on the C terminus end, the CendR element
can remain functional with a glycine on the C terminus side of the
lysine. However, it is not necessary to have glycine on the end in
order for the lysine residue to be functional as a C-terminal
element, so that lysine can be present without glycine and still be
functional. The converse is not true, however, in that glycine
cannot function as a C-terminal element without the presence of
lysine adjacent to it. Arginine does not require either lysine or
glycine to function as a C-terminal element, as long as it remains
in the furthest C-terminal position.
[0487] Type 2 CendR elements are C-terminal histidine and amino
acid sequences having the sequence X.sub.1X.sub.2X.sub.3X.sub.4,
where X.sub.1 can be R, K or H, where X.sub.4 can be R, K, H, or
KG, and where X.sub.2 and X.sub.3 can each be, independently, any
amino acid. Such CendR elements can be referred to as type 2 CendR
elements. The X.sub.2 and X.sub.3 amino acids can be selected for
specific purposes. For example, X.sub.2, X.sub.3, or both can be
chosen to form all or a portion of a protease recognition sequence.
This would be useful, for example, to specify or enable cleavage of
a peptide having the CendR element as a latent or cryptic CendR
element that is activated by cleavage following the X.sub.4 amino
acid. The X.sub.1, X.sub.2 and X.sub.3 amino acids can also be
selected, for example, to recruit additional proteins to NRP-1
molecules at the cell surface. This can be applied, for example, to
modulate the selectivity and internalization and/or tissue
penetration potency of CendR elements (and the compositions,
conjugates, proteins, and peptides containing CendR elements). The
X.sub.2 and X.sub.3 amino acids can also be selected to prevent
protease cleavage within the X.sub.1-X.sub.4 motif. For example,
X.sub.2 and/or X.sub.3 can be proline, which reduces or eliminates
protease cleavage, such as by carboxypeptidase, between the proline
and the next downstream amino acid. As another example, one or more
of the bonds between X.sub.1, X.sub.2, X.sub.3, and/or X.sub.4 can
be modified to reduce or eliminate protease cleavage at those
bonds. Optionally, certain amino acids can also be excluded from
use for X.sub.2, X.sub.3, or both. For example, if desired, G and D
can be excluded from simultaneous use as X.sub.2 and X.sub.3,
respectively. Some type 2 CendR elements can also be described as
R/K/HXXR/K/H (SEQ ID NO:20), R/KXXR/K (SEQ ID NO:23), and R/K/HXXKG
(SEQ ID NO:21).
[0488] For the sake of convenience, amino acid motifs that would
constitute a CendR element if an arginine, lysine, lysine-glycine
pair, or histidine were at the C-terminus and where the exposure in
the future of the arginine, lysine, lysine-glycine pair, or
histidine at the C-terminus is planned or intended, can be referred
to as CendR elements or latent CendR elements.
[0489] CendR elements are described in U.S. Patent Application
Publication Nos. 20090226372, 20090226372, 20090246133, and
20100322862. U.S. Patent Application Publication Nos. 20090226372,
20090226372, 20090246133, and 20100322862 are hereby incorporated
herein by reference in their entirety, and specifically for their
description of CendR elements.
EXAMPLES
[0490] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
disclosure. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
A. Example 1
Targeting of Pulmonary Arterial Hypertension with CAR Pepetides
[0491] 1. Introduction
[0492] Pulmonary arterial hypertension (PAH) is a disease of the
pulmonary vasculature defined by an elevated pulmonary vascular
resistance, which eventually leads to right heart failure and
premature death. The cause of this disease remains unknown. PAH can
be idiopathic (IPAH) or associated with other conditions or
exposures (secondary pulmonary hypertension), including connective
tissue diseases, HIV infection, portal hypertension, and
anorexigenic drug ingestion.
[0493] Most cases of severe PAH, especially IPAH, are associated
with aberrant proliferation of pulmonary arterial endothelial cells
and smooth muscle cells, leading to narrowing or even obliteration
of the precapillary pulmonary vessel lumen (Humbert, M. et al.
Cellular and molecular pathobiology of pulmonary arterial
hypertension. J Am Coll Cardiol 43, 13S-24S (2004)). Thus, intimal
and medial thickening of resistant arteries is typically found in
the lesions. The adventitia is often markedly remodeled in patients
with certain forms of collagen vascular diseases associated with
severe PAH, most notably scleroderma (Cool, C. D., et al. Pulmonary
hypertension: cellular and molecular mechanisms. Chest 128,
565S-571S (2005)). Inflammatory mechanisms appear to play a
significant role in pathogenesis and progression of PAH. The
involvement of leukocytes, such as macrophages and lymphocytes, in
complex lesions of PAH has been described (Tuder, et al. Exuberant
endothelial cell growth and elements of inflammation are present in
plexiform lesions of pulmonary hypertension. Am J Pathol 144,
275-85 (1994); Dorfmuller, P., et al. Inflammation in pulmonary
arterial hypertension. Eur Respir J 22, 358-63 (2003)).
[0494] The current therapeutic approaches for PAH consist of the
administration of a variety of systemic vasodilators, which reduce
pulmonary vascular resistance in some patients. However, the
systemic use of vasodilators can produce adverse effects such as
hypotension, impaired intrapulmonary gas exchange, and depressed
cardiac function and even death (McLaughlin, V. V. et al. ACCF/AHA
2009 expert consensus document on pulmonary hypertension: a report
of the American College of Cardiology Foundation Task Force on
Expert Consensus Documents and the American Heart Association:
developed in collaboration with the American College of Chest
Physicians, American Thoracic Society, Inc., and the Pulmonary
Hypertension Association. Circulation 119, 2250-94 (2009)). Drugs
such as endothelin receptor antagonist may cause serious liver
damage (McLaughlin, V. V. et al. ACCF/AHA 2009 expert consensus
document on pulmonary hypertension: a report of the American
College of Cardiology Foundation Task Force on Expert Consensus
Documents and the American Heart Association: developed in
collaboration with the American College of Chest Physicians,
American Thoracic Society, Inc., and the Pulmonary Hypertension
Association. Circulation 119, 2250-94 (2009)). These limitations of
the current PAH regimens necessitate the development of
target-specific interventions (Rubin, L. J. Primary pulmonary
hypertension. N Engl J Med 336, 111-7 (1997)).
[0495] Delivering drugs to a diseased tissue by coupling the drug
to a compound that specifically binds at the target tissue
(synaphic targeting) can overcome the limitations of non-selective
drug activity (Ruoslahti, E. Vascular zip codes in angiogenesis and
metastasis. Biochem Soc Trans 32, 397-402 (2004); Ruoslahti, E., et
al. Targeting of drugs and nanoparticles to tumors. J Cell Biol
188, 759-68 (2010)). Such a technology would provide significant
therapeutic advantages such as concentrating the drug at the
targeted site, which increases efficacy while decreasing side
effects in other tissues. However, no compounds that would
specifically bind to the blood vessels in PAH lungs are known.
[0496] In vivo screening of phage peptide libraries has been used
to identify specific molecular markers in the vasculature in
different organs and diseased tissues. The `molecular zip codes`
revealed by these screens can be used in organ-specific or
lesion-specific delivery of systemically administered diagnostics
and therapeutics (Ruoslahti, E. Vascular zip codes in angiogenesis
and metastasis. Biochem Soc Trans 32, 397-402 (2004)). For
instance, .alpha..sub.v integrins are highly expressed in tumor
vasculature, where they have been accessed with peptides containing
the RGD integrin recognition motif to deliver drugs, biologicals,
viruses, and nanoparticles to tumor vasculature (Ruoslahti, E., et
al. Targeting of drugs and nanoparticles to tumors. J Cell Biol
188, 759-68 (2010)).
[0497] Previously, a cyclic peptide, CARSKNKDC, (CAR peptide; SEQ
ID NO:147) was identified via in vivo screening of phage peptide
library for homing to angiogenic blood vessels during wound repair
(Jarvinen, T. A. & Ruoslahti, E. Molecular changes in the
vasculature of injured tissues. Am J Pathol 171, 702-11 (2007)).
CAR peptide accumulates in tendon and skin wounds in rats and mice
with excellent selectivity, indicating that the peptide targets the
vasculature of injured tissues/lesions (Jarvinen, T. A. &
Ruoslahti, E. Molecular changes in the vasculature of injured
tissues. Am J Pathol 171, 702-11 (2007); Jarvinen, T. A. H. &
Ruoslahti, E. Target seeking anti-fibrotic compound enhances wound
healing and suppresses scar formation. Proc Natl Acad Sci USA,
(2010)). The CAR peptide specifically binds to heparin and its
binding to angiogenic endothelial cells and tumor cells requires
the glycosaminoglycan heparan sulfate (Jarvinen, T. A. &
Ruoslahti, E. Molecular changes in the vasculature of injured
tissues. Am J Pathol 171, 702-11 (2007)), indicating that this
peptide recognizes a specific form of heparan sulfate in the target
tissues. As endothelial activation is associated with PAH
(Dorfmuller, P., et al. Inflammation in pulmonary arterial
hypertension. Eur Respir J 22, 358-63 (2003); Kato, G. J. et al.
Levels of soluble endothelium-derived adhesion molecules in
patients with sickle cell disease are associated with pulmonary
hypertension, organ dysfunction, and mortality. Br J Haematol 130,
943-53 (2005)) the CAR peptide is useful for targeting vascular
lesions in the diseased lung. Here, it is shown that CAR enables
highly effective and selective targeting of PAH lesions.
[0498] 2. Materials and Methods
[0499] i. Animals
[0500] a. Monocrotaline-Induced PAH Model.
[0501] Monocrotaline (Sigma-Aldrich, St. Louis, Mo.) was dissolved
in 0.3 M hydrochloride solution and neutralized with 0.3 M sodium
hydroxide solution, and adjusted pH around 7.0. Adult male
Sprague-Dawley rats (150-200 g, Harlan Laboratories, Indianapolis,
Ind.) were administered with a single subcutaneous injection of
monocrotaline solution at a dose of 60 mg/kg body weight, while
control rats were administered with 0.9% saline (Bader, M. Rat
models of cardiovascular diseases. Methods Mol Biol 597, 403-14
(2010)). Rats were randomly selected and studied for peptide
targeting studies on 1, 3, 7, 14 or 21 days after the treatment of
monocrotaline.
[0502] b. SU5416/Hypoxia-Induced PAH Model.
[0503] Adult male Sprague-Dawley rats (approx. 200 g) were injected
subcutaneously with SU5416 (20 mg/kg body weight; SUGEN Inc, South
San Francisco, Calif.), which was suspended in
carboxymethylcellulose (0.5% carboxymethylcellulose sodium, 0.9%
sodium chloride, 0.4% polysorbate, 0.9% benzyl alcohol in deionized
water). The rats were then exposed to chronic hypoxia in a
hypobaric chamber (barometric pressure, 410 mm Hg: inspired O.sub.2
tension, 76 mm Hg) for 3 weeks followed by an additional 6 weeks of
reexposure to normoxia. Rats were examined at 9 weeks after the
injection of SU5416 when they develop severe occlusive pulmonary
arterial lesions (Abe, K. et al. Formation of plexiform lesions in
experimental severe pulmonary arterial hypertension. Circulation
121, 2747-54 (2010)).
[0504] ii. Peptide Targeting Study
[0505] The following peptides were labeled with
5-carboxyfluorescein (FAM) and used for the lung targeting studies:
CAR, CARSKNKDC (SEQ ID NO:147); VCAM1, CVHSPNKKCGGSK (SEQ ID
NO:13); CG7C control CGGGGGGGC (SEQ ID NO:14); CAR mutant (CAR-M),
CAQSNNKDC (SEQ ID NO:15). Peptides were dissolved in PBS at the
concentrations of 0.5 mg/mL. Hypertensive rats were injected with
peptide solution through tail vein (3.3 mg/kg). At 2 hours after
the injection, rats were perfused with PBS containing 1% bovine
serum albumin under the deep anesthesia and tissues were fixed by
systemic perfusion with 10% buffered formalin. The organs were
excised and fixed for additional 24 hours and processed for
anti-fluorescein immunohistochemistry.
[0506] iii. Immunohistochemistry
[0507] To determine the localization of the peptides,
paraffin-embedded tissue sections were immunostained with rabbit
anti-fluoroscein isothiocyanate (FITC) antibody (Invitrogen,
Carlsbad, Calif.) followed by horseradish peroxidase-labeled
anti-rabbit IgG secondary antibody. The peptide localization was
then visualized by DAB. Sections were counter stained with
hematoxylin to visualize the region of cells. An automated staining
system, Discovery XT (Ventana, Tucson, Ariz.) was used for the
staining. To examine the co-localization of the peptides with
macrophages or alveolar type II cells, tissue sections were doubly
stained with rabbit anti-FITC antibody and mouse anti-rat-CD68
antibody; EDI (Serotec, Raleigh, N.C.) or rabbit anti-Prosurfactant
Protein C (proSP-C, Millipore, Temecula, Calif.), respectively. For
FITC/proSP-C double staining, FITC staining was completed first.
The tissues sections were then treated withcitrate buffer (pH 6.0)
at 98.degree. C. for 10 min before proceeding to proSP-C staining.
Biotinylated species-specific secondary antibodies and alkaline
phosphatase-labeled streptavidin (Vector Laboratories, Burlingame,
Calif.) were used to visualize macrophages and alveolar type II
cells with Permanent Red (Dako, Carpinteria, Calif.).
[0508] iv. Data Analysis
[0509] Images of stained slides were observed and captured with
ECLIPSE 90i or 80i microscope with CCD camera DS-5M (Nikon
Instruments, Melville, N.Y.). To quantify the targeting efficiency
of the peptides to the lung, the immunostained sections were
scanned by Aperio Scanscope XT and analyzed using ImageScope
software (Aperio Technologies, Vista, Calif.). For the
quantification of area and density of peptide staining, the Aperio
Color Deconvolution tool was used to compare total peptide positive
area and intensity of peptide staining in each animal. The
thresholds were set empirically for strong, medium, and weak
staining. The area of each level of staining was presented as
percent of such area in the total cell area. The total stained area
was normalized by hematoxylin stained area of same regions.
[0510] 3. Results
[0511] i. Specific Accumulation of CAR Peptide in
Monocrotaline-Induced PAH Lesions
[0512] PAH was induced in the rat by subcutaneous injection of
monocrotaline (MCT), which resulted in considerable remodeling of
the pulmonary vasculature 3.about.4 weeks later.
Fluorescein-labeled CAR peptide (FAM-CAR) or the control FAM-CG7C
peptide was intravenously injected into these animals to determine
peptide homing to the lung lesions. The strong green auto
fluorescence of the lungs made it difficult to accurately interpret
the data of direct FITC imaging. For this reason, and to preserve
the histology, immunohistochemical staining with anti-FITC
antibodies was used to detect the FITC-labeled peptides.
[0513] In this analysis, extensive accumulation of CAR peptide was
found in the PAH lungs (FIG. 1). CAR accumulated in the endothelium
and medial smooth muscle of pulmonary arteries. The accumulation of
CAR was also prominent at the adventitia of pulmonary arteries. In
addition, capillary vessel walls and infiltrating macrophages were
strongly positive for CAR staining. Furthermore, CAR accumulated
significantly in the extravascular space (interstitial space and
alveolar lumen) of the PAH lungs, indicating that CAR penetrates
through the vessel wall and binds to macrophages and extracellular
matrix (ECM) components deposited in the injured lung; the putative
receptors for CAR are heparan sulfate proteoglycans upregulated at
the site of tissue injury (Jarvinen, T. A. & Ruoslahti, E.
Molecular changes in the vasculature of injured tissues. Am J
Pathol 171, 702-11 (2007)). The staining with a Type II alveolar
cell marker indicates that these cells are also positive for CAR in
MCT treated hypertensive rats. In comparison, little accumulation
of CAR was detected in the healthy normal lung of non-MCT-treated
rats (FIG. 1). Moreover, only very weak staining was detected for
the control CG7C peptide in the MCT-treated lungs (FIG. 1). These
results demonstrate the specificity of CAR targeting to the PAH
lung lesions.
[0514] PAH targeting activity of VCAM-1-binding peptide (VHSPNKK
(amino acids 2-8 of SEQ ID NO:13)) was also examined (Kelly, K. A.,
et al. In Vivo Phage Display Selection Yields Atherosclerotic
Plaque Targeted Peptides for Imaging. MolImaging Biol (2006);
Kelly, K. A. et al. Detection of vascular adhesion molecule-1
expression using a novel multimodal nanoparticle. Circ Res 96,
327-36 (2005)) to compare with that of CAR. This peptide has been
shown to target atherosclerotic plaques in ApoE.sub.-/- mice.
VCAM-1 is highly expressed in inflammatory endothelium (Cybulsky,
M. I. et al. Endothelial expression of a mononuclear leukocyte
adhesion molecule during atherogenesis. Science 251, 788-91 (1991);
Libby, P. Inflammation in atherosclerosis. Nature 420, 868-74
(2002)). Clinical data indicate that this is also true for the
activated endothelium in PAH (Kato, G. J. et al. Levels of soluble
endothelium-derived adhesion molecules in patients with sickle cell
disease are associated with pulmonary hypertension, organ
dysfunction, and mortality. Br J Haematol 130, 943-53 (2005)),
indicating that VCAM-1 may be useful in PAH targeting. VCAM-1
binding peptide accumulated in the PAH lungs to a similar extent to
CAR (FIG. 1). However, the majority of the peptide accumulation was
in infiltrating macrophages and/or Type II alveolar cells, and the
vasculature was less positive. Importantly, a fair amount of the
VCAM-1-binding peptide also accumulated in normal healthy lungs,
indicating the lack of specificity to the diseased lung, which was
in contrast with the results of CAR (FIG. 1). These observations
demonstrate the excellent PAH selectivity and targeting ability of
the CAR peptide.
[0515] ii. Spatiotemporal Pattern of PAH Targeting by CAR
[0516] During the course of PAH lesion development, weak to
moderate accumulation of CAR was detected in a few small discrete
areas in the injured lung at 3 days post injury. This accumulation
was absent at 7 days post injury, indicating that CAR homes to
acute lung lesions. More significant and broad accumulation of CAR
to the pulmonary vasculature was detected at 2 weeks post injury,
and the intensity of the accumulation significantly increased in
the chronic lesions in the 3.sub.rd week. The temporal pattern of
CAR homing coincided with the progressive remodeling of the
pulmonary vasculature and with an increase in the number of
infiltrating macrophages (Bader, M. Rat models of cardiovascular
diseases. Methods Mol Biol 597, 403-14 (2010)). These observations
demonstrate that the lung targeting by CAR is dependent on the
formation of the lung lesions. Despite the extensive homing of CAR
peptide to the injured lung, accumulation in other organs was
absent or very weak, except for the kidney. Intravenously
administered peptides get excreted through the kidney. Thus, CAR
accumulation in the kidney was observed in both MCT-treated PAH
rats and untreated healthy animals. The control peptide also
accumulated in the kidney. Little accumulation in healthy lungs and
other organs further supports that CAR is useful for selectively
targeting the PAH lesions.
[0517] iii. CAR Targets VEGFR Inhibition/Hypoxia-Induced PAH
[0518] To examine whether CAR peptide also exhibits selective
targeting to PAH lesions induced by a different mechanism, another
rat PAH model was employed, severe occlusive disease induced by the
VEGF receptor blocker SU5416 and hypoxia (Abe, K. et al. Formation
of plexiform lesions in experimental severe pulmonary arterial
hypertension. Circulation 121, 2747-54 (2010)). After a single
subcutaneous injection of SU5416, rats were exposed to hypoxia for
3 weeks followed by an additional 6 weeks of reexposure to
normoxia. Rats were examined at 9 weeks after the injection of
SU5416 when they develop severe occlusive pulmonary arterial
lesions. In this model, sustained pulmonary hypertension is
accompanied by the formation of complex plexiform lesions in
addition to the medial wall thickening and neointima formation,
thus well recapitulating the pulmonary lesion development in the
pulmonary arteriopathy of human PAH (Abe, K. et al. Formation of
plexiform lesions in experimental severe pulmonary arterial
hypertension. Circulation 121, 2747-54 (2010)). The intravenous CAR
administration resulted in prominent accumulation of the peptide
throughout the PAH lungs. Notably, CAR accumulation was detected at
high intensity in all layers of remodeled pulmonary arteries, i.e.,
endothelium, neointima, media, and adventitia. Furthermore, the CAR
positive lesions included not only the endothelium and thickening
medial wall but plexiform-like lesions and occluded arterioles. In
contrast, only negligible signal was detected in the PAH lesion
when CAR-M, an inactive CAR derivative with site-directed mutations
(CAQSNNKDC (SEQ ID NO:15)), was tested, demonstrating the
specificity of the CAR homing. Thus, CAR peptide exhibited a
specific targeting to various forms of pulmonary arterial lesions
in two distinct PAH models.
[0519] iv. Binding and Penetration of CAR into Human Cells
[0520] To assess the potential utility of CAR in targeting human
PAH, binding of CAR to human endothelial cells (HUVEC) was tested
in culture. When grown in culture, these endothelial cells (EC) are
highly activated and mitotic with elevated expression of angiogenic
genes, recapitulating the characteristics of the ECs of
pathologically regenerating blood vessels (Chappey, O., et al.
Endothelial cells in culture: an experimental model for the study
of vascular dysfunctions. Cell Biol Toxicol 12, 199-205 (1996);
Schoenfeld, J. et al. Bioinformatic analysis of primary endothelial
cell gene array data illustrated by the analysis of transcriptome
changes in endothelial cells exposed to VEGF-A and PlGF.
Angiogenesis 7, 143-56 (2004)). CAR peptide specifically bound to
the growing ECs in culture and was internalized into the cells.
Reinforcing the ability of CAR to target human cells in vivo,
intravenously injected CAR was shown to home to human tumor
xenografts in mice with high selectivity and efficiency.
Significantly, the peptide was found associated with the human
tumor cells. These results indicate expression of CAR receptor in
human cells, indicating that the application of CAR targeting to
human PAH is feasible.
[0521] 4. Discussion
[0522] In this study, highly selective PAH-targeting and
tissue-penetration abilities of a recently described cyclic
peptide, CAR (CARSKNKDC (SEQ ID NO:147)) were demonstrated. This is
the first report on a technology that enables selective targeting
of the life-threatening lung disorder, PAH. It was found that CAR
homes to the pulmonary arterial endothelium and smooth muscle of
the diseased lung, which are the primary tissue targets of current
PAH interventions (Girgis, R. E. Emerging drugs for pulmonary
hypertension. Expert Opin Emerg Drugs 15, 71-85 (2010)). It is
noteworthy that CAR also accumulates in the adventitia of pulmonary
arteries and in interstitial macrophages. The remodeling of the
adventitia is important in the development of PAH, and macrophages
recruited to the PAH lesion play a crucial role in the pathogenesis
of the disease through mediating inflammatory responses. The
CAR-targeting of the lung lesions may therefore offer opportunities
to target multiple cell types important in the pathogenesis of
PAH.
[0523] As inflammation is an element in the PAH models we used,
there could be a correlation between inflammation in the lungs and
CAR accumulation. However, the inflammatory response in the lungs
is significantly stronger in MCT-induced PAH than in the
SU5416/hypoxia model, or in human IPAH (Abe, K. et al. Formation of
plexiform lesions in experimental severe pulmonary arterial
hypertension. Circulation 121, 2747-54 (2010)). Nonetheless, CAR
effectively targets the PAH lungs in both rat models. Moreover, CAR
showed excellent specificity for the lungs in the MCT model,
although this treatment is known to cause inflammation in the heart
and liver as well (Campian, M. E. et al. Early inflammatory
response during the development of right ventricular heart failure
in a rat model. Eur J Heart Fail 12, 653-8; Copple, B. L., et al.
Liver inflammation during monocrotaline hepatotoxicity. Toxicology
190, 155-69 (2003)). Therefore, inflammation alone does not appear
to account for the selective CAR homing to the hypertensive
lungs.
[0524] The molecular target of CAR homing in the PAH lesion is
unknown. A BLAST analysis revealed a high homology of CAR sequence
with the heparin-binding motif of bone morphogenetic protein-4
(BMP-4) (Jarvinen, T. A. & Ruoslahti, E. Molecular changes in
the vasculature of injured tissues. Am J Pathol 171, 702-11
(2007)). Whether CAR has any effect on BMP-4 signaling is unknown.
In agreement with its putative heparin-binding sequence, cell
binding of CAR is dependent on heparan sulfate expression of the
cells (Jarvinen, T. A. & Ruoslahti, E. Molecular changes in the
vasculature of injured tissues. Am J Pathol 171, 702-11 (2007)),
indicating that the mechanism for the vascular homing to the site
of hypertensive lesions may be due to the expression of a unique
glycosaminoglycan in the diseased lung. Thus, PAH vasculature
appears to express a molecular zip code not present in normal lung
vasculature. The identification of the receptor for CAR will be an
important goal of future work that could further advance PAH
targeting technology.
[0525] Currently, there are several pharmacological agents for the
treatment of PAH, some of which hold promise for improved efficacy
(McLaughlin, V. V. et al. ACCF/AHA 2009 expert consensus document
on pulmonary hypertension: a report of the American College of
Cardiology Foundation Task Force on Expert Consensus Documents and
the American Heart Association: developed in collaboration with the
American College of Chest Physicians, American Thoracic Society,
Inc., and the Pulmonary Hypertension Association. Circulation 119,
2250-94 (2009); Girgis, R. E. Emerging drugs for pulmonary
hypertension. Expert Opin Emerg Drugs 15, 71-85 (2010)). However,
the lack of pulmonary vascular selectivity and associated systemic
adverse effects imposes significant obstacles to successful
outcomes of these therapies. When conjugated with CAR, a
systemically administered fluorescence probe (FAM) was successfully
delivered to and concentrated in the lung lesions. This indicates
that CAR will also be useful for delivering other imaging probes
and therapeutic compounds to PAH lesions. CAR accumulated
significantly in the interstitial space of the PAH lungs,
indicating that CAR, having specifically bound to the vasculature
of the diseased lungs, extravasates and penetrates into lung
parenchyma. This deep tissue penetration can make it possible to
overcome the additional problem of inadequate drug penetration into
the target tissues. The targeting technology described herein can
be useful to develop drugs specifically engineered for targeted
treatment of PAH.
B. Example 2
Truncated CAR Peptide Mediates Heparin Sulfate (HS)-Dependent Tumor
Homing
[0526] Several CAR peptides have been used in the present studies
(Table 1).
TABLE-US-00003 TABLE 1 Nomenclature of CAR peptides Peptide
sequence Name CAR SKN KDC CAR (SEQ ID NO: 147) CAR SKN K tCAR (SEQ
ID NO: 4) AR SKN K ARSKNK (SEQ ID NO: 18) KNKSRAC-CARSKNK tCAR
dimer (SEQ ID NO: 4)
[0527] Multivalent complexes of the CendR peptide RPARPAR (SEQ ID
NO:9) were used to block the neuropilin-1 binding site for
peptides. RPARPAR inhibits the binding and internalization of
RPARPAR phage (positive control) to cultured CHO-K cells, whereas
the RPARPAR complexes have no effect on the binding and
internalization of the CAR or tCAR phage (FIG. 2). These results
show that the CAR and tCAR peptides do not use the CendR pathway
(Teesalu et al., 2009) in their entry into cells.
[0528] The phage internalize into CHO-K cells, whereas there is
much less uptake into pgsA-745 mutant CHO-K cells, which are
incapable of synthesizing glycosaminoglycans, including heparan
sulfate (FIG. 3), and have previously shown not bind CAR (Jarvinen
and Ruoslahti, 2008). The tCAR phage was more effectively
internalized into the CHO-K cells than the CAR phage. These results
show that like CAR, tCAR requires glycosaminoglycans, likely
heparan sulfate, to be able to bind to cells and become
internalized into them.
[0529] In vivo tumor homing of the CAR peptide was tested. Mice
bearing orthotopic tumors generated with 4T1 mouse breast cells
were intravenously injected with the 200 .mu.L of 1 mM
fluorescamine (FAM)-conjugated CAR peptide. The circulation time
was 1 hour. The peptide accumulated in the tumor tissue and traces
were also seen in the lungs. The heart muscle showed faint, uniform
fluorescence. The kidneys are always positive regardless of what
peptide is injected because peptides are excreted into the
urine.
[0530] Homing of tCAR peptide to 4T1 tumors was examined. Confocal
images of sections of the tumor and normal organs from mice
injected with the 200 .mu.L of 1 mM FAM-tCAR peptide. The
circulation time was 2 hours. The tCAR peptide shows wider
spreading within tumor tissue than CAR, and tCAR there was not
detectable accumulation of tCAR in normal tissues.
[0531] A lack of homing of ARSKNK peptide (SEQ ID NO:18) to 4T1
tumors was determined. Confocal images of sections of the tumor and
normal organs from mice injected with the 200 .mu.L of 1 mM
FAM-ARSKNK peptide. The circulation time was 1 hour. Little ARSKNK
peptide accumulated in the tumor tissue, but some fluorescence was
detected in the heart. These results indicate that, surprisingly,
the N-terminal Cys residue is required for the tumor homing.
[0532] Homing of dimeric tCAR peptide to 4T1 tumors. Confocal
images of sections of the tumor and normal organs from mice
injected with the 200 .mu.L of 1 mM FAM-KNKSRAC-CARSKNK peptide
(SEQ ID NO:4). The circulation time was 1 hour. The dimeric peptide
shows less tumor accumulation than the monomeric tCAR peptide. The
inferior tumor homing of the dimeric tCAR shows that dimerization
of tCAR through the free sulfhydryl group is not the basis of the
strong tumor homing of tCAR.
[0533] In vivo tumor homing of nanoparticles coated with the tCAR
peptide to 4T1 tumors was examined. Confocal images of tumors from
mice injected with tCAR iron oxide nanoworms (Park et al., 2009); 5
mg/kg of iron in 130 .mu.L were intravenously injected into the
tumor mice. The circulation times were as shown. The tCAR nanoworms
were initially found in and around the blood vessels, but over time
extravasated and spread in the tumor tissue.
[0534] The lack of homing of tCAR nanoparticles to normal tissues
in 4T1 tumor mice was determined. Confocal images of tumors from
mice injected with tCAR iron oxide nanoworms (Park et al., 2009); 5
mg/kg of iron in 130 .mu.L were intravenously injected into the
tumor mice. The circulation time was 2 hours. The tCAR nanoworms
accumulated in the liver and spleen because nanoparticles are
cleared via the reticuloendothelial system (RES) in the liver and
spleen.
[0535] A candidate receptor for the tCAR peptides was identified.
Affinity chromatography of 4T1 tumor extracts on tCAR peptide
immobilized onto iodoacetyl-modified agarose beads. Tumor tissue
was extracted with a 200 mM glucopyranoside buffer, and the extract
was incubated beads coated with tCAR peptide, followed by extensive
washing with 50 mM glucopyranoside, and elution with 2 mM tCAR
peptide solution. The fractions eluted from the washed affinity
matrix with the tCAR peptide show the presence of a band at 98, 55,
40, and 36-kDa.
C. Example 3
Monocrotaline Pulmonary Hypertension Model
[0536] 1. Animal Model
[0537] A rat model of monocrotaline (MCT)-induced pulmonary
arterial hypertension was used for this study. Briefly, male
Sprague-Dawley rats (150-200 g, Harlan Laboratories, IN) were
administered with a single subcutaneous injection of monocrotaline
at 60 mg/kg (Sigma-Aldrich, MO), while control rats administered
0.9% saline. Rats were randomly selected and studied for peptide
distribution studies on 1, 3, 7, 14 or 21 days after the treatment
of monocrotaline.
[0538] 2. Peptides
[0539] The following peptides were labeled with
5-carboxyfluorescein (5FAM) and used for the lung targeting
studies: CAR, 5FAM-CARSKNKDC; VCAM1, CVHSPNKKCGGSK-5FAM; Control,
5FAM-CGGGGGGGC. All peptides were synthesized by Anaspec (Anaspec
Inc., CA). Peptides were resolved in PBS at the concentrations of
0.5 mg/mL.
[0540] 3. Peptide Targeting Study
[0541] MCT-treated or untreated rats were injected with peptide
solution at a dose of 3.3 mg/kg body weight via the tail vein. At
two hours after the injection, rats were perfused with PBS
containing 1% bovine serum albumin under the deep anesthesia with
isofluorane at a rate of 3.0% and euthanized. Tissues were fixed by
systemic perfusion with 10% buffered formalin via right ventricle.
The lung was inflated by injection of 10% formalin through the
trachea. Various organs were excised from the rat and fixed for
additional twenty four hours and processed for
immunohistochemistory.
[0542] 4. Immunohistochemistry
[0543] To determine the localization of the peptides,
paraffin-embedded tissue sections were immunostained with either
hematoxylin and eosin or rabbit anti-fluoroscein isothiocyanate
(FITC) antibody (Invitrogen, CA) followed by horseradish
peroxidase-labeled anti-rabbit IgG secondary antibody. The peptide
localization was then visualized by diaminobenzidine (DAB). An
automated staining system, Discovery XT (Ventana, Az.) was used. To
quantify the targeting efficiency of the peptides to the lung, the
immunostained sections were scanned by Aperio Scanscope XT and
analyzed using ImageScope software (Aperio Technologies, CA).
D. Example 4
Targeted Vasodilation in SU5416/Hypoxia/Normoxia-Exposed Severe
Occlusive Pulmonary Hypertension
[0544] 1. Animal Model
[0545] Adult male Sprague-Dawley rats weighing approximately 200 g
are injected subcutaneously with SU5416 (20 mg/kg; SUGEN Inc),
which is suspended in carboxymethylcellulose (0.5%
[wt/vol]carboxymethylcellulose sodium, 0.9% [wt/vol] NaCl, 0.4%
[vol/vol] polysorbate, 0.9% [vol/vol] benzyl alcohol in deionized
water). The rats are then exposed to chronic hypoxia in a hypobaric
chamber (10% O.sub.2) for 3 weeks and are returned to normoxia (21%
O.sub.2) for an additional 2 to 10 weeks.
[0546] 2. Catheterized Rats
[0547] Rats are anesthetized with intramuscular pentobarbital
sodium (30 mg/kg). The rats are placed on controlled heating pads.
Hemodynamic measurements are performed in anesthetized animals
under normoxic conditions. Polyvinyl catheters (PV-1, internal
diameter: 0.28 mm) are inserted into the right jugular vein for
measurement of right ventricular systolic pressure (RVSP) and into
the left jugular vein for drug administration. A microtip P-V
catheter (SPR-838, Millar Instruments) is inserted into the right
carotid artery and advanced into the left ventrical (LV). The
signals are continuously recorded by MPVS-300 system with
PowerLab/4SP, A/D converter (AD Instruments), and a personal
computer. RVSP, heart rate, maximal left ventriclar systolic
pressure, left ventricular end-diastolic pressure (LVEDP), mean
arterial pressure (MAP), cardiac output, and stroke volume are
measured. If the heart rate falls below 300 beats/min, the
measurements are excluded from analysis. At the end of each
hemodynamic study, the rat is sacrificed by an overdose of
pentobarbital sodium, and organs are removed for various
measurements and analyses.
[0548] After baseline hemodynamic measurements, a simple mixture of
CAR (1 mg/300 g rat), or control peptide CARM, and fasudil (0.1,
0.3, 1, or 3 mg/kg) or each agent alone is injected intravenously,
and all hemodynamic parameters are continuously monitored.
[0549] 3. Immunohistochemical Staining
[0550] Organs (lung, heart, liver, spleen, and kidney) are
collected after blood is flushed with 30 ml phosphate buffered
saline (PBS). Lungs are inflated via trachea with 10% formalin at a
constant pressure of 20 cm H.sub.2O. After 24 hour-fixation with
10% formalin, all organs are embedded in paraffin, and sectioned at
5 mm thickness. After deparaffinization, tissue sections are
pretreated with 3% hydrogen peroxidase for 10 minutes and blocked
with normal horse serum for 1 hour. They are then incubated for 1
hour with an anti-fluorescein antibody (1:200; Invitrogen) as a
primary antibody. After washing with PBS, the sections were
incubated with biotinylated secondary antibodies, washed with PBS,
and incubated in ABC Regent for 5 minutes. Diaminobenzidine was
used as a substrate for the immunoperoxidase reaction. Sections
were lightly counterstained with hematoxylin, and analyzed light
microscopically. CAR (but not CARM) was detected in high intensity
in all layers of severely remodeled arteries from lung tissue.
Neither CAR nor CARM was found in other organs except for the
kidney.
E. Example 5
Bleomycin-Induced Acute Lung Injury and Pulmonary Fibrosis
Model
[0551] The bleomycin (BL) model is usually considered a model of
pulmonary fibrosis, but its administration is also associated with
features of acute lung injury (ALI). Bleomycin is an antineoplastic
antibiotic drug isolated in 1966 from the actinomycete Streptomyces
verticillus. Bleomycin forms a complex with oxygen and metals such
as Fe2+, leading to the production of oxygen radicals, DNA breaks,
and ultimately cell death. Bleomycin can be inactivated by
bleomycin hydrolase, a cysteine protease that shows variable levels
of expression in the lungs. The susceptibility of the lungs to
bleomycin-induced toxicity is largely dependent on the levels of
expression of bleomycin hydrolase in the lungs; species with high
levels of expression, such as rabbits, are relatively resistant to
bleomycin-induced toxicity, whereas species with low levels of
expression, such as C57BL/6 mice, are sensitive. In addition to
species-related differences in bleomycin susceptibility, there are
also differences in strain susceptibility, with C57BL/6 mice being
highly sensitive.
[0552] 1. Animal Model
[0553] A mouse model of bleomycin induced acute lung injury and
pulmonary fibrosis was used for this study. Briefly, 6 WT C57B1/6
male mice, 8-12 weeks were weighed and anesthetized, and given
bleomycin (BL) intratracheally at 4 U/kg. At 3 days (acute lung
injury model) and 14 days (pulmonary fibrosis model) after BL
injection, peptides were injected via the tail vein.
[0554] 2. Peptides
[0555] The following peptides were labeled with
5-carboxyfluorescein (5FAM) and used for the lung targeting
studies: CAR, 5FAM-CARSKNKDC; VCAM1, CVHSPNKKCGGSK-5FAM; Control,
5FAM-CGGGGGGGC. All peptides were synthesized by Anaspec (Anaspec
Inc., CA). Peptides were resolved in PBS at the concentrations of
0.5 mg/mL.
[0556] 3. Peptide Targeting Study
[0557] BL-treated mice were injected with peptide solution at a
dose of 3.3 mg/kg body weight via the tail vein. At two hours after
the injection, mice were perfused with PBS containing 1% bovine
serum albumin under the deep anesthesia with isofluorane at a rate
of 3.0% and euthanized. Tissues were fixed by systemic perfusion
with 10% buffered formalin via right ventricle. The lung was
inflated by injection of 10% formalin through the trachea. Various
organs were excised from the rat and fixed for additional twenty
four hours and processed for immunohistochemistory.
[0558] 4. Immunohistochemistry
[0559] To determine the localization of the peptides,
paraffin-embedded tissue sections were immunostained with rabbit
anti-fluoroscein isothiocyanate (FITC) antibody (Invitrogen, CA)
followed by horseradish peroxidase-labeled anti-rabbit IgG
secondary antibody. The peptide localization was then visualized by
diaminobenzidine (DAB). An automated staining system, Discovery XT
(Ventana, Az.) was used. To quantify the targeting efficiency of
the peptides to the lung, the immunostained sections were scanned
by Aperio Scanscope XT and analyzed using ImageScope software
(Aperio Technologies, CA).
[0560] 5. Blood Pressure Tracing
[0561] To measure the acute effects of fasudil with and without CAR
administration on the right and left ventricular systolic pressure,
blood pressure measurements were performed on catheterized
SU5416/hypoxia/normoxia-exposed rats with PAH (FIG. 4).
Surprisingly, co-administered CAR enhanced the blood pressure
lowering effect of fasudil on RVSP with only a minor reduction in
LVSP, as compared to fasudil alone. Of additional importance,
continuous infusion of CAR+fasudil resulted in a sustained,
pulmonary-specific effect even after the cessation of the infusion.
An alternative analysis was conducted, observing the same
pulmonary-specific effects when comparing pressure in the RVSP to
systolic aortic pressure (SAP). While the selective decrease in
pulmonary pressure as measured in the RVSP is present, there is no
increased CAR effect systemically when co-administered with
fasudil.
F. Example 6
CAR Variant+Fasudil Analysis
[0562] 1. Animal Model
[0563] Severe occlusive PAH rat model was used. Animals were
injected with SU5416 (20 mg/kg; SUGEN Inc), followed by 3 weeks
hypoxia, then followed by 2-10 weeks normoxia.
[0564] 2. Peptides
[0565] The peptide administered was a 7 amino acid variant to the
CAR peptide used in previous examples. This variant (tCAR; CARK)
consisted of the following sequence: CARSKNK (SEQ ID NO:4). In
these experiments, tCAR was administered at a dose of 3 mg/kg and
fasudil administered at 1 mg/kg. CARK is an alternative designation
for the truncated CAR peptide CARSKNK (SEQ ID NO:4).
[0566] 3. Blood Pressure Tracing
[0567] To measure the acute effects of fasudil with and tCAR
administration on the right and left ventricular systolic pressure
(or systolic aortic pressure), blood pressure measurements were
performed on catheterized SU5w/hypoxia/normoxia-exposed rats with
PAH. Similar to CAR, tCAR co-administration enhanced the blood
pressure lowering effect of fasudil on RVSP with only a minor
reduction in SAP and LVSP, as compared to fasudil alone.
Interestingly, administration of 10 mg/kg of fasudil 30 minutes
after cessation of tCAR infusion still resulted in a sustained,
pulmonary-specific effect.
[0568] Table 2 shows the results of a blood tracing experiment with
fasudil co-administered with CARK. Fasudil was dosed at 1 mg/kg and
CARK dosed at 3 mg/kg. Pressure measurements were observed at RVSP
(mmHg) and SAP (mmHg). Table 3 shows the results of blood tracing
experiments with fasudil co-administered with CARK. Fasudil was
dosed at 1 mg/kg and CARK dosed at 3 mg/kg. Pressure measurements
were observed at RVSP (mmHg) and LVSP (mmHg). Table 4 shows the
results of a blood tracing experiment with fasudil administered
after cessation of CARK infusion. Fasudil was dosed at 10 mg/kg.
Pressure measurements were observed at RVSP (mmHg) and LVSP
(mmHg).
TABLE-US-00004 TABLE 2 tCAR 3 mg/kg + fasudil Baseline 1 mg/kg RVSP
(mmHg) 89.9 66.9 SAP (mmHg) 134.9 146.1
TABLE-US-00005 TABLE 3 tCAR 3 mg/kg + fasudil tCAR 3 mg/kg +
Baseline 1 mg/kg-1 fasudil 1 mg/kg-2 RVSP (mmHg) 84.6 69.6 87.9
.fwdarw. 63.6 LVSP (mmHg) 140.6 123.9 136.9 .fwdarw. 128.9
TABLE-US-00006 TABLE 4 Baseline Fasudil 10 mg/kg RVSP (mmHg) 84.6
27.2 LVSP (mmHg) 140.6 107.7
G. Example 7
CAR+Imatinib Analysis
[0569] 1. Animal Model
[0570] Severe occlusive PAH rat model was used. Animals were
injected with SU5416 (20 mg/kg; SUGEN Inc), followed by 3 weeks
hypoxia, then followed by 2-10 weeks normoxia.
[0571] 2. Peptides
[0572] The peptide administered was CAR, CARSKNKDC (SEQ ID NO:147).
In this experiment, CAR was administered at a dose of 3 mg/kg and
imatinib administered at 10 mg/kg.
[0573] 3. Blood Pressure Tracing
[0574] To measure the acute effects of imatinib with CAR
administration on the right and left ventricular systolic pressure,
blood pressure measurements were performed on catheterized
SU5w/hypoxia/normoxia-exposed rats with PAH. Similar to fasudil,
CAR co-administration enhanced the blood pressure lowering effect
of imatinib on RVSP with only a minor reduction in LVSP.
H. Example 8
Altered Levels of Gene Expression of Enzymes involved in Heparan
Sulfate Proteoglycan Biosynthesis Found in a Progressive Porcine
Surgical Shunt Model of PAH
[0575] Heparan sulfate biosynthetic enzymes are key components in
generating a myriad of distinct heparan sulfate structures that
carry out multiple biologic activities. To determine whether CAR or
any variants utilized the heparan sulfate pathway, an analysis was
first performed to identify differential gene expression in the PAH
model since CAR displayed both homing and selective therapeutic
efficacy in models of PAH.
[0576] It was discovered that in the surgical shunt model of PAH, a
large increase in gene expression was identified in a select group
of genes, all of which are related to the heparan sulfate
biosynthetic pathway. The heparan sulfate 2-O-sulfotransferase 1
(HS2ST1) gene, which encodes an enzyme responsible for catalyzing
the transfer of sulfate to the C2 position of selected hexuronic
acid residues within the maturing heparan sulfate, was found to be
greatly increased over time in the PAH pig model.
[0577] Another gene which showed a selective increase in expression
in the PAH model was exostosin 1 (EXT1), a glycosyltransferase
required for the biosynthesis of heparan sulfate. Specifically,
EXT1 encodes an endoplasmic reticulum-resident type II
transmembrane glycosyltranferase involved in the chain elongation
step of heparan sulfate biosynthesis.
[0578] Other genes identified as exhibiting an increase in
expression in the PAH model were glycosyltransferase 8 domain
containing 2 (GLT8D2), heparan sulfate
N-deacetylase/N-sulfotransferase (NDST1) and O-linked
N-acetylglucosamine transferase (OGT).
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[0599] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0600] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a peptide" includes a plurality of such
peptides, reference to "the peptide" is a reference to one or more
peptides and equivalents thereof known to those skilled in the art,
and so forth.
[0601] "Optional" or "optionally" means that the subsequently
described event, circumstance, or material may or may not occur or
be present, and that the description includes instances where the
event, circumstance, or material occurs or is present and instances
where it does not occur or is not present.
[0602] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, also specifically contemplated and
considered disclosed is the range from the one particular value
and/or to the other particular value unless the context
specifically indicates otherwise. Similarly, when values are
expressed as approximations, by use of the antecedent "about," it
will be understood that the particular value forms another,
specifically contemplated embodiment that should be considered
disclosed unless the context specifically indicates otherwise. It
will be further understood that the endpoints of each of the ranges
are significant both in relation to the other endpoint, and
independently of the other endpoint unless the context specifically
indicates otherwise. Finally, it should be understood that all of
the individual values and sub-ranges of values contained within an
explicitly disclosed range are also specifically contemplated and
should be considered disclosed unless the context specifically
indicates otherwise. The foregoing applies regardless of whether in
particular cases some or all of these embodiments are explicitly
disclosed.
[0603] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed method and compositions
belong. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present method and compositions, the particularly useful
methods, devices, and materials are as described. Publications
cited herein and the material for which they are cited are hereby
specifically incorporated by reference. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such disclosure by virtue of prior invention.
No admission is made that any reference constitutes prior art. The
discussion of references states what their authors assert, and
applicants reserve the right to challenge the accuracy and
pertinency of the cited documents. It will be clearly understood
that, although a number of publications are referred to herein,
such reference does not constitute an admission that any of these
documents forms part of the common general knowledge in the
art.
[0604] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps.
[0605] It is understood that the disclosed method and compositions
are not limited to the particular methodology, protocols, and
reagents described as these may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims.
[0606] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the method and
compositions described herein. Such equivalents are intended to be
encompassed by the following claims.
Sequence CWU 1
1
16815PRTArtificial Sequencesynthetic construct; homing peptide 1Cys
Gly Lys Arg Lys1 526PRTArtificial Sequencesynthetic construct;
homing peptide 2Cys Arg Lys Asp Lys Cys1 5314PRTArtificial
Sequencesynthetic construct; membrane perturbing molecule 3Lys Leu
Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys1 5
1047PRTArtificial Sequencesynthetic construct; homing peptide 4Cys
Ala Arg Ser Lys Asn Lys1 5514PRTArtificial Sequencesynthetic
construct; membrane perturbing molecule 5Lys Leu Ala Lys Lys Leu
Ala Lys Leu Ala Lys Lys Leu Ala1 5 10614PRTArtificial
Sequencesynthetic construct; membrane perturbing molecule 6Lys Ala
Ala Lys Lys Ala Ala Lys Ala Ala Lys Lys Ala Ala1 5
10721PRTArtificial Sequencesynthetic construct; membrane perturbing
molecule 7Lys Leu Gly Lys Lys Leu Gly Lys Leu Gly Lys Lys Leu Gly
Lys Leu1 5 10 15Gly Lys Lys Leu Gly 20824PRTArtificial
Sequencesynthetic construct; homing peptide 8Cys Ala Arg Ser Lys
Asn Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 2097PRTArtificial Sequencesynthetic construct;
homing peptide 9Arg Pro Ala Arg Pro Ala Arg1 5109PRTArtificial
Sequencesynthetic construct; LyP-1 homing peptide 10Cys Gly Asn Lys
Arg Thr Arg Gly Cys1 5119PRTArtificial Sequencesynthetic construct;
cyclic homing peptide 11Cys Arg Gly Asp Xaa Gly Pro Xaa Cys1
5125PRTArtificial Sequencesynthetic construct; homing peptide 12Cys
Arg Glu Lys Ala1 51313PRTArtificial Sequencesynthetic construct;
VCAM1 targeting peptide 13Cys Val His Ser Pro Asn Lys Lys Cys Gly
Gly Ser Lys1 5 10149PRTArtificial Sequencesynthetic construct;
control peptide 14Cys Gly Gly Gly Gly Gly Gly Gly Cys1
5159PRTArtificial Sequencesynthetic construct; CAR mutant - homing
peptide 15Cys Ala Gln Ser Asn Asn Lys Asp Cys1 5169PRTArtificial
Sequencesynthetic construct; heart homing peptide 16Cys Arg Ser Thr
Arg Ala Asn Pro Cys1 5179PRTArtificial Sequencesynthetic construct;
heart homing peptide 17Cys Pro Lys Thr Arg Arg Val Pro Cys1
5186PRTArtificial Sequencesynthetic construct; homing peptide 18Ala
Arg Ser Lys Asn Lys1 5195PRTArtificial Sequencesynthetic construct;
heart homing peptide 19Arg Gly Ser Ser Ser1 5204PRTArtificial
Sequencesynthetic construct; homing peptide 20Xaa Xaa Xaa
Xaa1215PRTArtificial Sequencesynthetic construct; homing peptide
21Xaa Xaa Xaa Lys Gly1 5225PRTArtificial Sequencesynthetic
construct; enterokinase cleavage sequence 22Asp Asp Asp Asp Lys1
5234PRTArtificial Sequencesynthetic construct; homing peptide 23Xaa
Xaa Xaa Xaa1246PRTArtificial Sequencesynthetic construct; homing
peptide 24Asn Gly Arg Ala His Ala1 5257PRTArtificial
Sequencesynthetic construct; gut homing peptide 25Phe Arg Val Arg
Gly Ser Pro1 5267PRTArtificial Sequencesynthetic construct; gut
homing peptide 26Arg Val Arg Gly Pro Glu Arg1 5277PRTArtificial
Sequencesynthetic construct; liver homing peptide 27Val Lys Ser Val
Cys Arg Thr1 5287PRTArtificial Sequencesynthetic construct; liver
homing peptide 28Trp Arg Gln Asn Met Pro Leu1 5297PRTArtificial
Sequencesynthetic construct; liver homing peptide 29Ser Arg Arg Phe
Val Gly Gly1 5307PRTArtificial Sequencesynthetic construct; liver
homing peptide 30Ala Leu Glu Arg Arg Ser Leu1 5317PRTArtificial
Sequencesynthetic construct; liver homing peptide 31Ala Arg Arg Gly
Trp Thr Leu1 5327PRTArtificial Sequencesynthetic construct;
prostate homing peptide 32Ser Met Ser Ile Ala Arg Leu1
5337PRTArtificial Sequencesynthetic construct; prostate homing
peptide 33Val Ser Phe Leu Glu Tyr Arg1 5347PRTArtificial
Sequencesynthetic construct; prostate homing peptide 34Arg Gly Arg
Trp Leu Ala Leu1 5357PRTArtificial Sequencesynthetic construct;
ovary homing peptide 35Glu Val Arg Ser Arg Leu Ser1
5367PRTArtificial Sequencesynthetic construct; ovary homing peptide
36Val Arg Ala Arg Leu Met Ser1 5377PRTArtificial Sequencesynthetic
construct; ovary homing peptide 37Arg Val Gly Leu Val Ala Arg1
5387PRTArtificial Sequencesynthetic construct; ovary homing peptide
38Arg Val Arg Leu Val Asn Leu1 5395PRTArtificial Sequencesynthetic
construct; heart homing peptide 39Cys Arg Pro Pro Arg1
5409PRTArtificial Sequencesynthetic construct; heart homing peptide
40Cys Gly Arg Lys Ser Lys Thr Val Cys1 5416PRTArtificial
Sequencesynthetic construct; heart homing peptide 41Cys Ala Arg Pro
Ala Arg1 5426PRTArtificial Sequencesynthetic construct; heart
homing peptide 42Cys Pro Lys Arg Pro Arg1 5436PRTArtificial
Sequencesynthetic construct; heart homing peptide 43Cys Lys Arg Ala
Val Arg1 5449PRTArtificial Sequencesynthetic construct; heart
homing peptide 44Cys Arg Asn Ser Trp Lys Pro Asn Cys1
5459PRTArtificial Sequencesynthetic construct; heart homing peptide
45Cys Ser Gly Met Ala Arg Thr Lys Cys1 5464PRTArtificial
Sequencesynthetic construct; homing peptide 46Xaa Asn Gly
Arg14710PRTArtificial Sequencesynthetic construct; homing peptide
47Cys Gln Ser Cys Asn Gly Arg Cys Val Arg1 5 104810PRTArtificial
Sequencesynthetic construct; homing peptide 48Cys Arg Thr Cys Asn
Gly Arg Cys Gln Val1 5 104910PRTArtificial Sequencesynthetic
construct; homing peptide 49Cys Val Gln Cys Asn Gly Arg Cys Ala
Leu1 5 105010PRTArtificial Sequencesynthetic construct; homing
peptide 50Cys Arg Cys Cys Asn Gly Arg Cys Ser Pro1 5
105110PRTArtificial Sequencesynthetic construct; homing peptide
51Cys Ala Ser Asn Asn Gly Arg Val Val Leu1 5 105210PRTArtificial
Sequencesynthetic construct; homing peptide 52Cys Gly Arg Cys Asn
Gly Arg Cys Leu Leu1 5 105310PRTArtificial Sequencesynthetic
construct; homing peptide 53Cys Trp Leu Cys Asn Gly Arg Cys Gly
Arg1 5 105410PRTArtificial Sequencesynthetic construct; homing
peptide 54Cys Ser Lys Cys Asn Gly Arg Cys Gly His1 5
105510PRTArtificial Sequencesynthetic construct; homing peptide
55Cys Val Trp Cys Asn Gly Arg Cys Gly Leu1 5 105610PRTArtificial
Sequencesynthetic construct; homing peptide 56Cys Ile Arg Cys Asn
Gly Arg Cys Ser Val1 5 105710PRTArtificial Sequencesynthetic
construct; homing peptide 57Cys Gly Glu Cys Asn Gly Arg Cys Val
Glu1 5 105810PRTArtificial Sequencesynthetic construct; homing
peptide 58Cys Glu Gly Val Asn Gly Arg Arg Leu Arg1 5
105910PRTArtificial Sequencesynthetic construct; homing peptide
59Cys Leu Ser Cys Asn Gly Arg Cys Pro Ser1 5 106010PRTArtificial
Sequencesynthetic construct; homing peptide 60Cys Glu Val Cys Asn
Gly Arg Cys Ala Leu1 5 10617PRTArtificial Sequencesynthetic
construct; heart homing peptide 61Gly Gly Gly Val Phe Trp Gln1
56213PRTArtificial Sequencesynthetic construct; tumor homing
peptide 62Cys Gly Arg Glu Cys Pro Arg Leu Cys Gln Ser Ser Cys1 5
106313PRTArtificial Sequencesynthetic construct; homing peptide
63Cys Asn Gly Arg Cys Val Ser Gly Cys Ala Gly Arg Cys1 5
10648PRTArtificial Sequencesynthetic construct; tumor homing
peptide 64Cys Leu Ser Gly Ser Leu Ser Cys1 5657PRTArtificial
Sequencesynthetic construct; tumor homing peptide 65Cys Gly Ser Leu
Val Arg Cys1 5669PRTArtificial Sequencesynthetic construct; homing
peptide 66Ala Leu Asn Gly Arg Glu Glu Ser Pro1 5679PRTArtificial
Sequencesynthetic construct; homing peptide 67Cys Val Leu Asn Gly
Arg Met Glu Cys1 5685PRTArtificial Sequencesynthetic construct;
homing peptide 68Cys Asn Gly Arg Cys1 56910PRTArtificial
Sequencesynthetic construct; homing peptide 69Cys Glu Met Cys Asn
Gly Arg Cys Met Gly1 5 107010PRTArtificial Sequencesynthetic
construct; homing peptide 70Cys Pro Leu Cys Asn Gly Arg Cys Ala
Leu1 5 107110PRTArtificial Sequencesynthetic construct; homing
peptide 71Cys Pro Thr Cys Asn Gly Arg Cys Val Arg1 5
107210PRTArtificial Sequencesynthetic construct; homing peptide
72Cys Gly Val Cys Asn Gly Arg Cys Gly Leu1 5 107310PRTArtificial
Sequencesynthetic construct; homing peptide 73Cys Glu Gln Cys Asn
Gly Arg Cys Gly Gln1 5 107410PRTArtificial Sequencesynthetic
construct; homing peptide 74Cys Arg Asn Cys Asn Gly Arg Cys Glu
Gly1 5 107510PRTArtificial Sequencesynthetic construct; homing
peptide 75Cys Val Leu Cys Asn Gly Arg Cys Trp Ser1 5
107610PRTArtificial Sequencesynthetic construct; homing peptide
76Cys Val Thr Cys Asn Gly Arg Cys Arg Val1 5 107710PRTArtificial
Sequencesynthetic construct; homing peptide 77Cys Thr Glu Cys Asn
Gly Arg Cys Gln Leu1 5 107810PRTArtificial Sequencesynthetic
construct; homing peptide 78Cys Arg Thr Cys Asn Gly Arg Cys Leu
Glu1 5 107910PRTArtificial Sequencesynthetic construct; homing
peptide 79Cys Glu Thr Cys Asn Gly Arg Cys Val Gly1 5
108010PRTArtificial Sequencesynthetic construct; homing peptide
80Cys Ala Val Cys Asn Gly Arg Cys Gly Phe1 5 108110PRTArtificial
Sequencesynthetic construct; homing peptide 81Cys Arg Asp Leu Asn
Gly Arg Lys Val Met1 5 108210PRTArtificial Sequencesynthetic
construct; homing peptide 82Cys Ser Cys Cys Asn Gly Arg Cys Gly
Asp1 5 108310PRTArtificial Sequencesynthetic construct; homing
peptide 83Cys Trp Gly Cys Asn Gly Arg Cys Arg Met1 5
108410PRTArtificial Sequencesynthetic construct; homing peptide
84Cys Pro Leu Cys Asn Gly Arg Cys Ala Arg1 5 108510PRTArtificial
Sequencesynthetic construct; homing peptide 85Cys Lys Ser Cys Asn
Gly Arg Cys Leu Ala1 5 108610PRTArtificial Sequencesynthetic
construct; homing peptide 86Cys Val Pro Cys Asn Gly Arg Cys His
Glu1 5 10878PRTArtificial Sequencesynthetic construct; homing
peptide 87Cys Val Leu Asn Gly Arg Met Glu1 58810PRTArtificial
Sequencesynthetic construct; homing peptide 88Cys Lys Val Cys Asn
Gly Arg Cys Cys Gly1 5 10899PRTArtificial Sequencesynthetic
construct; homing peptide 89Xaa Xaa Cys Asn Gly Arg Cys Xaa Xaa1
59011PRTArtificial Sequencesynthetic construct; homing peptide
90Cys Xaa Xaa Xaa Asn Gly Arg Xaa Xaa Xaa Cys1 5
109111PRTArtificial Sequencesynthetic construct; homing peptide
91Cys Asn Gly Arg Cys Xaa Xaa Xaa Xaa Xaa Xaa1 5 10928PRTArtificial
Sequencesynthetic construct; lung homing peptide 92Val Gly Val Gly
Glu Trp Ser Val1 5937PRTArtificial Sequencesynthetic construct;
intestine homing peptide 93Tyr Ser Gly Lys Trp Gly Trp1
5947PRTArtificial Sequencesynthetic construct; uterus homing
peptide 94Gly Leu Ser Gly Gly Arg Ser1 5957PRTArtificial
Sequencesynthetic construct; adrenal gland homing peptide 95Leu Met
Leu Pro Arg Ala Asp1 5967PRTArtificial Sequencesynthetic construct;
adrenal gland homing peptide 96Leu Pro Arg Tyr Leu Leu Ser1
5979PRTArtificial Sequencesynthetic construct; retina homing
peptide 97Cys Ser Cys Phe Arg Asp Val Cys Cys1 5989PRTArtificial
Sequencesynthetic construct; retina homing peptide 98Cys Arg Asp
Val Val Ser Val Ile Cys1 5997PRTArtificial Sequencesynthetic
construct; gut homing peptide 99Tyr Ser Gly Lys Trp Gly Lys1
51008PRTArtificial Sequencesynthetic construct; gut homing peptide
100Gly Ile Ser Ala Leu Val Leu Ser1 51017PRTArtificial
Sequencesynthetic construct; gut homing peptide 101Ser Arg Arg Gln
Pro Leu Ser1 51027PRTArtificial Sequencesynthetic construct; gut
homing peptide 102Met Ser Pro Gln Leu Ala Thr1 51037PRTArtificial
Sequencesynthetic construct; gut homing peptide 103Met Arg Arg Asp
Glu Gln Arg1 51047PRTArtificial Sequencesynthetic construct; gut
homing peptide 104Gln Val Arg Arg Val Pro Glu1 51057PRTArtificial
Sequencesynthetic construct; gut homing peptide 105Val Arg Arg Gly
Ser Pro Gln1 51067PRTArtificial Sequencesynthetic construct, gut
homing peptide 106Gly Gly Arg Gly Ser Trp Glu1 51077PRTArtificial
Sequencesynthetic construct; heart homing peptide 107His Gly Arg
Val Arg Pro His1 51089PRTArtificial Sequencesynthetic construct;
tumor homing peptide 108Cys Ser Arg Pro Arg Arg Ser Glu Cys1
51099PRTArtificial Sequencesynthetic construct; tumor homing
peptide 109Cys Ser Arg Pro Arg Arg Ser Val Cys1 51109PRTArtificial
Sequencesynthetic construct; tumor homing peptide 110Cys Ser Arg
Pro Arg Arg Ser Trp Cys1 511131PRTArtificial Sequencesynthetic
construct; tumor homing peptide 111Lys Asp Glu Pro Gln Arg Arg Ser
Ala Arg Leu Ser Ala Lys Pro Ala1 5 10 15Pro Pro Lys Pro Glu Pro Lys
Pro Lys Lys Ala Pro Ala Lys Lys 20 25 3011210PRTArtificial
Sequencesynthetic construct; tumor homing peptide 112Pro Gln Arg
Arg Ser Ala Arg Leu Ser Ala1 5 1011310PRTArtificial
Sequencesynthetic construct; tumor homing peptide 113Pro Lys Arg
Arg Ser Ala Arg Leu Ser Ala1 5 101149PRTArtificial
Sequencesynthetic construct; brain homing peptide 114Cys Asn Ser
Arg Leu His Leu Arg Cys1 51159PRTArtificial Sequencesynthetic
construct; brain homing peptide 115Cys Glu Asn Trp Trp Gly Asp Val
Cys1 511621PRTArtificial Sequencesynthetic construct; brain homing
peptide 116Trp Arg Cys Val Leu Arg Glu Gly Pro Ala Gly Gly Cys Ala
Trp Phe1 5 10 15Asn Arg His Arg Leu 201179PRTArtificial
Sequencesynthetic construct; brain homing peptide 117Cys Leu Ser
Ser Arg Leu Asp Ala Cys1 51188PRTArtificial Sequencesynthetic
construct; brain homing peptide 118Cys Val Leu Arg Gly Gly Arg Cys1
51199PRTArtificial Sequencesynthetic construct; brain homing
peptide 119Cys Asn Ser Arg Leu Gln Leu Arg Cys1 51207PRTArtificial
Sequencesynthetic construct; brain homing peptide 120Cys Gly Val
Arg Leu Gly Cys1 51218PRTArtificial Sequencesynthetic construct;
brain homing peptide 121Cys Lys Asp Trp Gly Arg Ile Cys1
51228PRTArtificial Sequencesynthetic construct; brain homing
peptide 122Cys Leu Asp Trp Gly Arg Ile Cys1 51238PRTArtificial
Sequencesynthetic construct; brain homing peptide 123Cys Thr Arg
Ile Thr Glu Ser Cys1 51247PRTArtificial Sequencesynthetic
construct; brain homing peptide 124Cys Glu Thr Leu Pro Ala Cys1
51258PRTArtificial Sequencesynthetic construct; brain homing
peptide 125Cys Arg Thr Gly Thr Leu Phe Cys1 51268PRTArtificial
Sequencesynthetic construct; brain homing peptide 126Cys Gly Arg
Ser Leu Asp Ala Cys1 51279PRTArtificial Sequencesynthetic
construct; brain homing peptide 127Cys Arg His Trp Phe Asp Val Val
Cys1 51288PRTArtificial Sequencesynthetic construct; brain homing
peptide 128Cys Ala Asn Ala Gln Ser His Cys1 51298PRTArtificial
Sequencesynthetic construct; brain homing peptide 129Cys Gly Asn
Pro Ser Tyr Arg Cys1 513020PRTArtificial Sequencesynthetic
construct; brain homing peptide 130Tyr Pro Cys Gly Gly Glu Ala Val
Ala Gly Val Ser Ser Val Arg Thr1 5 10 15Met Cys Ser Glu
2013120PRTArtificial Sequencesynthetic construct; brain homing
peptide 131Leu Asn Cys Asp Tyr Gln Gly Thr Asn Pro Ala Thr Ser Val
Ser Val1 5 10 15Pro Cys Thr Val 201327PRTArtificial
Sequencesynthetic construct; kidney homing peptide 132Cys Leu Pro
Val Ala Ser Cys1 51337PRTArtificial Sequencesynthetic construct;
kidney homing peptide 133Cys Gly Ala Arg Glu Met Cys1
51348PRTArtificial Sequencesynthetic construct; kidney homing
peptide 134Cys Lys Gly Arg Ser Ser Ala Cys1 51358PRTArtificial
Sequencesynthetic construct; kidney homing peptide 135Cys Trp Ala
Arg Ala Gln Gly Cys1 51368PRTArtificial Sequencesynthetic
construct; kidney homing peptide 136Cys Leu Gly Arg Ser Ser Val
Cys1
51378PRTArtificial Sequencesynthetic construct; kidney homing
peptide 137Cys Thr Ser Pro Gly Gly Ser Cys1 51388PRTArtificial
Sequencesynthetic construct; kidney homing peptide 138Cys Met Gly
Arg Trp Arg Leu Cys1 51398PRTArtificial Sequencesynthetic
construct; kidney homing peptide 139Cys Val Gly Glu Cys Gly Gly
Cys1 51407PRTArtificial Sequencesynthetic construct; kidney homing
peptide 140Cys Val Ala Trp Leu Asn Cys1 51417PRTArtificial
Sequencesynthetic construct; kidney homing peptide 141Cys Arg Arg
Phe Gln Asp Cys1 51427PRTArtificial Sequencesynthetic construct;
kidney homing peptide 142Cys Leu Met Gly Val His Cys1
51438PRTArtificial Sequencesynthetic construct; kidney homing
peptide 143Cys Lys Leu Leu Ser Gly Val Cys1 51448PRTArtificial
Sequencesynthetic construct; kidney homing peptide 144Cys Phe Val
Gly His Asp Leu Cys1 51457PRTArtificial Sequencesynthetic
construct; kidney homing peptide 145Cys Arg Cys Leu Asn Val Cys1
51467PRTArtificial Sequencesynthetic construct; kidney homing
peptide 146Cys Lys Leu Met Gly Glu Cys1 51479PRTArtificial
Sequencesynthetic construct; skin homing peptide 147Cys Ala Arg Ser
Lys Asn Lys Asp Cys1 51487PRTArtificial Sequencesynthetic
construct; heart homing peptide 148Val Val Leu Val Thr Ser Ser1
514913PRTArtificial Sequencesynthetic construct; skin homing
peptide 149Cys Val Ala Leu Cys Arg Glu Ala Cys Gly Glu Gly Cys1 5
1015013PRTArtificial Sequencesynthetic construct; skin homing
peptide 150Cys Ser Ser Gly Cys Ser Lys Asn Cys Leu Glu Met Cys1 5
101518PRTArtificial Sequencesynthetic construct; skin homing
peptide 151Cys Ile Gly Glu Val Glu Val Cys1 51529PRTArtificial
Sequencesynthetic construct; skin homing peptide 152Cys Lys Trp Ser
Arg Leu His Ser Cys1 51539PRTArtificial Sequencesynthetic
construct; skin homing peptide 153Cys Trp Arg Gly Asp Arg Lys Ile
Cys1 51549PRTArtificial Sequencesynthetic construct; skin homing
peptide 154Cys Glu Arg Val Val Gly Ser Ser Cys1 51559PRTArtificial
Sequencesynthetic construct; skin homing peptide 155Cys Leu Ala Lys
Glu Asn Val Val Cys1 515613PRTArtificial Sequencesynthetic
construct; lung homing peptide 156Cys Gly Phe Glu Cys Val Arg Gln
Cys Pro Glu Arg Cys1 5 101578PRTArtificial Sequencesynthetic
construct; lung homing peptide 157Cys Gly Phe Glu Leu Glu Thr Cys1
51588PRTArtificial Sequencesynthetic construct; lung homing peptide
158Cys Thr Leu Arg Asp Arg Asn Cys1 51598PRTArtificial
Sequencesynthetic construct; heart homing peptide 159Cys Leu His
Arg Gly Asn Ser Cys1 516012PRTArtificial Sequencesynthetic
construct; heart homing peptide 160Cys Arg Ser Trp Asn Lys Ala Asp
Asn Arg Ser Cys1 5 101618PRTArtificial Sequencesynthetic construct;
lung homing peptide 161Cys Gly Lys Arg Tyr Arg Asn Cys1
51628PRTArtificial Sequencesynthetic construct; lung homing peptide
162Cys Leu Arg Pro Tyr Leu Asn Cys1 516312PRTArtificial
Sequencesynthetic construct; lung homing peptide 163Cys Thr Val Asn
Glu Ala Tyr Lys Thr Arg Met Cys1 5 1016412PRTArtificial
Sequencesynthetic construct; lung homing peptide 164Cys Arg Leu Arg
Ser Tyr Gly Thr Leu Ser Leu Cys1 5 1016512PRTArtificial
Sequencesynthetic construct; lung homing peptide 165Cys Arg Pro Trp
His Asn Gln Ala His Thr Glu Cys1 5 101669PRTArtificial
Sequencesynthetic construct; pancreas homing peptide 166Ser Trp Cys
Glu Pro Gly Trp Cys Arg1 51677PRTArtificial Sequencesynthetic
construct; pancreas homing peptide 167Cys Lys Ala Ala Lys Asn Lys1
51687PRTArtificial Sequencesynthetic construct; pancreas homing
peptide 168Cys Lys Gly Ala Lys Ala Arg1 5
* * * * *