U.S. patent application number 14/696081 was filed with the patent office on 2015-10-22 for tusc2 therapies.
This patent application is currently assigned to BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. The applicant listed for this patent is Board of Regents, The University of Texas System. Invention is credited to Charles LU, Maria I. NUNEZ, Jack ROTH, David STEWART, Ignacio I. WISTUBA, Shaoyu YAN.
Application Number | 20150297631 14/696081 |
Document ID | / |
Family ID | 45816009 |
Filed Date | 2015-10-22 |
United States Patent
Application |
20150297631 |
Kind Code |
A1 |
ROTH; Jack ; et al. |
October 22, 2015 |
TUSC2 THERAPIES
Abstract
A method for predicting a subject's response to a TUSC2 therapy
is provided. In particular, a subject's response is predicted based
on the proportion of cancers cells that are apoptotic. Also
provided is a method of treating a subject previously predicted to
have a favorable response with a TUSC2 therapy. Methods for
treating cancer by administration of a TUSC2 therapeutic in
conjunction with an EGFR inhibitor and/or a protein kinase
inhibitor are also disclosed. Kits and reagents for use in TUSC2
therapy are provided.
Inventors: |
ROTH; Jack; (Houston,
TX) ; STEWART; David; (Ottawa, CA) ; LU;
Charles; (Houston, TX) ; WISTUBA; Ignacio I.;
(Houston, TX) ; YAN; Shaoyu; (Pearland, TX)
; NUNEZ; Maria I.; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System |
Austin |
TX |
US |
|
|
Assignee: |
BOARD OF REGENTS, THE UNIVERSITY OF
TEXAS SYSTEM
Austin
TX
|
Family ID: |
45816009 |
Appl. No.: |
14/696081 |
Filed: |
April 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14480341 |
Sep 8, 2014 |
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14696081 |
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13410811 |
Mar 2, 2012 |
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14480341 |
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61448463 |
Mar 2, 2011 |
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61472530 |
Apr 6, 2011 |
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61513244 |
Jul 29, 2011 |
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61603686 |
Feb 27, 2012 |
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Current U.S.
Class: |
424/450 ;
424/172.1; 514/24 |
Current CPC
Class: |
C07K 16/2863 20130101;
A61K 9/127 20130101; G01N 2800/52 20130101; A61K 48/005 20130101;
A61K 31/517 20130101; A61K 39/39558 20130101; G01N 2510/00
20130101; A61K 31/7088 20130101; A61K 31/7135 20130101; A61K
39/3955 20130101; A61K 45/06 20130101; C07K 14/47 20130101; C07K
16/3023 20130101; A61K 38/17 20130101; A61K 2039/505 20130101; A61K
38/1709 20130101; A61P 35/00 20180101; G01N 33/5011 20130101; A61K
48/0033 20130101 |
International
Class: |
A61K 31/7135 20060101
A61K031/7135; A61K 39/395 20060101 A61K039/395; A61K 9/127 20060101
A61K009/127; A61K 45/06 20060101 A61K045/06; A61K 31/7088 20060101
A61K031/7088; A61K 31/517 20060101 A61K031/517 |
Goverment Interests
[0002] This invention was made with Government support under grant
nos. CA-016672, CA-070907 and CA-113450 awarded by the National
Institutes of Health. The Government has certain rights in the
invention.
Claims
1-78. (canceled)
79. A method for treating a subject having a cancer, comprising
administering to the subject an effective amount of a TUSC2
therapy, an epidermal growth factor receptor (EGFR) inhibitor and
an anti-inflammatory agent to treat the cancer.
80. The method of claim 79, wherein the TUSC2 therapy is
administered after the epidermal growth factor receptor (EGFR)
inhibitor or the anti-inflammatory agent.
81. The method of claim 79, wherein the TUSC2 therapy is
administered before or essentially simultaneously with the
epidermal growth factor receptor (EGFR) inhibitor or the
anti-inflammatory agent.
82. The method of claim 79, wherein the cancer was determined to
express an EGFR.
83. The method of claim 79, wherein the TUSC2 therapy comprises
administration of a TUSC2 expression vector.
84. The method of claim 83, wherein the TUSC2 expression vector is
plasmid DNA.
85. The method of claim 83, wherein the TUSC2 expression vector is
provided in a liposome.
86. The method of claim 85, wherein the liposome is a
DOTAP:cholesterol liposome.
87. The method of claim 79, wherein the TUSC2 therapy comprises
administration of a TUSC2 polypeptide.
88. The method of claim 87, wherein the TUSC2 polypeptide is
myristoylated.
89. The method of claim 87, wherein the TUSC2 polypeptide is
comprised in a nanoparticle.
90. The method of claim 79, wherein the EGFR inhibitor is a
tyrosine kinase inhibitor.
91. The method of claim 79, wherein the EGFR inhibitor is an EGFR
binding antibody.
92. The method of claim 79, wherein the EGFR inhibitor is
erlotinib, gefitinib, cetuximab, matuzumab, panitumumab, AEE788;
CI-1033, HKI-272, HKI-357 or EKB-569.
93. The method of claim 92, wherein the EGFR inhibitor is
erlotinib.
94. The method of claim 79, wherein the anti-inflammatory agent is
an anti-rheumatic agent, non-steroidal anti-inflammatory agent or a
gold salt.
95. The method of claim 94, wherein the gold salt is auranofin,
gold sodium thiomalate or aurothioglucose.
96. The method of claim 95, wherein the anti-inflammatory agent is
auranofin.
97. The method of claim 79, further comprising administering a
further anti-cancer therapy to the subject.
98. The method of claim 97, wherein the second anti-cancer therapy
is chemotherapy, radiotherapy, gene therapy, surgery, hormonal
therapy, anti-angiogenic therapy or cytokine therapy.
99. The method of claim 79, wherein the cancer is lung cancer.
100. The method of claim 99, wherein the lung cancer is non-small
cell lung cancer.
101. The method of claim 99, wherein the lung cancer is a
metastatic lung cancer.
102. The method of claim 79, wherein the cancer is EGFR inhibitor
or chemotherapy resistant.
103. A method for treating a subject having a cancer, comprising
administering to the subject a TUSC2 therapy, erlotinib and
auranofin in a therapeutically effective amount to treat the
cancer.
104. A pharmaceutical composition comprising a TUSC2 therapeutic,
erlotinib, and auranofin in pharmaceutically acceptable
formulation.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 14/480,341, filed Sep. 8, 2014, which is a divisional of U.S.
application Ser. No. 13/410,811, filed Mar. 2, 2012, which claims
the benefit of U.S. Provisional Patent Application No. 61/448,463,
filed Mar. 2, 2011; 61/472,530, filed Apr. 6, 2011; 61/513,244,
filed Jul. 29, 2011; and 61/603,686, filed Feb. 27, 2012, each of
which is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present embodiments provided herein relate generally to
the fields of molecular biology and cancer therapies.
[0005] 2. Description of Related Art
[0006] As the molecular and genetic mechanisms of oncogenesis
become better elucidated, the focus of cancer therapy has shifted
from the tissue to the genetic level (Bishop, 1991). Mutations in
two major classes of genes, oncogenes and tumor suppressor genes
(TSGs), play central roles in the oncogenic process. TSGs appear to
require homozygous deletion or mutation for inactivation, and
restoration of TSG expression is feasible in human tumors (Lowe et
al., 2004; Roth, 2006). Intratumoral injection of retroviral or
adenoviral vectors expressing the wildtype TSG p53 have been
performed in patients with locally advanced non-small cell lung
cancer and head and neck cancer (Swisher et al., 1999; Roth et al.,
1996; Clayman et al., 1998). These studies have demonstrated that
viral vectors expressing the TSG p53 can be safely injected into
tumors repetitively and can mediate tumor regression. However,
because of the systemic immune response, current viral vectors are
limited to intratumoral administration, which does not have an
effect on tumor metastases, the primary cause of cancer-related
death. Thus development of therapies for intravenous, systemic TSG
replacement would represent a significant advance.
[0007] Homozygous deletions in the 3p21.3 region in lung cancer
cell lines and primary lung tumors have lead to the identification
of multiple genes with tumor suppressor activity from this region
(Lerman et al., 2000).
SUMMARY OF THE INVENTION
[0008] In a first embodiment, there is provided a method for
predicting a response to a TUSC2 (also known as FUS1) therapy in a
subject having a cancer, wherein the subject is being evaluated as
a candidate for TUSC2 therapy, comprising assessing apoptosis in
cancer cells of the subject, wherein if 10% or more of the cancer
cells are apoptotic, then the subject is predicted to have a
favorable response to TUSC2 therapy. For example, in certain
aspects, the subject is predicted to have a favorable response to a
TUSC2 therapy if at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60% or
more of the cancer cells are apoptotic. Conversely, in certain
aspects, if fewer than 10% of the cancer cells the subject are
apoptotic (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less), the
subject is predicted to have a poor response to the TUSC2 therapy.
For example, a favorable response to TUSC2 therapy can comprise a
reduction in tumor size or burden, blocking of tumor growth,
reduction in tumor-associated pain, reduction in cancer associated
pathology, reduction in cancer associated symptoms, cancer
non-progression, increased disease free interval, increased time to
progression, induction of remission, reduction of metastasis, or
increased patient survival.
[0009] In a further embodiment there is provided a method of
selecting a subject having a cancer for a TUSC2 therapy comprising
assessing apoptosis in cancer cells of the subject, wherein if 10%
or more of the cancer cells are apoptotic, then the subject is
selected for the TUSC2 therapy. For example, in certain aspects,
the subject is selected for a TUSC2 therapy if at least 20%, 25%,
30%, 35%, 40%, 45%, 50%, 60% or more of the cancer cells are
apoptotic. On the other hand, if fewer than 10% of the cancer cells
the subject are apoptotic (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
1%, or less), the subject is not selected for the TUSC2
therapy.
[0010] In certain embodiments, assessing apoptosis in cancer cells
or a sample of cancer cells comprises testing the cells for a
marker of apoptosis. A variety of apoptotic markers are known in
the art and can be used to assess apoptosis in cancer cells. For
example, apoptosis can be assessed by testing for caspase
activation, membrane blebbing, loss of mitochondrial membrane
integrity, or DNA fragmentation. Various techniques may be used for
testing cells to assess apoptosis and the testing method will
depend upon the marker that is being used. For example, testing for
apoptosis may comprise performing an ELISA, an immunoassay, a
radioimmunoassay (RIA), an immunoradiometric assay, a
fluoroimmunoassay, a chemiluminescent assay, a bioluminescent
assay, a gel electrophoresis, a Western blot analysis, a southern
blot, flow cytometry, in situ hybridization, positron emission
tomography (PET), single photon emission computed tomography
(SPECT) imaging or a microscopic assay. Thus, in certain aspects,
cancer cells are tested for an apoptotic marker in vivo (e.g., by
PET or SPECT imaging).
[0011] In certain embodiments, testing cells for a marker of
apoptosis comprises contacting the cancer cells with a reagent that
labels cells comprising a marker of apoptosis. Examples of reagents
that can be used to label apoptotic cells include, but are not
limited to, antibodies, small molecules, stains, enzymes nucleic
acid probes and aptamers. For instance, in certain cases, apoptosis
may be assessed by detecting DNA fragmentation, such as by terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling
(TUNEL). In this case a subject would be predicted to have a
favorable response to a TUSC2 therapy, if 10% or more of the cancer
cells in a sample from the patient are TUNEL positive.
[0012] The types of cancer cell samples that are assessed for
apoptosis will depend upon the type of cancer involved. For
example, in the case of a cancer that presents as one or more solid
tumor, the sample may be tumor biopsy sample from a primary cancer
site or a metastatic site. Cancer cells may also be comprised in
other body samples, such as, serum, stool, urine and sputum. In
certain aspects, wherein a sample comprises a large number of
non-cancer cells, assessing cancer cells for apoptosis may
additionally comprise identifying the cancer cells and assessing
the identified cancer cells for apoptosis.
[0013] In a further embodiment, there is provided a method for
treating a subject having a cancer, wherein it was previously
determined (or previously estimated) that at least 10% of the cells
of said cancer are apoptotic, the method comprising administering a
TUSC2 therapy to the subject. For example, in certain aspects, at
least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60% or more of the cancer
cells of the subject were previously determined to be apoptotic. As
used herein a TUSC2 therapy can be any type of therapy that
provides or causes expression of a TUSC2 polypeptide in a cancer
cell (see, e.g., U.S. Pat. No. 7,902,441, incorporated herein by
reference). For example, a TUSC2 therapy may comprise delivery of a
TUSC2 polypeptide or TUSC2 expression vector to cancer cell. A
therapy may, for instance, be delivered via nanoparticles, or in
the case of nucleic acid expression vectors, through the use of a
viral vector.
[0014] In certain embodiments, administration of a TUSC2 therapy
comprises administration of a TUSC2 expression vector, such a DNA
plasmid encoding TUSC2. An expression vector for use according to
the embodiments provided herein will generally comprise control
elements for the expression of the TUSC2 coding sequence. For
example, a vector can comprise a promoter and enhancer element that
are effective for expression in cancer cell of interest. In certain
aspects, for instance, TUSC2 expression is provided by a CMV
promoter or recombinant version thereof, such as the CMV promoter
construct described in U.S. Patent Publn. No. 20070092968,
incorporated herein by reference. In certain embodiments, a vector
provided herein comprises a modified CMV promoter. In certain
embodiments, a vector provided herein comprises a mini-CMV
promoter. Additional expression control elements can be included
such as, for example, an intron, a drug response element, a RNA
stabilizing or destabilizing sequence, a cellular localization
signal, a polyadenylation signal sequence and/or an optimized
translation start codon. Plasmid DNA vectors may also comprise
sequences that help facilitate DNA production, such as, a bacterial
origin of replication and/or a drug resistance marker. In certain
specific aspects, the TUSC2 expression vector is the
pLJ143/KGB2/FUS1 plasmid (SEQ ID NO: 1).
[0015] Methods for delivery of an expression vector to cells (e.g.,
in vivo delivery) are well known in the art and include, without
limitation, nanoparticles (e.g., liposome nanoparticles), lipid
conjugates and viral vectors. In certain aspects, a TUSC2
expression vector is administered in a nanoparticle, such as
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTAP):cholesterol liposome nanoparticle. A skilled artisan will
recognize that various properties of liposomes can be adjusted to
optimize vector delivery. For example, the liposomes may be
adjusted to have a certain size range and/or a particular ratio of
DNA to lipid; DNA to cholesterol; or lipid to cholesterol. For
instance, in the case of a DOTAP:cholesterol liposome, the
DOTAP:cholesterol ratio can be defined as between about 1.5:1 and
1:1.5, such as about 10:9. In further aspects, a TUSC2 expression
vector is provided in a liposome nanoparticle, wherein the
nanoparticles comprise an average particle size of between about 50
and about 500 nm (e.g., 200-500 nm). In still further aspects, a
TUSC2-nanoparticle formulation can be defined by their optical
density (OD), such as having OD.sub.400 of between about 0.65 and
0.95.
[0016] In still further embodiments a TUSC2 therapy can comprise
administration of a TUSC2 polypeptide. Methods for administration
of TUSC2 polypeptide are described for example in U.S. Publn. Nos.
20060251726 and 20090023207, incorporated herein by reference. A
TUSC2 polypeptide may be modified to enhance its activity and/or
ability to enter cancer cells. For instance, the polypeptide can be
modified with a lipid moiety (e.g., myristoylated). In certain
aspects a TUSC2 in provided as a nanoparticle (e.g., a lipid-based
nanoparticle) such as, a superparamagnetic nanoparticle, a
nanoshell, a semiconductor nanocrystal, a quantum dot, a
polymer-based nanoparticle, a silicon-based nanoparticle, a
silica-based nanoparticle, a metal-based nanoparticle, a fullerene
or a nanotube.
[0017] A TUSC2 therapy according to the embodiments provided herein
is typically formulated in a pharmaceutically acceptable carrier.
Such a therapy may be delivered, for example, intravenously,
intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostaticaly,
intrapleurally, intratracheally, intranasally, intravitreally,
intravaginally, intrarectally, topically, intratumorally,
intramuscularly, intraperitoneally, subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially,
intraumbilically, intraocularally, orally, topically, locally, via
inhalation (e.g. aerosol inhalation), by injection or by infusion,
and the route of delivery can depend upon the type of cancer to be
treated. For example, a TUSC2 expression vector complexed with
DOTAP:cholesterol liposome can be administer via intravenous
infusion. In certain specific aspects, a TUSC2 therapy is
administered intravenously in a dose of from about 0.01 mg/kg to
about 0.10 mg/kg, such as a dose of about 0.02, 0.03, 0.04, 0.05,
0.06, 0.07, 0.08 0.09 or 0.10 mg/kg. In further aspects, a TUSC2
therapy, can be administer two or more times (e.g., 3, 4, 5, 6, 7,
8, 9 or 10 times). The timing between doses of such a therapy can
be varied and can include, without limitation, about 1, 2 or 3
days, about 1, 2, or 3 weeks or 1 month or more between doses.
[0018] In yet a further embodiment, there is provided a method for
treating a subject having a cancer, comprising administering a
TUSC2 therapy to the subject in conjunction with one or more
anti-inflammatory agent. For example, the anti-inflammatory agent
may be administered before, after or during a TUSC2 therapy. In a
further aspects, more than one anti-inflammatory agent is
administered, such as administration of an antihistamine and a
corticosteroid. Thus, in certain specific aspects the
anti-inflammatory for use in conjunction with a TUSC2 therapy is
diphenhydramine and/or dexamethasone.
[0019] In certain embodiments, a cancer for treatment or assessment
may present as a tumor, such as primary or metastatic tumor. A
cancer may be an early stage cancer, or may be a metastatic or late
stage cancer. In certain aspects, the cancer is an oral cancer,
oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, a
urogenital cancer, a gastrointestinal cancer, a central or
peripheral nervous system tissue cancer, an endocrine or
neuroendocrine cancer, a hematopoietic cancer, a glioma, a sarcoma,
a carcinoma, a lymphoma, a melanoma, a fibroma, a meningioma, brain
cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer,
biliary cancer, prostatic cancer, pheochromocytoma, pancreatic
islet cell cancer, a Li-Fraumeni tumor, thyroid cancer, parathyroid
cancer, pituitary tumors, adrenal gland tumors, osteogenic sarcoma
tumors, multiple neuroendrcine type I and type II tumors, breast
cancer, lung cancer (e.g., a non-small cell lung cancer (NSCLC) or
small cell lung cancer (SCLC)), head & neck cancer, prostate
cancer, esophageal cancer, tracheal cancer, skin cancer brain
cancer, liver cancer, bladder cancer, stomach cancer, pancreatic
cancer, ovarian cancer, uterine cancer, cervical cancer, testicular
cancer, colon cancer, rectal cancer or skin cancer. In further
aspects a cancer may be defined as a cancer that is resistant to
one or more anticancer therapy, such a chemotherapy resistant
cancer. For example, the cancer may be a cancer that is resistant
to a platinum-based chemotherapeutic, such as cisplatin.
[0020] In further embodiments, a method provided herein further
comprises administering at least a second anticancer therapy. For
example, a method can comprise treating a subject having a cancer,
wherein it was previously determined that at least 10% of the cells
of said cancer are apoptotic, comprising administering a TUSC2
therapy and at least a second anticancer agent to the subject. The
second anticancer therapy may be, without limitation, a surgical
therapy, chemotherapy (e.g., administration of a protein kinase
inhibitor or a EGFR-targeted therapy), radiation therapy,
cryotherapy, hyperthermia treatment, phototherapy, radioablation
therapy, hormonal therapy, immunotherapy, small molecule therapy,
receptor kinase inhibitor therapy, anti-angiogenic therapy,
cytokine therapy or a biological therapies such as monoclonal
antibodies, siRNA, antisense oligonucleotides, ribozymes or gene
therapy. Without limitation the biological therapy may be a gene
therapy, such as tumor suppressor gene therapy, a cell death
protein gene therapy, a cell cycle regulator gene therapy, a
cytokine gene therapy, a toxin gene therapy, an immunogene therapy,
a suicide gene therapy, a prodrug gene therapy, an anti-cellular
proliferation gene therapy, an enzyme gene therapy, or an
anti-angiogenic factor gene therapy.
[0021] In still a further embodiments provided herein is a kit
comprising a TUSC2 therapeutic. For example, in some aspects, a kit
provided herein comprises a TUSC2 therapeutic and a reagent for
testing cells for a marker of apoptosis, such as a TUNEL reagent.
In further aspects, a kit comprises a TUSC2 therapeutic and one or
more anti-inflammatory agents. In still further aspects the kit may
comprise one more additional components including, but not limited
to, a reagent for assessing apoptosis in a cell sample, an
anti-inflammatory agent, pharmaceutically acceptable dilution
agent, a syringe, an infusion bag, an infusion line, and/or a set
of instruction for use of the kit.
[0022] In yet a further embodiment provided herein are
compositions, therapies, and methods for treating a subject having
a cancer, comprising administering to the subject a TUSC2 therapy
(e.g., a TUSC2 polypeptide or a TUSC2 expression vector) in
conjunction with a second anticancer agent, such as a
chemotherapeutic. For example, the chemotherapeutic can be a
protein kinase inhibitor, such as a Src or Akt kinase inhibitor. In
some aspects, the chemotherapeutic is a epidermal growth factor
receptor (EGFR) inhibitor.
[0023] In certain embodiments, a method is provided for treating a
subject having a cancer, comprising administering to the subject a
TUSC2 therapy in conjunction with a protein kinase inhibitor. For
instance, the TUSC2 therapy can be administered, before, after or
essentially concomitantly with the protein kinase inhibitor. Thus,
in some embodiments, a composition is provided comprising a TUSC2
therapeutic and a protein kinase inhibitor in a therapeutically
effective amount to treat a cancer. Protein kinase inhibitors for
use according to the embodiments include, without limitation, EGFR,
VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K,
Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF
inhibitors. For example, the protein kinase inhibitor can be
Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib,
Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib,
Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib,
Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib,
Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, CAL101,
PX-866, LY294002, rapamycin, temsirolimus, everolimus,
ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244,
Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or a
mixture thereof. In certain aspects, the protein kinase inhibitor
is an AKT inhibitor (e.g., MK-2206, GSK690693, A-443654, VQD-002,
Miltefosine or Perifosine).
[0024] EGFR-targeted therapies for use in accordance with the
embodiments include, but are not limited to, inhibitors of
EGFR/ErbB1/HER, ErbB2/Neu/HER2, ErbB3/HER3, and/or ErbB4/HER4. A
wide range of such inhibitors are known and include, without
limitation, tyrosine kinase inhibitors active against the
receptor(s) and EGFR-binding antibodies or aptamers. For instance,
the EGFR inhibitor can be gefitinib, erlotinib, cetuximab,
matuzumab, panitumumab, AEE788; CI-1033, HKI-272, HKI-357 or
EKB-569. In certain embodiments, the compositions and therapies
provided herein are administered systemically or locally. In one
embodiment, the compositions and therapies provided herein are
administered systemically. In certain aspects, an EGFR inhibitor is
administered to a patient before, after or essentially
concomitantly with a TUSC2 therapy. For example, the therapies may
be co-administered, such as by co-administration in an intravenous
infusion. In certain embodiments, TUSC2 and EGFR inhibitors can be
administered in any amount effective to treat cancers. In certain
embodiments, the compositions, therapies, and methods provided
herein comprise administering TUSC2 and EGFR inhibitors in lower
doses than either composition administered alone. In certain
embodiments, the compositions, therapies, and methods comprise
administering TUSC2 and EGFR inhibitors in lower doses that reduce
side effects. In certain embodiments, the compositions, therapies,
and methods comprise administering TUSC2 and EGFR inhibitors in
doses effective to provide additive, cooperative, or synergistic
effect than that provided by either composition administered alone.
In certain aspects, cancers for treatment with such therapies can
be any of those described herein, such as lung cancers (e.g.,
non-small cell lung cancer). In certain preferred aspects, a cancer
for treatment with a combination therapy is an EGFR-expressing
cancer. In certain embodiments, the EGFR-expressing cancer
comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
tumor cells expressing EGFR.
[0025] In yet still a further embodiment provided herein is a
method for treating a subject having a cancer, wherein it was
previously determined that the cancer expresses an EGFR, the method
comprising administering to the subject a TUSC2 therapy in
conjunction with a EGFR inhibitor. In certain embodiments, provided
herein is a method for treating a subject having a cancer
comprising the step of determining whether the cancer expresses an
EGFR, and administering to the subject a TUSC2 and an EGFR
inhibitor. Methods for assessing the EGFR-expression status of a
cancer have been described, for example in U.S. Patent Publn. No.
20110052570, incorporated herein by reference. In certain aspects,
the EGFR-expressing cancer can be a cancer that expresses a mutant
EGFR, such as a cancer expressing an EGFR having a L858R and/or
T790M mutation. In certain embodiments, the compositions and
therapies provided herein are administered to the patient that have
an EGFR-expressing cancer that comprises at least 1%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% tumor cells expressing EGFR. In
still further aspects, the subject for treatment has a cancer that
was previously determined to express an EGFR and in which at least
10% of the cells of the cancer are apoptotic. In certain
embodiments, the methods provided herein further comprise
determining whether at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% of the cells of the EGFR-expressing cancer are apoptotic.
[0026] It is contemplated that any method or composition described
herein can be implemented with respect to any other method or
composition described herein. Likewise, aspects of the present
embodiments discussed in the context of a method for treating a
subject are equally applicable to a method of predicting response
in a subject and vise versa.
[0027] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0028] Certain aspects of the embodiments concern selecting a
subject having a cancer for a TUSC2 therapy or for predicting a
response to a TUSC2 therapy in a subject. In this context, "a poor
response" to a TUSC2 therapy means that administration of a TUSC2
therapy, either alone or in combination with a further anticancer
agent, is predicted to result in no significant treatment of a
cancer (e.g., as measured by reduction of tumor mass, number of
metastases, or rate of cancer cell proliferation) or symptoms of a
cancer. On the other hand, "a favorable response" means that
administration of a TUSC2 therapy, either alone or in combination
with a further anticancer agent, is predicted to result in
significant treatment of a cancer (e.g., as measured by reduction
of tumor mass, number of metastases, or rate of cancer cell
proliferation) or cancer symptoms. For example, a favorable
response can be a significantly increased period of relapse-free
remission in a subject.
[0029] Other objects, features and advantages of the present
embodiments will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments provided herein, are given by way of illustration only,
since various changes and modifications within the spirit and scope
of the present embodiments will become apparent to those skilled in
the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present embodiments. The present embodiments may be
better understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0031] FIG. 1A-C: FIG. 1A, A schematic representation of a the
TUSC2 expression vector pLJ143/KGB2/FUS1 (SEQ ID NO: 1). FIG. 1B,
Change in apoptosis pathway mRNAs analyzed in pre- and
post-treatment biopsy specimens from patient 31 using SA Apoptosis
Signaling Nano-scale PCR Array. Genes in post-treatment samples
that differed from pretreatment controls by >3 fold are shown as
a scatter plot of log 10 post-treatment values vs log 10
pretreatment values. Factors for which mRNA expression increased
>3-fold post treatment appear above the line, while those that
decreased by >3 fold appear below the line. Exogenous TUSC2 mRNA
expression was detected in the post-treatment biopsy from this
patient. FIG. 1C, Canonical apoptosis pathway gene expression
pertubations following TUSC2-nanoparticle treatment as detected by
SA PRC Array and IPA Analysis. Molecules are represented as nodes,
and the biological relationship between two nodes is represented as
an edge (Line). The asterisks indicate up- (single asterisk) or
down- (double asterisk) regulation. Nodes are displayed using
various shapes that represent the functional class of the gene
products. Edges are displayed with various labels that describe the
nature of the relationship between the nodes (e.g., P for
phosphorylation, T for transcription). The identified nodes
indicate perturbation of elements of the intrinsic and extrinsic
apoptotic pathways following treatment with DOTAP:chol-TUSC2.
[0032] FIG. 2A-B: FIG. 2A, In situ Proximity Ligation Assay (PLA)
for TUSC2 protein in tumor biopsies. A synthetic oligopeptide
(GASGSKARGLWPFASAA; SEQ ID NO: 2) derived from the N-terminal
amino-acid sequence of the TUSC2 protein was used to develop the
anti-TUSC2 polyclonal antibody in rabbits used in this study. Red
denotes TUSC2 positivity. DAPI nuclear staining is blue. All panels
represent overlays of TUSC2 antibody and DAPI staining Detailed
methods are provided in the Supplementary Methods. Pre- and
post-treatment biopsies from patients 13, 26, and 31 were tested.
Magnification is .times.40. Panels: (1) anti-TUSC2 antibody; (2)
anti-TUSC2 antibody pre-absorbed with non-specific control peptide
(NSP); (3) anti-TUSC2 antibody pre-absorbed with TUSC2 peptide
(FP); (4) non-specific control antibody; (5) hematoxylin and eosin.
FIG. 2B, Quantitation of PLA signals for pre- and post-treatment
samples from patients 13, 26, and 31. The anti-TUSC2 antibody was
tested under the conditions described in A). The upper panels show
PLA signals from the respective patient biopsies as detected by the
anti-TUSC2 antibody with 400.times. magnification. The lower panel
presents quantitative comparisons of six independent fields from
each biopsy treated under the specified conditions. TUSC2
expression was significantly increased in post-treatment samples
compared to pretreatment samples. TUSC2 expression was not
significantly altered by anti-TUSC2 antibody pre-absorption with
non-specific control peptide (NSP), but was significantly decreased
by pre-absorption with TUSC2 peptide (FP). * p<0.05 compared to
corresponding pretreatment sample; .box-solid. p<0.05 compared
to post-treatment samples unabsorbed or pre-absorbed with NSP. All
comparisons are by two-tailed unpaired Student's t-test assuming
equal variances as determined by F test.
[0033] FIG. 3A-B: DOTAP:chol-TUSC2 metabolic tumor response in a
metastatic lung cancer patient. The patient is a 54 year old female
with a large cell neuroendocrine carcinoma. She had received six
prior chemotherapy regimens. Prior to entry in the protocol, two
hepatic metastases were progressing on gemcitabine. The patient
also had a metastasis in the head of the pancreas and a
peripancreatic lymph node (indicated by arrows). FIG. 3A,
Pretreatment PET scan. The dose of Fluorodeoxyglucose (.sup.18F)
was 8.8mCi. FIG. 3B, Post-treatment PET scan performed 20 days
following the fourth dose of DOTAP:chol-TUSC2. The dose of
Fluorodeoxyglucose (.sup.18F) was 9.0mCi. All scans were performed
within a 60 to 90 minute window after injection.
[0034] FIG. 4: TUSC2 expression was determined by
immunohistochemistry. The dashed line indicates the level of TUSC2
expression in a biopsy of normal bronchial epithelium from one
patient. The asterisks indicate patients who showed stable disease
or minor response following treatment with DOTAP:chol-TUSC2
nanoparticles. No associations between the IHC marker with
treatment outcome was observed.
[0035] FIG. 5: Apoptotic index was determined by TUNEL staining.
The asterisks indicate patients who showed stable disease or minor
response following treatment with DOTAP:chol-TUSC2 nanoparticles. A
maximum pretreatment apoptotic index of greater than 10% was
associated with stable disease or minor response following
treatment with DOTAP:chol-TUSC2 nanoparticles.
[0036] FIG. 6: Fus1 and Erlotinib combined treatment effect on
colony formation of H1299 cells. Graph shows the results of colony
formation assays as change in total colony area relative to control
for each treatment condition. "EV" indicates empty vector; Fus1
indicates a vector containing Fus1; numerical values following "+"
indicate .mu.g of Erlotinib; PBS indicates Phosphate-Buffer Saline
control.
[0037] FIG. 7: Fus1 and Erlotinib combined treatment effect on
colony formation of H322 cells. Graph shows the results of colony
formation assays as change in total colony area relative to control
for each treatment condition. "EV" indicates empty vector; Fus1
indicates a vector containing Fus1; numerical values following "+"
indicate .mu.g of Erlotinib; PBS indicates Phosphate-Buffer Saline
control.
[0038] FIG. 8: Fus1 and Erlotinib combined treatment effect on
colony formation of A549 cells. Graph shows the results of colony
formation assays as change in total colony area relative to control
for each treatment condition. "EV" indicates empty vector; Fus1
indicates a vector containing Fus1; numerical values following "+"
indicate .mu.g of Erlotinib; PBS indicates Phosphate-Buffer Saline
control.
[0039] FIG. 9: Fus1 and Erlotinib combined treatment effect on
colony formation of H460 cells. Graph shows the results of colony
formation assays as change in total colony area relative to control
for each treatment condition. "EV" indicates empty vector; Fus1
indicates a vector containing Fus1; numerical values following "+"
indicate .mu.g of Erlotinib; PBS indicates Phosphate-Buffer Saline
control.
[0040] FIG. 10: Fus1 and Erlotinib combined treatment effect on
colony formation of H1975 cells (H1975 cells have two EGFR
mutations, L858R/T790M). Graph shows the results of colony
formation assays as change in total colony area relative to control
for each treatment condition. "EV" indicates empty vector; Fus1
indicates a vector containing Fus1; numerical values following "+"
indicate .mu.g of Erlotinib; PBS indicates Phosphate-Buffer Saline
control.
[0041] FIG. 11A-B: FACS analysis was used to measure intracellular
levels of TNF-a, IL-15, IL-6, IL1b, IFNg, and IL-8 in peripheral
blood monocytes and lymphocytes in pretreatment and posttreatment
samples 24 hours after administration of the DOTAP:chol-TUSC2. For
one patient peripheral blood mononuclear cells (PBMC) were obtained
14 months following 12 treatments (Post 2). Only IL-15 showed
detectable levels in lymphocytes and monocytes. No statistically
significant increases in the post-treatment samples were observed
for any cytokine. All comparisons are by two-tailed paired
Student's t-test. FIG. 11A, are results from peripheral blood
monocytes (Mo). FIG. 11B, are results from peripheral blood
lymphocytes (Ly).
[0042] FIG. 12A-D: Effects of combination treatment of FUS1 and
gefitinib ("Gef" or "G") and erlotinib ("Erl" or "E") on tumor cell
growth and PTK activities in NSCLC cells in vitro and in vivo. FIG.
12A, Effects on induction of apoptosis using TUNEL reaction by
FACS. FIG. 12B, Effects of FUS1 and erlotinib on tumor cell growth
in resistant H322, H1299. FIG. 12C, Evaluation of therapeutic
efficacy and induction of apoptosis by systemic injection of FUS1
nanoparticles and oral administration of gefitinib in human H322
orthotopic lung tumors in nude mice. Fresh frozen tumors were
stained for apoptosis by in situ TUNEL staining FIG. 12D, Effects
on EGFR, AKT, and ERK activities by western blot analysis.
[0043] FIG. 13: FUS1 nanoparticle and Erlotinib combination therapy
on A549 Lung Colonies. Mice (5-6 wk old nu/nu) were injected in the
tail vein with 10.sup.6 A549 cells. Ten days later treatment was
begun with erlotinib 30 mg/kg orally daily for 7 days and FUS1
nanoparticles (25 .mu.g) intravenously on days 10, 13, and 16. Mice
were killed on day 36 and lung tumors counted. Erlo=erlotinib;
EV=empty vector. The FUS1+erlotinib group is significantly less
than all other groups by the two independent sample Wilcoxon rank
sum test (p<0.0005).
[0044] FIG. 14A-C: Effect of Conditioned Medium (CM) from
FUS1-nanoparticle Treated H1299 Cells on H1299 Tumor Cell Growth
(FIG. 14A) and apoptosis (FIG. 14B). FIG. 14C, Protein profiles of
CMs on ProteinChip Array by SELDI-MS.
[0045] FIG. 15A-B: Bystander effects induced by
FUS1-nanoparticle-mediated gene transfer in NSCLC H1299 cells by
FACS analysis (FIG. 15A and FIG. 15B). The populations of
dead/apoptotic cells are represented by both PI (upper left
quadrant)-positive and PI/GFP ("R+G") positive cells.
FUS1-transfected H1299 cells were used as effecter cells and
Ad-GFP-transduced H1299 cells as target cells and mixed at a ratio
of 1:1.
[0046] FIG. 16: Effect of FUS1 nanoparticles alone on lung cancer
cell lines. Top panel is a representation of Western blot to detect
FUS1 expression in cancer cell lines HCC366, H322, A549 or H2887.
.beta.-actin a was used as a loading control. Bottom panel are
graphs that show the total number of viable cells for each of the
four cell lines calculated at 24, 48 and 72 hours upon treatment
with FUS1 nanoparticles or empty vector (EV).
[0047] FIG. 17: Single drug treatment of MK2206 on lung cancer
cells. Graph shows the inhibitory concentration 50 (IC.sub.50) for
AKT inhibitor MK2206 on various cancer cells. Cell lines that were
further analyzed are indicated by arrows.
[0048] FIG. 18: FUS1/MK2206-induced cell death in lung cancer cell
lines. Graphs show the relative survival rates for the indicated
cancer cells (y-axis) when contacted with empty vector (EV) or FUS1
nanoparticles in the presence of increasing concentrations of
MK2206 (x-axis).
[0049] FIG. 19: FUS1/MK2206 inhibit colony formation in lung cancer
cell lines. Graphs show the relative percent of colony formation by
the indicated cells upon treatment with empty vector (EV); FUS1
nanoparticles (FUS1); MK2206; empty vector+MK2206 (EV+MK); or FUS1
nanoparticles+MK2206 (FUS1+MK). * indicates a statistically
significant difference in the amount of colony formation between
the two treatments.
[0050] FIG. 20: FUS1/MK2206 induced apoptosis in lung cancer cell
lines. The effects of empty vector (EV); FUS1 nanoparticles (FUS1);
MK2206; empty vector+MK2206 (EV+MK2206); or FUS1
nanoparticles+MK2206 (FUS1+MK2206) on the cell cycle were examined
in the four indicated cancer cell line. Treated cells were stained
by propidium iodide (PI) and analyzed by flow cytometry. Histograms
show cell count (y-axis) versus PI intensity as a measure of DNA
content. The horizontal bar in each histogram indicates apoptotic
cells as assessed by PI staining of DNA.
[0051] FIG. 21: Immunoblot of p-AKT, p-AMPK and p-mTOR in
FUS1/MK2206-treated cell lines. Phosphorylation specific antibodies
were used to assess expression of phosphorylated AMPK, AKT, mTOR
and S6K in HCC366 or H322 cells. Cells were treated with empty
vector (EV); FUS1 nanoparticles (FUS1); MK2206; empty vector+MK2206
(EV+MK); or FUS1 nanoparticles+MK2206 (FUS1+MK) prior to assessment
for phosphorylated protein expression. Immunoblot of .beta.-actin
was used as a loading control.
[0052] FIG. 22: The effect of AMP-activated protein kinase
(AMPK)-specific siRNA on FUS1/MK2206-induced cell death. Cell
survival was assessed in HCC366 and H322 cells treated with FUS1
nanoparticles and various concentrations of MK2206 in the presence
or absence of siRNA targeted to AMPK. Top panels are
representations of Western blots confirming effective knock-down of
AMPK expression upon introduction of siRNA. Bottom panels are
graphs showing relative cell survival (y-axis) at various
concentrations of MK2206 (x-axis) with and without AMPK siRNA as
indicated.
[0053] FIG. 23: The effect of AMPK inhibitor on FUS1/MK2206-induced
cell death. Cell survival was assessed in HCC366 and H322 cells
treated with FUS1 nanoparticles and various concentrations of
MK2206 in the presence or absence of AMPK inhibitor Compound C.
Graphs show relative cell survival (y-axis) at various
concentrations of MK2206 (x-axis) with and without AMPK inhibitor
as indicated.
[0054] FIG. 24: Combination effects of FUS1 and MK2206 in H322
xenograft mouse model. The chart shows the effects of empty vector
(EV); FUS1 nanoparticles (FUS1); MK2206; empty vector+MK2206
(EV+MK2206); or FUS1 nanoparticles+MK2206 (FUS1+MK2206) on H322
tumor growth in vivo. Total tumor volume (y-axis) is plotted as
function of time (x-axis). Expression of FUS1 and activity of
MK2206 (as evidenced by reduced p-AKT expression) was
histologically confirmed in samples from the mice.
[0055] FIG. 25: Proposed mechanism of FUS1/MK2206-induced cell
death through AKT/AMPK/mTOR pathway. Schematic shows example
members of a signaling pathway modulated by FUS1 and MK2206
treatment.
[0056] FIG. 26A-B: Afatinib synergistically inhibits colony
formation when used in conjunction with TUSC2 nanopraticles.
Results are shown for colony formation assays in H1299 (FIG. 26A)
or H322 (FIG. 26B) cells. TUSC2 nanoparticle treatment is indicated
by "FUS1" versus control treatment "301." These treatments were
applied in conjunction with control treatment "CTR"; 0.5 or 1.0
.mu.g of Erlotinib ("Erlo-0.5" or "Erlo-1.0"); or 0.5, 1.0 or 2.0
.mu.g of Afatinib ("Afa-0.5", "Afa-1.0" or "Afa-2.0") as
indicated.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0057] The development of cancer involves the deregulation of
number of cellular pathways that control normal cell growth.
Crucially, healthy cells express a number of tumor suppressor
genes, which act as molecular gatekeepers and prevent uncontrolled
cell division. A necessary step in the development of a cancer
cells, therefore, is disruption of tumor suppressor signaling
pathways. In view of this, one promising avenue for cancer therapy
involves expression of tumor suppressor genes in cancer cells to
restore normal cellular growth controls. Such therapies may prove
less toxic than standard radiation and chemotherapeutic regimes, as
normal, noncancerous cells, naturally express the suppressor
genes.
[0058] The studies detailed herein demonstrate the therapeutic
delivery of TUSC2 was safe, resulted in disease stabilization in a
number of the study patients and shows promise for clinical effect.
In the study, thirty-one human patients were treated at 6 dose
levels ranging from 0.01 to 0.09 milligrams per kilogram or
DOTAP:chol-TUSC2. The therapy resulted increased expression of
TUSC2 in post-treatment tumor specimens but not in pretreatment
specimens or peripheral blood lymphocyte controls. Likewise, TUSC2
protein expression was effectively detected in post-treatment
tissues and expression was shown to alter the regulation of a
number of genes involved in both the intrinsic and extrinsic
apoptotic pathways (see, FIG. 1C). Five patients achieved achieving
stable disease (2.6-10.8 months, including 2 minor responses). One
patient with stable disease had a metabolic response on positron
emission tomography (PET) imaging (FIG. 3). Thus, the studies
demonstrate the safety of TUSC2 therapy and indicate that the
therapy may be effective to improve patient outcome.
[0059] Owing to the fact that all cancer cells develop differently,
one crucial impediment to anticancer therapy is that certain
cancers respond to a given therapy while others prove recalcitrant.
Therefore, in order to provide effective therapy, methods are
needed to identify subjects that will favorably respond to a given
therapy. The studies detailed here demonstrate for the first time
effective methods to identify cancer patients who are responders to
a TUSC2 therapy regime. Counter intuitively, it has been
demonstrated that cancers exhibiting a high proportion of apoptotic
cells are more susceptible to therapy. Specifically, patients that
respond favorably to the TUSC2 therapy had cancers, wherein about
10% or more of the cells were identified as apoptotic by a TUNEL
assay to detect DNA fragmentation (see, e.g., FIG. 5). Accordingly,
provided herein is a method for predicting whether a subject will
have a favorable response to a TUSC2 therapy by testing the cancer
cells of the subject to determine the proportion of the cells that
are apoptotic. Likewise, methods for treating subjects who are
previously determined to have a cancer with a high proportion
(e.g., 10% or greater) of apoptotic cells are provided. These
methods will allow for identification and treatment of populations
of cancer patients who will likely response to TUSC2 therapies
thereby improving the efficacy of the therapy.
[0060] Despite the relatively low toxicity exhibited by TUSC2
therapeutics, minor adverse responses were initially noted in the
clinical studies described here. It was found, however, that such
adverse reactions could be nearly completely ablated by the use of
an anti-inflammatory regime in conjunction with the TUSC2 therapy.
Specifically, the administration of an antihistamine
(diphenhydramine) and a corticosteroid (dexamethasone) immediately
preceding and immediately following the TUSC2 administration
protected patients from adverse reactions and allowed for higher
doses of the TUSC2 therapeutic to be administered. This is an
important finding given the possible need to provide higher doses
of the therapy for effective clinical benefit. Thus, a methods is
provided for treating a patient with a TUSC2 therapeutic comprising
administering the therapy in conjunction with one or more
anti-inflammatory agent. Accordingly, the two therapies can be
included in combined therapeutic regime to increase anti-cancer
efficacy. Likewise, when combined the dose of one or both therapies
could be reduced while still maintaining effectiveness, thereby
potentially reducing the side effects of the combined therapy. Such
combined regimes may also show particular effect in specific
patient populations, such as those having cancers that are EGFR
positive, demonstrate increased apoptotic activity and/or exhibit
increased kinase (e.g., AKT) activity.
[0061] The embodiments and working examples provided herein
demonstrate for the first time that TUSC2 therapies show increased
effectiveness when combined with EGFR-targeted therapies.
Specifically, studies presented here show that erlotinib, an EGFR
tyrosine kinase inhibitor, is significantly more effective in
reducing cancer cell growth (as measured by colony formation) when
applied in conjunction with a TUSC2 therapeutic (see, e.g., FIGS.
6-10 and Tables 5-8). In all five cancer cell lines that were
tested application of TUSC2 nanoparticles was able to sensitize the
cancer cell to the effects of Erlotinib. The combined treatment was
able to achieve a similar level of inhibition of colony formation
while using less than half of the amount of a erlotinib (1.0 .mu.g
versus 2.3 .mu.g) treatment when provided alone. Moreover, at the
higher erlotinib treatment levels (2.3 .mu.g) the combination
treatment far exceeded the amount of inhibition that was achievable
with either agent alone. These results were confirmed using an in
vivo murine tumor explants model. Results shown in FIG. 13
demonstrate that the combination of TUSC2 therapy and erlotinib
significantly reduced the number of tumor nodules in the lungs of
treated animals as compared to either treatment alone. Further
studies in four different cancer cell lines confirmed that TUSC2
therapy likewise was able to sensitize cells to killing by a second
EGFR-targeted agent, gefitinib (FIG. 12). Moreover, the tyrosine
kinase inhibitor afatinib, which likewise acts to inhibit EGFR
signaling, synergistically inhibited cancer cell colony formation
when combined with TUSC2 treatment (FIG. 26A-B). Thus, TUSC2
therapy can be used to sensitize cancer cells to the effects of
EGFR-targeted therapies (such as erlotinib, afatinib or gefitinib)
and thereby reduce the effective amount of the EGFR-targeted
therapy required for effective treatment of a cancer.
[0062] Yet further studies presented here demonstrate that TUSC2
therapeutics are also able to sensitized cancer cells to the
effects of protein kinase inhibitors. For example, as shown in FIG.
18, the cancer cell killing effect of AKT kinase inhibitor MK2206
were greatly increased when the inhibitor was used in conjunction
with TUSC2 nanoparticles. Interestingly, TUSC2 treatment was able
to render cells that were otherwise highly resistant to the AKT
inhibitor (such as HCC366 cells) susceptible to MK2206 treatment.
Combination treatment was also found to be significantly more
effective than either treatment alone at reducing colony formation
in cancer cells (FIG. 19) and in inducing apoptosis in these cells
(FIG. 20). The ability of TUSC2 therapy to sensitize cells to AKT
protein kinase therapy was specifically quantified in Table 13,
which shows that TUSC2 treatment reduced the effective IC.sub.50 of
MK2206 at least 5-fold and, it some cases, by as much as 16-fold.
Furthermore the combined effectiveness of the therapies was
confirmed in vivo using a murine tumor explants model. As shown in
FIG. 24, the combined administration of TUSC2 nanoparticles and
MK2206 was far more effective than each therapy in isolation at
preventing tumor growth and tumor growth in co-treated animals was
infect very minimal. Thus, the TUSC2 therapies described here can
be combined with protein kinase inhibitor therapies to further
increase the effectiveness of these inhibitors and even to reverse
resistance to such agents in cancers.
[0063] I. Assessing Apoptosis
[0064] As detailed above, methods for determining the proportion of
apoptotic cancer cells in a subject can be useful predicting a
response to TUSC2 therapy. Assessment of apoptosis may be performed
on a sample of cancer cells from the subject or in vivo assessment
may be performed (e.g., by imaging). For example, methods for in
vivo assessment of apoptosis were recently review by Blankenberg
2008 and Zhao 2009 (both of which are incorporated herein by
reference). A wide range of methods may be employed to identify
apoptotic cells, ranging from simple light microscopy to molecular
assays that detect changes in cellular membrane integrity, changes
in cellular gene expression, activation proteases and DNA
fragmentation.
[0065] In certain aspects, the proportion (i.e., percentage) of
cells in a sample that are apoptotic. However, it will be
recognized that not all methods for determining apoptosis provide
an assessment on a cell-by-cell basis. Thus, in certain aspects, a
level of apoptosis is determined for a sample, wherein the level
correlates with a particular portion of apoptotic cells (e.g., at
least about 10% apoptotic cells). For example, level of apoptosis
may be determine for the cancer cells of patient (e.g., the
intensity of in vivo Annexin V staining) and the level correlated
to a percentage of apoptotic cells to determine with the subject
will response favorably to a TUSC2 therapy.
[0066] a. DNA Fragmentation
[0067] During apoptosis nuclear DNA is fragmented and these changes
can be detected to assess apoptosis in a sample. Fragmented DNA may
be detected, for example, by light microscopy, which can reveal
condensation and margination of chromatin. Fragmentation of DNA can
also be directly assessed using a separative method, e.g.,
chromatography or electrophoresis, to size fractionate the sample.
For example, DNA fragmentation, characteristic of apoptosis, will
be visualized as "ladders" containing a wide range of fragments.
Use of such methods, however, may not provide the best quantitative
assessment of apoptosis.
[0068] Apoptotic cells can also be detected by end labeling of
fragmented DNA. For instance, apoptosis can be assayed using
terminal deoxytransferase-mediated (TdT) dUTP biotin nick
end-labeling (TUNEL; Gavriel et al., J. Cell Biol. 119:493 (1992);
Gorczyca et al., Int. J. Oncol. 1:639 (1992). TUNEL labeling is
effected by incorporation of labeled nucleotides into the 3'
hydroxyl termini of the DNA breaks characteristic of apoptosis
using the enzyme terminal transferase. The incorporated nucleotide
may be labeled by a wide variety of techniques. A typical approach
is to incorporate a ligand such as fluorescein, biotin or
digoxigenin into the nucleotide. If the ligand itself is not
capable of yielding a signal, typically fluorescence, it can be
reacted with a second moiety such as an appropriate antibody or
other receptor which does carry a signal generator after
incorporation of the nucleotide into the DNA terminal. Typical of
such an approach is the use of a digoxigenin carrying nucleotide
with the later reaction with an anti-digoxigenin antibody carrying
Rhodamine, or a bromolated nucleotide with the later reaction with
an appropriate antibody carrying fluorescein.
[0069] A similar labeling method is know as in situ end-labeling
(INSEL). For INSEL, labeling is effected in a similar manner to
TUNEL labeling except that the labeled nucleotide is incorporated
using the enzyme DNA polymerase I or its Klenow fragment. It is
general this method may be somewhat less sensitive and specific
than TUNEL labeling.
[0070] Both TUNEL and INSEL labeling require that certain steps be
taken in order to have the labeled nucleotides access the nuclear
DNA of the cells being analyzed. These steps are well known and
included in the instructions accompanying the commercial kits. In
general they involve rendering the cell walls of the cells being
analyzed permeable to the labeled nucleotide and incorporating
enzyme and removing any protein masking by appropriate protein
digestion such as with pepsin.
[0071] b. Lose of Membrane Integrity
[0072] During apoptosis membrane integrity of the plasma membrane
and mitochondrial membrane is altered and these alterations can be
detected to identify apoptotic cells. For example, light microscopy
may be used to determine the presence of one or more morphological
characteristics of apoptosis such as condensed or rounded
morphology, shrinking and blebbing of the cytoplasm. Likewise,
certain membrane constituents can become exposed to the exterior of
the cell and detected as an indicator of apoptosis.
[0073] Detection of phosphatidylserine on the exterior of cells can
be indicative of apoptosis. For example, commercial kits are
available for the detection of phosphatidylserine via Annexin V
binding (see, e.g., the FITC Annexin V Apoptosis Kit available from
BD Pharmingen.TM.) Labeled Annexin V, such as radiolabeled Annexin
V, may also be used for in vivo imaging of cancer cells to assess
apoptosis (see, e.g., Blankenberg 2008).
[0074] Permeablization of the mitochondrial membrane is also an
indicator of apoptosis. Once, mitochondrial membrane integrity is
lost certain proteins are released to the cytoplasm and detection
of such proteins may be use to assess apoptosis. For example,
detection of cytochrome c (Cyt c) release is a commonly used
apoptotic indicator.
[0075] c. Caspase Activation
[0076] Members of the caspase family of proteins are major
effectors of cellular apoptosis. Caspases are cysteine proteases
that exist within the cell as inactive pro-forms or so-called
"zymogens." The zymogens are cleaved to form active enzymes
following the induction of apoptosis either via the death
receptor-mediated pathway or the mitochondrial pathway of
apoptosis. Depending upon the apoptotic pathway, different caspases
initiate the apoptotic process, with Caspase-8 and -10 initiating
the death receptor pathway, and Caspase-9 initiating the
mitochondrial pathway. Active initiator caspases then activate
(i.e., cleave) effector caspases, for example, Caspase-3, -6, and
-7, to induce apoptosis. These effector caspases cleave key
cellular proteins that lead to the typical morphological changes
observed in cells undergoing apoptosis. Thus, in certain aspects,
apoptosis can be detected by directly detecting caspase activity
(e.g., by use of fluorescently labeled peptides with a caspase
cleavage site) or indirectly detecting the activated enzymes by
detecting a cleaved target polypeptide.
[0077] One protein often used to indirectly detect caspase activity
is poly(ADP-ribose) polymerase (PARP-1). PARD-1 is a DNA-binding
protein that is specifically cleaved during apoptosis. Active
PARP-1 catalyzes the addition of poly(ADP-ribose) chains to some
nuclear proteins and is thought to play a critical role in DNA
damage repair. PARP-1 is rapidly activated during cellular
stresses, such as heat shock, ionizing radiation, exposure to
carcinogens, and treatment with chemotherapy agents. During
apoptosis, activated (i.e., cleaved) caspase-3 in turn cleaves
PARP-1. Thus, the presence and indeed the level of cleaved PARP-1
can be used to assess apoptosis in a sample.
[0078] d. Changes in Gene Expression
[0079] A variety of additional changes in cellular gene expression
occur during apoptosis and can be detected as indicators of
apoptosis. For example, the expression of pro-apoptotic proteins
such as Bid, Bim, Bik, Bmf, Bad, Hrk, BNIP3, Bax, Bak, and Bok may
be used as an apoptotic marker.
[0080] II. Nucleic Acid and Polypeptide Complexes
[0081] In certain aspects, concerns compositions and methods for
delivering a nucleic acid or a polypeptide to a cell. In
particular, provided herein are nanoparticle-nucleic acid or
nanoparticle-polypeptide complexes and methods of administering
such complexes to a subject. The complexes comprise a TUSC2
polypeptide and/or nucleic acid in association with a nanoparticle.
As used herein, "association" means a physical association, a
chemical association or both. For example, an association can
involve a covalent bond, a hydrophobic interaction, encapsulation,
surface adsorption, or the like.
[0082] Polypeptides and nucleic acids typically have difficulty
crossing cellular membranes. Both types of molecules include
charged residues, which hinder membrane binding and membrane
transport into cells. The present embodiments overcome this
difficulty by, providing nanoparticle complexes that facilitate
cellular uptake.
[0083] In accordance with the present embodiments, a polypeptide
and/or nucleic acid may be associated with a nanoparticle to form
nanoparticle complex. In some embodiments, the nanoparticle is a
liposomes or other lipid-based nanoparticle such as a lipid-based
vesicle (e.g., a DOTAP:cholesterol vesicle). As used in cancer
therapy, liposomes take advantage of the increased fenestrations in
the cancer neovasculature to enhance liposome concentration at
tumor sites.
[0084] In other embodiments, the nanoparticle is a non-lipid
nanoparticle, such as an iron-oxide based superparamagnetic
nanoparticles. Superparamagnetic nanoparticles ranging in diameter
from about 10 to 100 nm are small enough to avoid sequestering by
the spleen, but large enough to avoid clearance by the liver.
Particles this size can penetrate very small capillaries and can be
effectively distributed in body tissues. Superparamagnetic
nanoparticles complexes can be used as MRI contrast agents to
identify and follow those cells that take up the therapeutic
complexes. In certain embodiments, the nanoparticle is a
semiconductor nanocrystal or a semiconductor quantum dot, both of
which can be used in optical imaging. In further embodiments, the
nanoparticle can be a nanoshell, which comprises a gold layer over
a core of silica. One advantage of nanoshells is that a
polypeptideor nucleic acid can be conjugated to the gold layer
using standard chemistry. In other embodiments, the nanoparticle
can be a fullerene or a nanotube (Gupta et al., 2005).
[0085] In accordance with the present embodiments, nanoparticle
complexes can be targeted to specific tissues and cells. This can
be accomplished by conjugating a cell targeting moiety to the
nanoparticle. The targeting moiety can be, but is not limited to, a
protein, peptide, lipid, steroid, sugar, carbohydrate or synthetic
compound. Cell targeting moieties such as ligands recognize and
bind to their cognate receptors on the surface of cells. Similarly,
antibody can act as cell targeting moieties by recognizing their
cognate antigens on the cell surface. In certain embodiments,
targeted nanoparticle complexes provided herein can enhance the
specificity of disease treatment and increase the amount of
therapeutic agent entering a targeted cell.
[0086] a. Nanoparticles
[0087] As used herein, the term "nanoparticle" refers to any
material having dimensions in the 1-1,000 nm range. In some
embodiments, nanoparticles have dimensions in the 50-500 nm range.
Nanoparticles used in the present embodiments include such
nanoscale materials as a lipid-based nanoparticle, a
superparamagnetic nanoparticle, a nanoshell, a semiconductor
nanocrystal, a quantum dot, a polymer-based nanoparticle, a
silicon-based nanoparticle, a silica-based nanoparticle, a
metal-based nanoparticle, a fullerene and a nanotube (Ferrari,
2005). The conjugation of polypeptide or nucleic acids to
nanoparticles provides structures with potential application for
targeted delivery, controlled release, enhanced cellular uptake and
intracellular trafficking, and molecular imaging of therapeutic
peptides in vitro and in vivo (West, 2004; Stayton et al., 2000;
Ballou et al., 2004; Frangioni, 2003; Dubertret et al., 2002;
Michalet et al., 2005; Dwarakanath et al., 2004.
[0088] 1. Lipid-Based Nanoparticles
[0089] Lipid-based nanoparticles include liposomes, lipid
preparations and lipid-based vesicles (e.g., DOTAP:cholesterol
vesicles). Lipid-based nanoparticles may be positively charged,
negatively charged or neutral. In certain embodiments, the
lipid-based nanoparticle is neutrally charged (e.g., a DOPC
liposome).
[0090] A "liposome" is a generic term encompassing a variety of
single and multilamellar lipid vehicles formed by the generation of
enclosed lipid bilayers or aggregates. Liposomes may be
characterized as having vesicular structures with a bilayer
membrane, generally comprising a phospholipid, and an inner medium
that generally comprises an aqueous composition. Liposomes provided
herein include unilamellar liposomes, multilamellar liposomes and
multivesicular liposomes. Liposomes provided herein may be
positively charged, negatively charged or neutrally charged. In
certain embodiments, the liposomes are neutral in charge.
[0091] A multilamellar liposome has multiple lipid layers separated
by aqueous medium. They form spontaneously when lipids comprising
phospholipids are suspended in an excess of aqueous solution. The
lipid components undergo self-rearrangement before the formation of
closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991). Lipophilic
molecules or molecules with lipophilic regions may also dissolve in
or associate with the lipid bilayer.
[0092] In specific aspects, a polypeptide or nucleic acids may be,
for example, encapsulated in the aqueous interior of a liposome,
interspersed within the lipid bilayer of a liposome, attached to a
liposome via a linking molecule that is associated with both the
liposome and the polypeptide/nucleic acid, entrapped in a liposome,
complexed with a liposome, or the like.
[0093] A liposome used according to the present embodiments can be
made by different methods, as would be known to one of ordinary
skill in the art. For example, a phospholipid (Avanti Polar Lipids,
Alabaster, Ala.), such as for example the neutral phospholipid
dioleoylphosphatidylcholine (DOPC), is dissolved in tert-butanol.
The lipid(s) is then mixed with a polypeptide, nucleic acid, and/or
other component(s). Tween 20 is added to the lipid mixture such
that Tween 20 is about 5% of the composition's weight. Excess
tert-butanol is added to this mixture such that the volume of
tert-butanol is at least 95%. The mixture is vortexed, frozen in a
dry ice/acetone bath and lyophilized overnight. The lyophilized
preparation is stored at -20.degree. C. and can be used up to three
months. When required the lyophilized liposomes are reconstituted
in 0.9% saline.
[0094] Alternatively, a liposome can be prepared by mixing lipids
in a solvent in a container, e.g., a glass, pear-shaped flask. The
container should have a volume ten-times greater than the volume of
the expected suspension of liposomes. Using a rotary evaporator,
the solvent is removed at approximately 40.degree. C. under
negative pressure. The solvent normally is removed within about 5
min. to 2 hours, depending on the desired volume of the liposomes.
The composition can be dried further in a desiccator under vacuum.
The dried lipids generally are discarded after about 1 week because
of a tendency to deteriorate with time.
[0095] Dried lipids can be hydrated at approximately 25-50 mM
phospholipid in sterile, pyrogen-free water by shaking until all
the lipid film is resuspended. The aqueous liposomes can be then
separated into aliquots, each placed in a vial, lyophilized and
sealed under vacuum.
[0096] The dried lipids or lyophilized liposomes prepared as
described above may be dehydrated and reconstituted in a solution
of a protein or peptide and diluted to an appropriate concentration
with an suitable solvent, e.g., DPBS. The mixture is then
vigorously shaken in a vortex mixer. Unencapsulated additional
materials, such as agents including but not limited to hormones,
drugs, nucleic acid constructs and the like, are removed by
centrifugation at 29,000.times.g and the liposomal pellets washed.
The washed liposomes are resuspended at an appropriate total
phospholipid concentration, e.g., about 50-200 mM. The amount of
additional material or active agent encapsulated can be determined
in accordance with standard methods. After determination of the
amount of additional material or active agent encapsulated in the
liposome preparation, the liposomes may be diluted to appropriate
concentrations and stored at 4.degree. C. until use. A
pharmaceutical composition comprising the liposomes will usually
include a sterile, pharmaceutically acceptable carrier or diluent,
such as water or saline solution.
[0097] In other alternative methods, liposomes can be prepared in
accordance with other known laboratory procedures (e.g., see
Bangham et al., 1965; Gregoriadis, 1979; Deamer and Uster, 1983;
Szoka and Papahadjopoulos, 1978, each incorporated herein by
reference in relevant part). Additional liposomes which may be
useful with the present embodiments include cationic liposomes, for
example, as described in WO02/100435A1, U.S. Pat. No. 5,962,016,
U.S. Application 2004/0208921, WO03/015757A1, WO04029213A2, U.S.
Pat. No. 5,030,453, and U.S. Pat. No. 6,680,068, all of which are
hereby incorporated by reference in their entirety without
disclaimer. A process of making liposomes is also described in
WO04/002453A1. Neutral lipids can be incorporated into cationic
liposomes (e.g., Farhood et al., 1995). Various neutral liposomes
which may be used in certain embodiments are disclosed in U.S. Pat.
No. 5,855,911, which is incorporated herein by reference. These
methods differ in their respective abilities to entrap aqueous
material and their respective aqueous space-to-lipid ratios.
[0098] The size of a liposome varies depending on the method of
synthesis. Liposomes in the present embodiments can be a variety of
sizes. In certain embodiments, the liposomes are small, e.g., less
than about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60
nm, or less than about 50 nm in external diameter. For example, in
general, prior to the incorporation of nucleic acid, a
DOTAP:cholesterol liposome for use according to the present
embodiments comprises a size of about 50 to 500 nm. Such liposome
formulations may also be defined by particle charge (zeta
potential) and/or optical density (OD). For instance, a
DOTAP:cholesterol liposome formulation will typically comprise an
OD.sub.400 of less than 0.45 prior to nucleic acid incorporation.
Likewise, the overall charge of such particles in solution can be
defined by a zeta potential of about 50-80 mV.
[0099] In preparing such liposomes, any protocol described herein,
or as would be known to one of ordinary skill in the art may be
used. Additional non-limiting examples of preparing liposomes are
described in U.S. Pat. Nos. 4,728,578, 4,728,575, 4,737,323,
4,533,254, 4,162,282, 4,310,505, and 4,921,706; International
Applications PCT/US85/01161 and PCT/US89/05040; U.K. Patent
Application GB 2193095 A; Mayer et al., 1986; Hope et al., 1985;
Mayhew et al. 1987; Mayhew et al., 1984; Cheng et al., 1987; and
Liposome Technology, 1984, each incorporated herein by
reference).
[0100] In certain embodiments, the lipid based nanoparticle is a
neutral liposome (e.g., a DOPC liposome). "Neutral liposomes" or
"non-charged liposomes", as used herein, are defined as liposomes
having one or more lipid components that yield an
essentially-neutral, net charge (substantially non-charged). By
"essentially neutral" or "essentially non-charged", it is meant
that few, if any, lipid components within a given population (e.g.,
a population of liposomes) include a charge that is not canceled by
an opposite charge of another component (i.e., fewer than 10% of
components include a non-canceled charge, more preferably fewer
than 5%, and most preferably fewer than 1%). In certain
embodiments, neutral liposomes may include mostly lipids and/or
phospholipids that are themselves neutral under physiological
conditions (i.e., at about pH 7).
[0101] Liposomes and/or lipid-based nanoparticles of the present
embodiments may comprise a phospholipid. In certain embodiments, a
single kind of phospholipid may be used in the creation of
liposomes (e.g., a neutral phospholipid, such as DOPC, may be used
to generate neutral liposomes). In other embodiments, more than one
kind of phospholipid may be used to create liposomes.
[0102] Phospholipids include, for example, phosphatidylcholines,
phosphatidylglycerols, and phosphatidylethanolamines; because
phosphatidylethanolamines and phosphatidyl cholines are non-charged
under physiological conditions (i.e., at about pH 7), these
compounds may be particularly useful for generating neutral
liposomes. In certain embodiments, the phospholipid DOPC is used to
produce non-charged liposomes. In certain embodiments, a lipid that
is not a phospholipid (e.g., a cholesterol) may be used
[0103] Phospholipids include glycerophospholipids and certain
sphingolipids. Phospholipids include, but are not limited to,
dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine
("EPC"), dilauryloylphosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoylphosphatidylcholine ("DPPC"),
distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoyl
phosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoyl
phosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoyl
phosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoyl
phosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol
("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dip
almitoylphosphatidylglycerol ("DPPG"),
distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin
("DSSP"), distearoylphophatidylethanolamine ("DSPE"),
dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic
acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl
phosphatidylethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine
("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain
phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"),
dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine
("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"),
1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"),
1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"),
dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl
phosphatidylcholine ("POPC"), palmitoyloeoyl
phosphatidylethanolamine ("POPE"), lysophosphatidylcholine,
lysophosphatidylethanolamine, and
dilinoleoylphosphatidylcholine.
[0104] Phospholipids may be from natural or synthetic sources.
However, phospholipids from natural sources, such as egg or soybean
phosphatidylcholine, brain phosphatidic acid, brain or plant
phosphatidylinositol, heart cardiolipin and plant or bacterial
phosphatidylethanolamine are not used, in certain embodiments, as
the primary phosphatide (i.e., constituting 50% or more of the
total phosphatide composition) because this may result in
instability and leakiness of the resulting liposomes.
[0105] 2. DOTAP:Cholesterol Nanoparticle
[0106] In certain embodiments, the lipid-based vesicle is a
DOTAP:cholesterol nanoparticle. DOTAP:cholesterol nanoparticles are
prepared by mixing the cationic lipid DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)-propane) with cholesterol.
Vesicles prepared with DNA can form a structure (called a
"sandwich`) where the DNA appears to be condensed between two lipid
bilayers (U.S. Pat. Nos. 6,770,291 and 6,413,544).
[0107] A DOTAP:cholesterol-nucleic acid complex can be prepared as
in the following non-limiting example. The DOTAP:cholesterol (DC)
nanoparticles (sized 50 to 500 nm) are synthesized as described
previously (U.S. Pat. Nos. 6,770,291 and 6,413,544; Templeton,
1997). Briefly, 420 mg of DOTAP and 208 mg of cholesterol are
measure and mixed together with 30 ml of chloroform. Mixture is
then allowed to dry on a rotary evaporator for 30 minutes and
freeze dry for 15 minutes. The dried mixture is reconstituted in 30
ml of D5W by swirling at 50.degree. C. for 45 minutes and
37.degree. C. for 10 minutes. The mixture is ten subjected to low
frequency sonication for five minutes to form liposomes.
DOTAP:cholesterol liposome are then heated to 50.degree. C. and
sequentially filtered through 1.0, 0.45, 0.2 and 0.1 .mu.m sterile
Whatman filters. The synthesized nanoparticles are stored at
4.degree. C. and used for preparing nanoparticle complexes. The
formulated DOTAP:cholesterol liposome should be evenly dispersed
with a particle size of 50-250 nm, an OD.sub.400 of less than 0.45
and zeta potential of 50-80 mV. Residual CHCl.sub.3 levels should
be less than 60 ppm.
[0108] To prepare DOTAP:cholesterol-nucleic acid nanoparticles, 240
.mu.l of liposomes (see above) are diluted in 360 .mu.l D5W at room
temperature. DNA (.about.5 mg/ml) is added to the mixture to a
total volume of 600 .mu.l. The mixture is moved up and down in a
pipet to mix. Once settled the mixture should have a an OD.sub.400
of between 0.65 and 0.95, a particle size of 200-500 nm and be
confirmed gram stain negative. The liposome complexes are stored at
between 3.degree. C. and 28.degree. C. and agitated as little as
possible.
[0109] b. Targeting of Nanoparticles
[0110] Targeted delivery is achieved by the addition of ligands
without compromising the ability of nanoparticles to deliver their
payloads. It is contemplated that this will enable delivery to
specific cells, tissues and organs. The targeting specificity of
the ligand-based delivery systems are based on the distribution of
the ligand receptors on different cell types. The targeting ligand
may either be non-covalently or covalently associated with a
nanoparticle, and can be conjugated to the nanoparticles by a
variety of methods as discussed herein.
[0111] Examples of proteins or peptides that can be used to target
nanoparticles include transferin, lactoferrin, TGF-.alpha., nerve
growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin,
as well as others (Gupta et al., 2005; Ferrari, 2005).
[0112] III. TUSC2 Expression Vectors
[0113] The term "vector" is used to refer to a carrier nucleic acid
molecule into which a nucleic acid sequence can be inserted for
introduction into a cell where it can be replicated. A nucleic acid
sequence can be "exogenous," which means that it is foreign to the
cell into which the vector is being introduced or that the sequence
is homologous to a sequence in the cell but in a position within
the host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, viruses (bacteriophage,
animal viruses, and plant viruses), and artificial chromosomes
(e.g., YACs). One of skill in the art would be well equipped to
construct a vector through standard recombinant techniques (see,
for example, Maniatis et al., 1989 and Ausubel et al., 1994, both
incorporated herein by reference).
[0114] The term "expression vector" refers to any type of genetic
construct comprising a nucleic acid coding for a RNA capable of
being transcribed. In some cases, RNA molecules are then translated
into a protein, polypeptide, or peptide. In other cases, these
sequences are not translated, for example, in the production of
antisense molecules or ribozymes. Expression vectors can contain a
variety of "control sequences," which refer to nucleic acid
sequences necessary for the transcription and possibly translation
of an operably linked coding sequence in a particular host cell. In
addition to control sequences that govern transcription and
translation, vectors and expression vectors may contain nucleic
acid sequences that serve other functions as well and are described
infra.
[0115] In certain embodiments, provided herein is the use of
nucleic acids TUSC2 coding sequence. For example, such vector can
be used for recombinant production of a TUSC2 polypeptide and/or
for the expression of TUSC2 in vivo in a subject. The sequences may
be modified, given the ability of several different codons to
encode a single amino acid, while still encoding for the same
protein or polypeptide. Optimization of codon selection can also be
undertaken in light of the particular organism used for recombinant
expression or may be optimized for maximal expression in human cell
(e.g., a cancer cell). Vector for use in accordance with the
present embodiments additionally comprise elements that control
gene expression and/or aid in vector production and
purification.
[0116] a. Promoters and Enhancers
[0117] A "promoter" is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
are controlled. It may contain genetic elements at which regulatory
proteins and molecules may bind, such as RNA polymerase and other
transcription factors, to initiate the specific transcription a
nucleic acid sequence. The phrases "operatively positioned,"
"operatively linked," "under control," and "under transcriptional
control" mean that a promoter is in a correct functional location
and/or orientation in relation to a nucleic acid sequence to
control transcriptional initiation and/or expression of that
sequence.
[0118] A promoter generally comprises a sequence that functions to
position the start site for RNA synthesis. The best known example
of this is the TATA box, but in some promoters lacking a TATA box,
such as, for example, the promoter for the mammalian terminal
deoxynucleotidyl transferase gene and the promoter for the SV40
late genes, a discrete element overlying the start site itself
helps to fix the place of initiation. Additional promoter elements
regulate the frequency of transcriptional initiation. Typically,
these are located in the region 30-110 bp upstream of the start
site, although a number of promoters have been shown to contain
functional elements downstream of the start site as well. To bring
a coding sequence "under the control of" a promoter, one positions
the 5' end of the transcription initiation site of the
transcriptional reading frame "downstream" of (i.e., 3' of) the
chosen promoter. The "upstream" promoter stimulates transcription
of the DNA and promotes expression of the encoded RNA.
[0119] The spacing between promoter elements frequently is
flexible, so that promoter function is preserved when elements are
inverted or moved relative to one another. In the tk promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription. A promoter may or may
not be used in conjunction with an "enhancer," which refers to a
cis-acting regulatory sequence involved in the transcriptional
activation of a nucleic acid sequence.
[0120] A promoter may be one naturally associated with a nucleic
acid sequence, as may be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment and/or exon. Such
a promoter can be referred to as "endogenous" or "homologous."
Similarly, an enhancer may be one naturally associated with a
nucleic acid sequence, located either downstream or upstream of
that sequence. Alternatively, certain advantages will be gained by
positioning the coding nucleic acid segment under the control of a
recombinant, exogenous or heterologous promoter, which refers to a
promoter that is not normally associated with a nucleic acid
sequence in its natural environment. A recombinant or heterologous
enhancer refers also to an enhancer not normally associated with a
nucleic acid sequence in its natural environment. Such promoters or
enhancers may include viral promoter and enhancers such as the CMV
promoter.
[0121] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the organelle, cell, tissue, organ, or organism chosen for
expression. Those of skill in the art of molecular biology
generally know the use of promoters, enhancers, and cell type
combinations for protein expression, (see, for example Sambrook et
al. 1989, incorporated herein by reference). The promoters employed
may be constitutive, tissue-specific, inducible, and/or useful
under the appropriate conditions to direct high level expression of
the introduced DNA segment, such as is advantageous in the
large-scale production of recombinant proteins and/or peptides. The
promoter may be heterologous or endogenous.
[0122] Additionally any promoter/enhancer combination (as per, for
example, the Eukaryotic Promoter Data Base EPDB,
www.epd.isb-sib.ch/) could also be used to drive expression. Use of
a T3, T7 or SP6 cytoplasmic expression system is another possible
embodiment. Eukaryotic cells can support cytoplasmic transcription
from certain bacterial promoters if the appropriate bacterial
polymerase is provided, either as part of the delivery complex or
as an additional genetic expression construct.
[0123] b. Translation Initiation Signals
[0124] A specific initiation signal also may be required for
efficient translation of coding sequences. These signals include
the ATG initiation codon or adjacent sequences. Exogenous
translational control signals, including the ATG initiation codon,
may need to be provided. One of ordinary skill in the art would
readily be capable of determining this and providing the necessary
signals. It is well known that the initiation codon must be
"in-frame" with the reading frame of the desired coding sequence to
ensure translation of the entire insert. The exogenous
translational control signals and initiation codons can be either
natural or synthetic. The efficiency of expression may be enhanced
by the inclusion of appropriate transcription enhancer
elements.
[0125] c. Multiple Cloning Sites
[0126] Vectors can include a multiple cloning site (MCS), which is
a nucleic acid region that contains multiple restriction enzyme
sites, any of which can be used in conjunction with standard
recombinant technology to digest the vector (see, for example,
Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997,
incorporated herein by reference). "Restriction enzyme digestion"
refers to catalytic cleavage of a nucleic acid molecule with an
enzyme that functions only at specific locations in a nucleic acid
molecule. Many of these restriction enzymes are commercially
available. Use of such enzymes is widely understood by those of
skill in the art. Frequently, a vector is linearized or fragmented
using a restriction enzyme that cuts within the MCS to enable
exogenous sequences to be ligated to the vector. "Ligation" refers
to the process of forming phosphodiester bonds between two nucleic
acid fragments, which may or may not be contiguous with each other.
Techniques involving restriction enzymes and ligation reactions are
well known to those of skill in the art of recombinant
technology.
[0127] d. Splicing Sites
[0128] Most transcribed eukaryotic RNA molecules will undergo RNA
splicing to remove introns from the primary transcripts. Vectors
containing genomic eukaryotic sequences may require donor and/or
acceptor splicing sites to ensure proper processing of the
transcript for protein expression (see, for example, Chandler et
al., 1997, herein incorporated by reference). Inclusion of such
splice sites also can enhance expression by averting non-sense
mediated decay of resulting RNA transcripts.
[0129] e. Termination Signals
[0130] The vectors or constructs of the present embodiments will
generally comprise at least one termination signal. A "termination
signal" or "terminator" is comprised of the DNA sequences involved
in specific termination of an RNA transcript by an RNA polymerase.
Thus, in certain embodiments a termination signal that ends the
production of an RNA transcript is contemplated. A terminator may
be necessary in vivo to achieve desirable message levels.
[0131] Terminators contemplated for use in the present embodiments
include any known terminator of transcription described herein or
known to one of ordinary skill in the art, including but not
limited to, for example, the termination sequences of genes, such
as for example the bovine growth hormone terminator or viral
termination sequences, such as for example the SV40 terminator. In
certain embodiments, the termination signal may be a lack of
transcribable or translatable sequence, such as due to a sequence
truncation.
[0132] f. Polyadenylation Signals
[0133] In expression, particularly eukaryotic expression, one will
typically include a polyadenylation signal to effect proper
polyadenylation of the transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the present embodiments, and any such
sequence may be employed. Preferred embodiments include the SV40
polyadenylation signal or the bovine growth hormone polyadenylation
signal, convenient and known to function well in various target
cells. Polyadenylation may increase the stability of the transcript
or may facilitate cytoplasmic transport.
[0134] g. Origins of Replication
[0135] In order to propagate a vector in a host cell, it may
contain one or more origins of replication sites (often termed
"ori"), which is a specific nucleic acid sequence at which
replication is initiated. Alternatively an autonomously replicating
sequence (ARS) can be employed if the host cell is yeast.
[0136] h. Selectable and Screenable Markers
[0137] In certain embodiments, cells containing a nucleic acid
construct provided herein may be identified in vitro or in vivo by
including a marker in the expression vector. Such markers would
confer an identifiable change to the cell permitting easy
identification of cells containing the expression vector.
Generally, a selectable marker is one that confers a property that
allows for selection. A positive selectable marker is one in which
the presence of the marker allows for its selection, while a
negative selectable marker is one in which its presence prevents
its selection. An example of a positive selectable marker is a drug
resistance marker.
[0138] Usually the inclusion of a drug selection marker aids in the
cloning and identification of transformants, for example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers. In
addition to markers conferring a phenotype that allows for the
discrimination of transformants based on the implementation of
conditions, other types of markers including screenable markers
such as GFP, whose basis is colorimetric analysis, are also
contemplated. Alternatively, screenable enzymes such as herpes
simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be utilized. One of skill in the art
would also know how to employ immunologic markers, possibly in
conjunction with FACS analysis. The marker used is not believed to
be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable and screenable markers are well
known to one of skill in the art.
[0139] i. Plasmid Vectors
[0140] In certain embodiments, a plasmid vector is contemplated for
use to transform a host cell. In general, plasmid vectors
containing replicon and control sequences which are derived from
species compatible with the host cell are used in connection with
these hosts. The vector ordinarily carries a replication site, as
well as marking sequences which are capable of providing phenotypic
selection in transformed cells. In a non-limiting example, E. coli
is often transformed using derivatives of pBR322, a plasmid derived
from an E. coli species. pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR plasmid, or other microbial
plasmid or phage must also contain, or be modified to contain, for
example, promoters which can be used by the microbial organism for
expression of its own proteins.
[0141] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, the phage lambda GEM.TM.-11 may be utilized in making a
recombinant phage vector which can be used to transform host cells,
such as, for example, E. coli LE392.
[0142] Further useful plasmid vectors include pIN vectors (Inouye
et al., 1985); and pGEX vectors, for use in generating glutathione
S-transferase (GST) soluble fusion proteins for later purification
and separation or cleavage. Other suitable fusion proteins are
those with .beta.-galactosidase, ubiquitin, and the like.
[0143] Bacterial host cells, for example, E. coli, comprising the
expression vector, are grown in any of a number of suitable media,
for example, LB. The expression of the recombinant protein in
certain vectors may be induced, as would be understood by those of
skill in the art, by contacting a host cell with an agent specific
for certain promoters, e.g., by adding IPTG to the media or by
switching incubation to a higher temperature. After culturing the
bacteria for a further period, generally of 2-24 hr, the cells are
collected by centrifugation and washed to remove residual
media.
[0144] j. Viral Vectors
[0145] The ability of certain viruses to infect cells or enter
cells via receptor-mediated endocytosis, and to integrate into host
cell genome and express viral genes stably and efficiently have
made them attractive candidates for the transfer of foreign nucleic
acids into cells (e.g., mammalian cells). Viruses may thus be
utilized that encode and express TUSC2. Non-limiting examples of
virus vectors that may be used to deliver a TUSC2 nucleic acid are
described below.
[0146] Adenoviral Vectors.
[0147] A particular method for delivery of the nucleic acid
involves the use of an adenovirus expression vector. Although
adenovirus vectors are known to have a low capacity for integration
into genomic DNA, this feature is counterbalanced by the high
efficiency of gene transfer afforded by these vectors. "Adenovirus
expression vector" is meant to include those constructs containing
adenovirus sequences sufficient to (a) support packaging of the
construct and (b) to ultimately express a tissue or cell-specific
construct that has been cloned therein. Knowledge of the genetic
organization or adenovirus, a 36 kb, linear, double-stranded DNA
virus, allows substitution of large pieces of adenoviral DNA with
foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
[0148] AAV Vectors.
[0149] The nucleic acid may be introduced into the cell using
adenovirus assisted transfection. Increased transfection
efficiencies have been reported in cell systems using adenovirus
coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992;
Curiel, 1994). Adeno-associated virus (AAV) has a high frequency of
integration and it can infect non-dividing cells, thus making it
useful for delivery of genes into mammalian cells, for example, in
tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host
range for infectivity (Tratschin et al., 1984; Laughlin et al.,
1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details
concerning the generation and use of rAAV vectors are described in
U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein by
reference.
[0150] Retroviral Vectors.
[0151] Retroviruses have the ability to integrate their genes into
the host genome, transferring a large amount of foreign genetic
material, infecting a broad spectrum of species and cell types and
of being packaged in special cell-lines (Miller, 1992). In order to
construct a retroviral vector, a nucleic acid (e.g., one encoding a
protein of interest) is inserted into the viral genome in the place
of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol, and env genes but without the
LTR and packaging components is constructed (Mann et al., 1983).
When a recombinant plasmid containing a cDNA, together with the
retroviral LTR and packaging sequences is introduced into a special
cell line (e.g., by calcium phosphate precipitation for example),
the packaging sequence allows the RNA transcript of the recombinant
plasmid to be packaged into viral particles, which are then
secreted into the culture media (Nicolas and Rubinstein, 1988;
Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally
concentrated, and used for gene transfer. Retroviral vectors are
able to infect a broad variety of cell types. However, integration
and stable expression require the division of host cells (Paskind
et al., 1975).
[0152] Lentiviruses are complex retroviruses, which, in addition to
the common retroviral genes gag, pol, and env, contain other genes
with regulatory or structural function. Lentiviral vectors are well
known in the art (see, for example, Naldini et al., 1996; Zufferey
et al., 1997; Blomer et al., 1997; U.S. Pat. Nos. 6,013,516 and
5,994,136). Some examples of lentivirus include the Human
Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian
Immunodeficiency Virus: SIV. Lentiviral vectors have been generated
by multiply attenuating the HIV virulence genes, for example, the
genes env, vif, vpr, vpu and nef are deleted making the vector
biologically safe.
[0153] Other Viral Vectors.
[0154] Other viral vectors may be employed as vaccine constructs in
the present embodiments. Vectors derived from viruses such as
vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar
et al., 1988), sindbis virus, cytomegalovirus and herpes simplex
virus may be employed. They offer several attractive features for
various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal
and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
[0155] Modified Viruses.
[0156] A nucleic acid to be delivered may be housed within an
infective virus that has been engineered to express a specific
binding ligand. The virus particle will thus bind specifically to
the cognate receptors of the target cell and deliver the contents
to the cell. A novel approach designed to allow specific targeting
of retrovirus vectors was developed based on the chemical
modification of a retrovirus by the chemical addition of lactose
residues to the viral envelope. This modification can permit the
specific infection of hepatocytes via sialoglycoprotein
receptors.
[0157] Another approach to targeting of recombinant retroviruses
was designed in which biotinylated antibodies against a retroviral
envelope protein and against a specific cell receptor were used.
The antibodies were coupled via the biotin components by using
streptavidin (Roux et al., 1989). Using antibodies against major
histocompatibility complex class I and class II antigens, they
demonstrated the infection of a variety of human cells that bore
those surface antigens with an ecotropic virus in vitro (Roux et
al., 1989).
[0158] IV. Pharmaceutical Formulations
[0159] Pharmaceutical compositions provided herein comprise an
effective amount of one or more TUSC2 therapeutic and, optionally,
an additional agent dissolved or dispersed in a pharmaceutically
acceptable carrier. The phrases "pharmaceutical or
pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, such as, for
example, a human, as appropriate. The preparation of a
pharmaceutical composition that contains at least TUSC2 nucleic
acid, peptide or a nanoparticle complex or additional active
ingredient will be known to those of skill in the art in light of
the present disclosure, as exemplified by Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference. Moreover, for animal (e.g.,
human) administration, it will be understood that preparations
should meet sterility, pyrogenicity, general safety and purity
standards as required by FDA Office of Biological Standards.
[0160] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (see, for example, Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated
herein by reference). Except insofar as any conventional carrier is
incompatible with the active ingredient, its use in the therapeutic
or pharmaceutical compositions is contemplated.
[0161] In certain embodiments, the pharmaceutical composition may
comprise different types of carriers depending on whether it is to
be administered in solid, liquid or aerosol form, and whether it
need to be sterile for such routes of administration as injection.
In certain embodiments, pharmaceutical compositions provided herein
can be administered intravenously, intradermally, intraarterially,
intraperitoneally, intralesionally, intracranially,
intraarticularly, intraprostaticaly, intrapleurally,
intratracheally, intranasally, intravitreally, intravaginally,
intrarectally, topically, intratumorally, intramuscularly,
intraperitoneally, subcutaneously, subconjunctival,
intravesicularlly, mucosally, intrapericardially, intraumbilically,
intraocularally, orally, topically, locally, inhalation (e.g.
aerosol inhalation), injection, infusion, continuous infusion,
localized perfusion bathing target cells directly, via a catheter,
via a lavage, in cremes, in lipid compositions (e.g., liposomes),
or by other method or any combination of the forgoing as would be
known to one of ordinary skill in the art (see, for example,
Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing
Company, 1990, incorporated herein by reference).
[0162] In certain embodiments, the pharmaceutical composition is
administered intraperitoneally. In further embodiments, the
pharmaceutical composition is administered intraperitoneally to
treat a cancer (e.g., a cancerous tumor). For example, the
pharmaceutical composition may be administered intraperitoneally to
treat gastrointestinal cancer. In certain embodiments it may be
desirable to administer the pharmaceutical composition into or near
a tumor.
[0163] In certain preferred embodiments, the pharmaceutical
composition is administered orally to treat a cancer (e.g., a
gastrointestinal cancer).
[0164] In certain embodiments, the actual dosage amount of a
composition administered to a patient can be determined by physical
and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic interventions, idiopathy of the patient and
on the route of administration. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject.
[0165] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein. In
other non-limiting examples, a dose may also comprise from about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about
10 microgram/kg/body weight, about 15 microgram/kg/body weight,
about 20 microgram/kg/body weight, about 25 microgram/kg/body
weight, about 30 microgram/kg/body weight, about 35
microgram/kg/body weight, about 0.04 milligram/kg/body weight,
about 0.05 milligram/kg/body weight, about 0.06 milligram/kg/body
weight, about 0.07 milligram/kg/body weight, about 0.08
milligram/kg/body weight, about 0.09 milligram/kg/body weight,
about 0.1 milligram/kg/body weight, about 0.2 milligram/kg/body
weight, to about 0.5 mg/kg/body weight or more per administration,
and any range derivable therein. In non-limiting examples of a
derivable range from the numbers listed herein, a range of about
0.01 mg/kg/body weight to about 0.1 mg/kg/body weight, about 0.04
microgram/kg/body weight to about 0.08 milligram/kg/body weight,
etc., can be administered, based on the numbers described
above.
[0166] In any case, the composition may comprise various
antioxidants to retard oxidation of one or more component.
Additionally, the prevention of the action of microorganisms can be
brought about by preservatives such as various antibacterial and
antifungal agents, including but not limited to parabens (e.g.,
methylparabens, propylparabens), chlorobutanol, phenol, sorbic
acid, thimerosal or combinations thereof.
[0167] The one or more peptides, nanoparticle complexes or
additional agent may be formulated into a composition in a free
base, neutral or salt form. Pharmaceutically acceptable salts,
include the acid addition salts, e.g., those formed with the free
amino groups of a proteinaceous composition, or which are formed
with inorganic acids such as for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric
or mandelic acid. Salts formed with the free carboxyl groups can
also be derived from inorganic bases such as for example, sodium,
potassium, ammonium, calcium or ferric hydroxides; or such organic
bases as isopropylamine, trimethylamine, histidine or procaine.
[0168] In embodiments where the composition is in a liquid form, a
carrier can be a solvent or dispersion medium comprising but not
limited to, water, ethanol, polyol (e.g., glycerol, propylene
glycol, liquid polyethylene glycol, etc.), lipids (e.g.,
triglycerides, vegetable oils, liposomes) and combinations thereof.
The proper fluidity can be maintained, for example, by the use of a
coating, such as lecithin; by the maintenance of the required
particle size by dispersion in carriers such as, for example liquid
polyol or lipids; by the use of surfactants such as, for example
hydroxypropylcellulose; or combinations thereof such methods. In
many cases, it will be preferable to include isotonic agents, such
as, for example, sugars, sodium chloride or combinations
thereof.
[0169] In other embodiments, one may use eye drops, nasal solutions
or sprays, aerosols or inhalants in the present embodiments. Such
compositions are generally designed to be compatible with the
target tissue type. In a non-limiting example, nasal solutions are
usually aqueous solutions designed to be administered to the nasal
passages in drops or sprays. Nasal solutions are prepared so that
they are similar in many respects to nasal secretions, so that
normal ciliary action is maintained. Thus, in preferred embodiments
the aqueous nasal solutions usually are isotonic or slightly
buffered to maintain a pH of about 5.5 to about 6.5. In addition,
antimicrobial preservatives, similar to those used in ophthalmic
preparations, drugs, or appropriate drug stabilizers, if required,
may be included in the formulation. For example, various commercial
nasal preparations are known and include drugs such as antibiotics
or antihistamines.
[0170] In certain embodiments the one or more polypeptide, nucleic
acid or nanoparticle complexes are prepared for administration by
such routes as oral ingestion. In these embodiments, the solid
composition may comprise, for example, solutions, suspensions,
emulsions, tablets, pills, capsules (e.g., hard or soft shelled
gelatin capsules), sustained release formulations, buccal
compositions, troches, elixirs, suspensions, syrups, wafers, or
combinations thereof. Oral compositions may be incorporated
directly with the food of the diet. Preferred carriers for oral
administration comprise inert diluents, assimilable edible carriers
or combinations thereof. In other aspects, the oral composition may
be prepared as a syrup or elixir. A syrup or elixir, and may
comprise, for example, at least one active agent, a sweetening
agent, a preservative, a flavoring agent, a dye, a preservative, or
combinations thereof.
[0171] In certain preferred embodiments an oral composition may
comprise one or more binders, excipients, disintegration agents,
lubricants, flavoring agents, and combinations thereof. In certain
embodiments, a composition may comprise one or more of the
following: a binder, such as, for example, gum tragacanth, acacia,
cornstarch, gelatin or combinations thereof; an excipient, such as,
for example, dicalcium phosphate, mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate or combinations thereof; a disintegrating agent, such as,
for example, corn starch, potato starch, alginic acid or
combinations thereof; a lubricant, such as, for example, magnesium
stearate; a sweetening agent, such as, for example, sucrose,
lactose, saccharin or combinations thereof; a flavoring agent, such
as, for example peppermint, oil of wintergreen, cherry flavoring,
orange flavoring, etc.; or combinations thereof the foregoing. When
the dosage unit form is a capsule, it may contain, in addition to
materials of the above type, carriers such as a liquid carrier.
Various other materials may be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules may be coated with shellac, sugar or both.
[0172] Additional formulations which are suitable for other modes
of administration include suppositories. Suppositories are solid
dosage forms of various weights and shapes, usually medicated, for
insertion into the rectum, vagina or urethra. After insertion,
suppositories soften, melt or dissolve in the cavity fluids. In
general, for suppositories, traditional carriers may include, for
example, polyalkylene glycols, triglycerides or combinations
thereof. In certain embodiments, suppositories may be formed from
mixtures containing, for example, the active ingredient in the
range of about 0.5% to about 10%, and preferably about 1% to about
2%.
[0173] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and/or the other ingredients. In the case of
sterile powders for the preparation of sterile injectable
solutions, suspensions or emulsion, the preferred methods of
preparation are vacuum-drying or freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered liquid medium
thereof. The liquid medium should be suitably buffered if necessary
and the liquid diluent first rendered isotonic prior to injection
with sufficient saline or glucose. The preparation of highly
concentrated compositions for direct injection is also
contemplated, where the use of DMSO as solvent is envisioned to
result in extremely rapid penetration, delivering high
concentrations of the active agents to a small area.
[0174] The composition must be stable under the conditions of
manufacture and storage, and preserved against the contaminating
action of microorganisms, such as bacteria and fungi. It will be
appreciated that endotoxin contamination should be kept minimally
at a safe level, for example, less that 0.5 ng/mg protein.
[0175] In particular embodiments, prolonged absorption of an
injectable composition can be brought about by the use in the
compositions of agents delaying absorption, such as, for example,
aluminum monostearate, gelatin or combinations thereof.
[0176] V. Combination Therapies
[0177] In order to increase the effectiveness of a nucleic acid,
polypeptide or nanoparticle complex of the present embodiments, it
may be desirable to combine these compositions with other agents
effective in the treatment of the disease of interest.
[0178] As a non-limiting example, the treatment of cancer may be
implemented with TUSC2 therapeutic of the present embodiments along
with other anti-cancer agents. An "anti-cancer" agent is capable of
negatively affecting cancer in a subject, for example, by killing
cancer cells, inducing apoptosis in cancer cells, reducing the
growth rate of cancer cells, reducing the incidence or number of
metastases, reducing tumor size, inhibiting tumor growth, reducing
the blood supply to a tumor or cancer cells, promoting an immune
response against cancer cells or a tumor, preventing or inhibiting
the progression of cancer, or increasing the lifespan of a subject
with cancer. More generally, these other compositions would be
provided in a combined amount effective to kill or inhibit
proliferation of the cell. This process may involve contacting the
cells with the anti-cancer peptide or nanoparticle complex and the
agent(s) or multiple factor(s) at the same time. This may be
achieved by contacting the cell with a single composition or
pharmacological formulation that includes both agents, or by
contacting the cell with two distinct compositions or formulations,
at the same time, wherein one composition includes the anti-cancer
peptide or nanoparticle complex and the other includes the second
agent(s). In particular embodiments, an anti-cancer peptide can be
one agent, and an anti-cancer nanoparticle complex can be the other
agent.
[0179] Treatment with the anti-cancer peptide or
nanoparticle-complex may precede or follow the other agent
treatment by intervals ranging from minutes to weeks. In
embodiments where the other agent and the anti-cancer peptide or
nanoparticle complex are applied separately to the cell, one would
generally ensure that a significant period of time did not expire
between the time of each delivery, such that the agent and the
anti-cancer peptide or nanoparticle complex would still be able to
exert an advantageously combined effect on the cell. In such
instances, it is contemplated that one may contact the cell with
both modalities within about 12-24 hours of each other and, more
preferably, within about 6-12 hours of each other. In some
situations, it may be desirable to extend the time period for
treatment significantly where several days (e.g., 2, 3, 4, 5, 6 or
7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks)
lapse between the respective administrations.
[0180] Likewise, in certain aspects a TUSC2 therapy is administered
in conjunction with an anti-inflammatory agent. For example, a
TUSC2 therapy may precede or follow the anti-inflammatory agent
treatment by intervals ranging from minutes to weeks. In certain
aspects, the anti-inflammatory agent is administered immediately
before the TUSC2 therapy and immediately after the TUSC2 therapy.
For example, the anti-inflammatory agent may be given less than a
day before and less than a day after the therapy. In still further
aspects more than one anti-inflammatory is administered, such
administration of a antihistamine (e.g., diphenhydramine) and a
corticosteroid (e.g., dexamethasone).
[0181] Various combinations may be employed, where the TUSC2
therapy is "A" and the secondary agent, such as radiotherapy,
chemotherapy or anti-inflammatory agent, is "B":
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0182] In certain embodiments, administration of the TUSC2 therapy
of the present embodiments to a patient will follow general
protocols for the administration of chemotherapeutics, taking into
account the toxicity, if any, of the vector. It is expected that
the treatment cycles would be repeated as necessary. It also is
contemplated that various standard therapies, as well as surgical
intervention, may be applied in combination with the described
hyperproliferative cell therapy.
[0183] a. Chemotherapy
[0184] Cancer therapies also include a variety of combination
therapies. In some aspects a TUSC2 therapeutic of the embodiments
is administered (or formulated) in conjunction with a
chemotherapeutic agent. For example, in some aspects the
chemotherapeutic agent is a protein kinase inhibitor such as a
EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3,
PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF
inhibitors. Nonlimiting examples of protein kinase inhibitors
include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab,
Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib,
Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib,
Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib,
Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib,
AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002,
Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin,
temsirolimus, everolimus, ridaforolimus, Alvocidib, Genistein,
Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839,
P276-00, GW572016 or a mixture thereof.
[0185] Yet further combination chemotherapies include, for example,
alkylating agents such as thiotepa and cyclosphosphamide; alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analogue topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogues); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammalI and calicheamicin omegaI1; dynemicin, including dynemicin
A; bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antiobiotic chromophores, aclacinomysins, actinomycin,
authrarnycin, azaserine, bleomycins, cactinomycin, carabicin,
carminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, pteropterin, trimetrexate;
purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as mitotane, trilostane; folic
acid replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;
PSK polysaccharide complex; razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g.,
paclitaxel and docetaxel gemcitabine; 6-thioguanine;
mercaptopurine; platinum coordination complexes such as cisplatin,
oxaliplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine;
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase
inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such
as retinoic acid; capecitabine; carboplatin, procarbazine,
plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase
inhibitors, transplatinum, and pharmaceutically acceptable salts,
acids or derivatives of any of the above. In certain embodiments,
the compositions provided herein may be used in combination with
gefitinib. In other embodiments, the present embodiments may be
practiced in combination with Gleevac (e.g., from about 400 to
about 800 mg/day of Gleevac may be administered to a patient). In
certain embodiments, one or more chemotherapeutic may be used in
combination with the compositions provided herein.
[0186] b. Radiotherapy
[0187] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on the
assembly and maintenance of chromosomes. Dosage ranges for X-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0188] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
composition and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0189] c. Immunotherapy
[0190] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells.
[0191] Immunotherapy, thus, could be used as part of a combined
therapy, in conjunction with a TUSC2 therapy of the present
embodiments. The general approach for combined therapy is discussed
below. Generally, the tumor cell must bear some marker that is
amenable to targeting, i.e., is not present on the majority of
other cells. Many tumor markers exist and any of these may be
suitable for targeting in the context of the present embodiments.
Common tumor markers include carcinoembryonic antigen, prostate
specific antigen, urinary tumor associated antigen, fetal antigen,
tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA,
MucB, PLAP, estrogen receptor, laminin receptor, erb B and
p155.
[0192] d. Gene Therapy
[0193] In yet another embodiment, the secondary treatment is a gene
therapy in which a therapeutic polynucleotide is administered
before, after, or at the same time as the therapeutic composition.
Viral vectors for the expression of a gene product are well known
in the art, and include such eukaryotic expression systems as
adenoviruses, adeno-associated viruses, retroviruses,
herpesviruses, lentiviruses, poxviruses including vaccinia viruses,
and papiloma viruses, including SV40. Alternatively, the
administration of expression constructs can be accomplished with
lipid based vectors such as liposomes or DOTAP:cholesterol
vesicles. All of these method are well known in the art (see, e.g.
Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).
[0194] Delivery of a vector encoding one of the following gene
products will have a combined anti-hyperproliferative effect on
target tissues. A variety of proteins are encompassed within the
present embodiments, some of which are described below.
[0195] i. Inhibitors of Cellular Proliferation
[0196] As noted above, the tumor suppressor oncogenes function to
inhibit excessive cellular proliferation. The inactivation of these
genes destroys their inhibitory activity, resulting in unregulated
proliferation.
[0197] Genes that may be employed as secondary treatment in
accordance with the present embodiments include p53, p16, Rb, APC,
DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1/PTEN,
DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions,
anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc,
neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes
involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1,
GDAIF, or their receptors), MCC and other genes listed in Table
IV.
[0198] ii. Regulators of Programmed Cell Death
[0199] Apoptosis, or programmed cell death, is an essential process
for normal embryonic development, maintaining homeostasis in adult
tissues, and suppressing carcinogenesis (Kerr et al., 1972). The
Bcl-2 family of proteins and ICE-like proteases have been
demonstrated to be important regulators and effectors of apoptosis
in other systems. The Bcl-2 protein, discovered in association with
follicular lymphoma, plays a prominent role in controlling
apoptosis and enhancing cell survival in response to diverse
apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, Proc.
Nat'l. Acad. Sci. USA, 82(21):7439-43, 1985; Cleary et al., 1986;
Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The
evolutionarily conserved Bcl-2 protein now is recognized to be a
member of a family of related proteins, which can be categorized as
death agonists or death antagonists.
[0200] Subsequent to its discovery, it was shown that Bcl-2 acts to
suppress cell death triggered by a variety of stimuli. Also, it now
is apparent that there is a family of Bcl-2 cell death regulatory
proteins which share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bcl-2 (e.g., Bcl.sub.XL, Bcl.sub.W, Bcl.sub.S,
Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell
death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
[0201] e. Surgery
[0202] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatments provided herein, chemotherapy,
radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or
alternative therapies.
[0203] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
miscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present embodiments may be used in
conjunction with removal of superficial cancers, precancers, or
incidental amounts of normal tissue.
[0204] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0205] f. Anti-Inflammatory Agents
[0206] In certain aspects TUSC2 therapies are administered in
conjuction with an anti-inflammatory agent. An anti-inflammatory
agent is defined herein to refer to an agent that is known or
suspected to be of benefit in the treatment or prevention of
inflammation in a subject. Corticosteroids are a major class of
anti-inflammatory agent. The corticosteroids may be short, medium,
or long acting, and may be delivered in a variety of methods. A
non-limiting list of corticosteroids contemplated in the present
embodiments include the oral corticosteroids such as: cortisone,
hydrocortisone, prednisone, and dexamethasone.
[0207] Another major class of anti-inflammatory agents are
non-steroidal anti-inflammatory agents. Non-steroidal
anti-inflammatory agents include a class of drugs used in the
treatment of inflammation and pain. The exact mode of action of
this class of drugs is unknown. Examples of members of this class
of agents include, but are not limited to, ibuprofen, ketoprofen,
flurbiprofen, nabumetone, piroxicam, naproxen, diclofenac,
indomethacin, sulindac, tolmetin, etodolac, flufenamic acid,
diflunisal, oxaprozin, rofecoxib, and celecoxib. One of ordinary
skill in the art would be familiar with these agents. Included in
this category are salicylates and derivates of salicylates, such as
acetyl salicylic acid, sodium salicylate, choline salicylate,
choline magnesium salicylate and diflunisal.
[0208] Other anti-inflammatory agents include anti-rheumatic
agents, such as gold salts (e.g., gold sodium thiomalate,
aurothioglucose, and auranofin), anti-rheumatic agents (e.g.,
chloroquine, hydroxychloroquine, and penicillamine), antihistamines
(e.g., diphenhydramine, chlorpheniramine, clemastine, hydroxyzine,
and triprolidine), and immunosuppressive agents (e.g.,
methotrexate, mechlorethamine, cyclophosphamide, chlorambucil,
cyclosporine, and azathioprine). Other immunosuppressive agent
contemplated by the present embodiments is tacrolimus and
everolimus. Tacrolimus suppresses interleukin-2 production
associated with T-cell activation, inhibits differentiation and
proliferation of cytotoxic T cells. Today, it is recognized
worldwide as the cornerstone of immunosuppressant therapy. One of
ordinary skill in the art would be familiar with these agents, and
other members of this class of agents, as well as the mechanism of
actions of these agents and indications for use of these
agents.
[0209] g. Other Agents
[0210] It is contemplated that other agents may be used in
combination with the compositions provided herein to improve the
therapeutic efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adehesion, or agents
that increase the sensitivity of the hyperproliferative cells to
apoptotic inducers. Immunomodulatory agents include tumor necrosis
factor; interferon alpha, beta, and gamma; IL-2 and other
cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta,
MCP-1, RANTES, and other chemokines. It is further contemplated
that the upregulation of cell surface receptors or their ligands
such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the
apoptotic inducing abilities of the compositions provided herein by
establishment of an autocrine or paracrine effect on
hyperproliferative cells. Increases intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the compositions provided herein to improve the
anti-hyerproliferative efficacy of the treatments Inhibitors of
cell adehesion are contemplated to improve the efficacy of the
present invention. Examples of cell adhesion inhibitors are focal
adhesion kinase (FAKs) inhibitors and Lovastatin. It is further
contemplated that other agents that increase the sensitivity of a
hyperproliferative cell to apoptosis, such as the antibody c225,
could be used in combination with the compositions provided herein
to improve the treatment efficacy.
[0211] In certain embodiments, hormonal therapy may also be used in
conjunction with the present embodiments or in combination with any
other cancer therapy previously described. The use of hormones may
be employed in the treatment of certain cancers such as breast,
prostate, ovarian, or cervical cancer to lower the level or block
the effects of certain hormones such as testosterone or estrogen.
This treatment is often used in combination with at least one other
cancer therapy as a treatment option or to reduce the risk of
metastases.
EXAMPLES
[0212] The following examples are included to demonstrate preferred
embodiments provided herein. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the present embodiments, and thus
can be considered to constitute preferred modes for its practice.
However, those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
present embodiments.
Example 1
Clinical Study Design
[0213] Eligible patients were required to have histologically
documented non-small cell lung cancer (NSCLC) or small cell lung
cancer (SCLC) not curable by standard therapies and previously
treated with platinum-based chemotherapy. The primary end point was
assessment of DOTAP:chol-TUSC2 toxicity during cycle 1 and
determination of the maximum tolerated dose (MTD). Cycles consisted
of a single intravenous infusion every 21 days. Secondary end
points included TUSC2 plasmid expression in pretreatment and 24
hour post treatment tumor specimens from subjects consenting to
tumor biopsies and tumor response. The presence of viable cancer
cells in the biopsied lesion was confirmed in all cases by
histopathological examination. Mandatory biopsies were explicitly
precluded by regulatory committees at the local and federal level.
Tumor response assessed by computed tomography (CT) scans was
determined in accordance with standard World Health Organization
(WHO) criteria (Miller et al., 1981). This study was approved by
the University of Texas MD Anderson Institutional Review Board, the
NIH Recombinant DNA Advisory Committee, and the FDA. All patients
provided written informed consent prior to entry into the
study.
[0214] Other eligibility criteria included: Eastern Cooperative
Oncology Group (ECOG) performance status .ltoreq.1; adequate
hematologic, hepatic, and renal function; prothrombin time and
partial thromboplastin time .ltoreq.1.25 times the upper limit of
normal; left ventricular ejection fraction >50%; forced
expiratory volume in 1 second (FEV1) and diffusing capacity of the
lung for carbon monoxide (DLCO) .gtoreq.40% of predicted; and
negative human immunodeficiency virus serology test. Exclusion
criteria included: prior gene therapy; brain metastases, unless
treated, asymptomatic, and not requiring steroid therapy;
chemotherapy within 21 days before enrollment; radiation therapy
within 30 days before enrollment; investigational therapies within
30 days before enrollment; active infection requiring antibiotic
therapy; myocardial infarction or angina within 6 months before
enrollment; and pregnancy or lactation.
[0215] A history and physical examination were performed before
every cycle. Adverse events were assessed and laboratory tests
performed prior to each cycle and on days 2, 3, and 8. Laboratory
tests included a complete blood count with differential, sodium,
potassium, chloride, calcium, albumin, total protein, blood urea
nitrogen, creatinine, alanine aminotransferase, aspartate
aminotransferase, alkaline phosphatase, lactate dehydrogenase, and
total bilirubin. Urinalysis and electrocardiograms were obtained
prior to each cycle.
[0216] The primary end point was assessment of DOTAP:chol-TUSC2
toxicity during cycle 1 and determination of the maximum tolerated
dose (MTD). Secondary end points included tumor response and TUSC2
plasmid expression in pretreatment and 24 hour posttreatment tumor
specimens from subjects consenting to tumor biopsies.
DOTAP:chol-TUSC2 was administered at escalating doses as a 30
minute infusion in a peripheral vein in a total volume of 100 mL of
5% dextrose solution. Patients received DOTAP:chol-TUSC2 every 21
days for up to 6 treatments. After the ninth patient was enrolled,
the protocol was amended to require diphenhydramine 50 mg orally or
intravenously 30 minutes prior to treatment and dexamethasone 8 mg
orally 24 and 12 hours before treatment, 20 mg intravenously 30
minutes prior to treatment, and 8 mg orally 12, 24, and 36 hours
after treatment.
[0217] The initial starting dose (0.02 mg/kg) was selected based on
toxicology studies in non-human primates. This dose was one tenth
the dose which resulted in no deaths in non-human primates. After
the sixth patient was enrolled, the starting dose was amended to
0.01 mg/kg. Dose escalation was based on a continuous reassessment
method (CRM) which allows the MTD to be periodically re-estimated
(O'Quigley et al., 1990). The MTD was defined as the highest dose
level in which no more than 10% of patients develop dose-limiting
toxicity (DLT), defined as grade 3 non-hematologic or hematologic
toxicity during cycle 1 judged by the investigator to be related to
DOTAP:chol-TUSC2. Patients entered at a given dose level were not
eligible for dose escalation or dose reduction. A cohort of 3
patients was treated at each dose level. After treating 3 patients
at a given dose level, the information of whether the patients
developed DLT was used to compute the posterior probability of
toxicity. Only toxicity during cycle 1 was used to determine the
next dose level. If no DOTAP:chol-TUSC2-related toxicities were
observed in any prior patient, the subsequent dose level was
increased by 100%. If only grade 1 or 2 toxicities were observed,
the subsequent dose level was increased by 50%. If any DLT was
observed, the CRM could lead to either escalation or reduction of
dose levels. If DLT occurred and the CRM resulted in a dose
escalation, the subsequent dose level was increased by 25%.
Toxicity was graded according to the National Cancer Institute
Common Toxicity Criteria, version 2.0. Tumor status was assessed at
baseline and after every two cycles of therapy with computed
tomography (CT) scans and/or positron emission tomography (PET)/CT
scans. Tumor response assessed by computed tomography (CT) scans
was determined in accordance with standard World Health
Organization (WHO) criteria. 10 Additional details on patient
selection and assessment are provided in the Supplementary Methods.
Dr. J. Jack Lee designed the clinical trial and analyzed the data.
This study was approved by the University of Texas MD Anderson
Institutional Review Board, the NIH Recombinant DNA Advisory
Committee, and the FDA. All patients provided written informed
consent prior to entry into the study.
Example 2
FUS1/TUSC2 Expression Vector
[0218] This pLJ143/KGB2/FUS1 plasmid vector (FIG. 1A) includes a
mammalian gene-expression cassette driven by a CMV minimum promoter
with an E1 enhancer at the 3' end and a BGH-poly A signal sequence
at the 5' end to ensure the efficient expression of the transgene
in vivo. The kanamycin-resistance gene was chosen as the selectable
marker to avoid development of antibiotic-resistance in patients. A
minimum pMB1 origin of replication (ori) sequence is used to drive
high-copy replication and production of the plasmid in the
bacterial host strain DH5a. The plasmid backbone is minimal to
ensure a higher yield of plasmid DNA production and a higher
concentration of recombinant plasmid DNA per plasmid DNA
preparation. The entire DNA sequence of the plasmid vector was
determined by automated DNA sequencing using the DNA Sequencing
Core Facility at the M.D. Anderson Cancer Center. The complete DNA
sequence of pLJ143/KGB2/FUS1 plasmid vector is provided as SEQ ID
NO: 1. Specific elements of the vector are detailed below.
[0219] E1 Enhancer (bases 4-473): The E1 enhancer is a
transcriptional enhancer for adenoviral gene E1 protein and is
derived from the adenoviral shuttle vector constructed by Grahm et
al. E1 enhancer is used to enhance transcription of gene of
interest under the control of CMV promoter in mammalian cells.
[0220] CMV Promoter (Bases 474-1171):
[0221] The CMV promoter is derived from the adenoviral shuttle
vector constructed by Grahm et al. The CMV promoter is covered
under U.S. Pat. Nos. 5,168,062 and 5,385,839, owned and licensed by
the University of Iowa Research Foundation (Iowa City, Iowa 52242).
The human cytomegalovirus (CMV) promoter has been cloned,
sequenced, and used to construct a series of mammalian cell
expression plasmid (Chapman et al., 1991). A high level of gene
expression can be achieved under the control of CMV promoter in
mammalian cells.
[0222] BGH Polyadenylation Signal (Bases 1652-1877):
[0223] BGH polyadenylation signal sequence is derived from the
adenoviral shuttle vector constructed by Grahm et al. The BGH
polyadenylation sequence is covered under U.S. Pat. No. 5,122,458
and licensed by Research Corporation Technologies (Tucson, Ariz.).
Transcriptional termination by RNA polymerase III at the 3' end of
eukyrotic genes requires two distinct cis-active elements, a
functional poly (A) signal and a downstream transcription pause
site. The BGH poly A signal has been widely used as a transcription
termination signal for mammalian gene expression in vitro and in
vivo (Eggermont et al., 1993; Goodwin et al., 1992)
[0224] Kanamycin Resistance Gene (Bases 2049-2934):
[0225] The antibiotics Kanamycin resistance gene is derived from
the pVAX1 plasmid vector from Invitrogen (Carlsbad, Calif.). The
kanamycin resistance gene is used as selective marker for plasmid
production in bacterial E. coli. in the presence of antibiotics
Kanamycin.
[0226] pMBJ Origin (Bases 2995-3734):
[0227] The high copy number plasmid pMB1 replication origin
sequence is derived from the pMG plasmid vector from Invivogen (San
Diego, Calif.). The minimal pMB1 origin is used to reduce plasmid
size and drive a high copy number replication of plasmid DNA in E.
coli.
Example 3
Plasmid Preparation
[0228] The pLJ143/KGB2/FUS1 plasmid vector was produced under GMP
conditions at the Baylor College of Medicine Center for Cell and
Gene Therapy (Houston, Tex.) and the Beckman Research Institute of
the City of Hope (Duarte, Calif.).
[0229] A 1 mL vial of the pLJ143 master cell bank stock was
aseptically inoculated into 500 mL sterile Terrific Broth with 1.6%
Glycerol (Teknova: 1.2% Tryptone, 2.4% yeast extract, 1.6%
glycerol. 1.times. phosphate buffer) supplemented with Kanamycin
(Sigma) and grown overnight (15-18 hours) at 37.degree. C. This was
then used to inoculate 20 L of Terrific Broth in a New Brunswick
scientific BioFlo IV 4500 fermentor, operating at 37.degree. C.,
250-300 rpm, 20-30% CO.sub.2. Cells are harvested by
centrifugation, washed once (1 mL buffer per g wet cell paste) with
Alkaline Lysis Solution I (Teknova: 50 mM Glucose, 25 mM Tris-HCl,
pH 8.0, 10 mM EDTA, pH 8.0, sterile solution) and frozen at
-80.degree. C.
[0230] The cell pastes were removed from -80.degree. C. storage and
thawed in a 4.degree. C. refrigerator overnight. The cell paste was
mixed with Alkaline Lysis Solution I at 8 ml per gram of wet cell
paste. The pLJ143 suspension was then mixed 1:1 (v/v) with Alkaline
Lysis Solution II (Teknova: 200 mM NaOH, 1.0% SDS, sterile
solution). After mixing, the material was allowed to lyse at room
temperature between 8 and 10 minutes. Two volumes of Alkaline Lysis
Solution III (Teknova: 3M potassium acetate, 1.18M formic acid, pH
5.5, sterile solution) were then added with the lysate, and mixed
on ice to ensure complete neutralization and precipitation of host
cell proteins, genomic host cell DNA and SDS. The neutralized cell
lysate was clarified using a bucket centrifuge at 4000 rpm for 30
minutes at 4.degree. C. The supernatant was decanted and clarified
through a 1.2 mM PP2 filter.
[0231] This four step purification process does not require RNase
enzyme, organic solvents, detergents, precipitants or animal
derived components. The entire process is controlled with an Aekta
Purifier (Amersham Bioscience) and Unicorn Software (Amersham
Bioscience). All columns and packing material are from Amersham
Bioscience. All column preparation and storage is as follows:
[0232] CIP: 0.5 M NaOH, 25.degree. C. for I hour contact time
[0233] Depyrogenation: 100 ppm sodium hypochlorite pH 10, then
0.1-0.5N NaOH, pH13
[0234] Storage: 20% ethanol (aqueous solution)
[0235] Preparation for Use (Sanitization): Cell culture grade water
(US Pharmacopia), then primed with applicable buffer
[0236] Step 1: Concentration Using Hollow Fiber Filter (HFF)
[0237] The clarified lysate was first concentrated approximately
10-fold and equilibrated with using a 300,000 kDa nominal molecular
weight cut-off (NMWCO) A/G Technology hollow fiber filter (HFF).
The HFF was flushed with 4-L of Alkaline Lysis Solution III (3M
potassium acetate, 1.18M formic acid, pH 5.5, sterile solution) and
pooled with the concentrated lysate. A final volume of
approximately 2-L is recovered, filtered with a 0.45 .mu.m filter,
and stored at 4.degree. C. until the next step.
[0238] Step 2: Size Exclusion
[0239] RNA removal and buffer exchange by group separation using
Sepharose 6 Fast Flow with BPG size exclusion column. UF
Concentrate is applied to the column in batches of 0.3 column
volumes (CV) to change the buffer to Buffer A (2M
(NH.sub.4)SO.sub.4, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0).
Simultaneously this procedure also removes RNA and other
contaminants. The void fractions are stored at 4.degree. C. and
then pooled for the next step.
[0240] Step 3: Selective Capture of Supercoiled Plasmid DNA by
Thiophilic Aromatic Adsorption Chromatography.
[0241] Supercoiled plasmid DNA is separated from open circular
plasmid DNA and remaining contaminants such as residual genomic DNA
and RNA. The pooled void fraction (from Step 2) is subsequently
applied on the XK50 Affinity column packed with PlasmidSelect and
equilibrated in the same Buffer A. The column is washed and
supercoiled plasmid DNA is eluted with Buffer B (1.4M NaCl, 2.0 M
(NH.sub.4)SO.sub.4, 10 mM EDTA, 100 mM Tris-HCl, pH7.0.). Fractions
are stored at 4.degree. C. prior to next step. Fractions are pooled
for step 4. then diluted with four volumes of water for the next
step.
[0242] Step 4: Polishing and Concentration with SOURCE 30Q
[0243] Endotoxins are further removed and at the same time, the
supercoiled plasmid DNA preparation is concentrated by ion exchange
chromatography. The fraction (from step 3) containing supercoiled
plasmid DNA is diluted with 4 volumes of pharmaceutical grade water
and loaded on a XK26 ion exchange column packed with SOURCE 30Q.
The column is equilibrated Buffer C (0.4 M NaCl, 10 mM EDTA, 100 mM
Tris-HCl, pH 7.0) and eluted with a linear gradient, Buffer D (1.0
M NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0). The fractions are
then pooled and filtered through a 0.22 .mu.m filter.
[0244] PlasmidSelect is the key protocol component since it
interacts differentially with nucleic acids by thiophilic aromatic
adsorption in the presence of water structuring salts. This enables
the topoisomere-selective purification of native supercoiled
plasmid DNA and removal of damaged, nicked or open circular DNA by
simple adjustment of chromatographic conditions. A group separation
for removal of RNA prior to application on the column optimizes the
capacity of PlasmidSelect for binding of the supercoiled form of
plasmids. Furthermore, group separation with Sepharose 6 Fast Flow
greatly reduces the risk of precipitation during addition of
ammonium sulfate and limits the variation in initial salt
concentration that can influence selectivity, thus giving the
process considerable robustness.
[0245] Ethanol precipitation was used to concentrate the pLJ143 to
5 mg/ml. A 3.0 M sterile NaCl solution was used to increase the
NaCl concentration of the pLJ143 solution to 0.15M. Ethanol was
added into the pLJ143 solution in a 2:1 ratio to give a final
ethanol concentration of 67%. The pLJ143 suspension was stored at
-20.degree. C. overnight to allow for complete precipitation. The
next morning, the pLJ143 was recovered by centrifugation. The
pLJ143 pellets were further washed with 70% ethanol and allowed to
air dry aseptically in a laminar flow hood for approximately one
hour. Dried pellets were frozen at -80.degree. C. until
purification of wet paste from all fermentation runs was complete.
The pLJ143 was then reconstituted at 5 mg/ml in sterile endotoxin,
RNAse, DNAse free water. All work is performed in a class 100
biosafety cabinet.
[0246] Product was filled into 1.2 ml crimp cap glass vials with
semi-automated dispensing pipette in a class 100 biosafety cabinet.
The fill volume is either 0.3 ml, 0.5 ml, or 1.0 ml. The target
plasmid DNA concentration was 5 mg/mL. The final product is stored
at -80.degree. C. Plamid purity tests and quality control standards
are shown in Table 1 below.
TABLE-US-00002 TABLE 1 Plasmid quality control specifications.
Characteristics TEST Specifications Appearance Clear, Colorless pH
pH meter 5.5 .ltoreq. pH .gtoreq. 8.0 DNA Identity DNA Homogeneity
.gtoreq.90% Appropriate form DNA Supercoiled DNA .gtoreq.95%
Restriction Map Restriction Digestion pattern Identical to the
reference Bio-activity by cell Positive for Transgene transfection
Expression and Western-blotting Purity A260/280 Ratio 1.7-2.0
A260/230 Ratio 2.0-2.2 Protein Contamination <10 .mu.g/mg DNA
Host E. coli <1% (w/w) of total detectable Genomic DNA nucleic
acid by qPCR Contamination Residual RNA <5% (w/w) of total
detectable Contamination nucleic acid (0.5 .phi.g load) by gel
Residual Isopropanol by GC analysis and ethanol Residual
Antibiotics Undetectable (<3.0 .phi.g/ml) (kanamycin) Ammonia
.ltoreq.1 mg/mL of final formulated plasmid DNA Sulfate .ltoreq.1
mg/mL of Final Formulated Plasmid DNA Endotoxin <5 EU/mg DNA by
LAL Assay Sterility Bacterial (CFR) Negative Fungal Negative In
vitro Negative Adventitious Virus Concentration O.D. At 260 nm 1 to
5 mg/mL/vial .+-. 0.5% per vial Biologic Activity Transfection in
Protein expression by Western H1299 cells Blot
Example 4
Nanoparticle Preparation
[0247] DOTAP GMP grade was purchased from Avanti Polar Lipids, Inc.
(Alabaster, Ala.) and cholesterol GMP grade was purchased from
Sigma-Aldrich (St. Louis, Mo.). A ratio of 20 mM DOTAP:18 mM
cholesterol was used for preparation of the nanoparticles. The
reagents were mixed and the dry lipids dissolved in purified GMP
grade chloroform. A Buchi rotary evaporator was used to form a dry
lipid film. Further drying was performed under a vacuum in a
Labconco Freeze dry system. The film was resuspended in sterile 5%
dextrose in water. After sonication the following day under aseptic
conditions the lipids are sequentially extruded through a series of
sterile Whatman filters from 1 .mu.m to 0.1 .mu.m in pore size.
[0248] The diluted plasmid DNA and diluted nanoparticle stock were
mixed in equal volumes to a final concentration of 4 mM DOTAP and
0.5 mg/ml of DNA. Prior to treatment the assigned dose was diluted
in 100 ml D5W. A negative gram stain was required prior to
treatment.
Example 5
Therapy Protocol and Results
[0249] Thirty-one patients were enrolled in the study at a single
institution. Patient characteristics are described in Table 2.
TABLE-US-00003 TABLE 2 Baseline Characteristics of Patients No. of
Patients (%) Characteristic (n = 31) Median age, years (range) 60
(43-76) Sex Male 16 (51.6%) Female 15 (48.4%) ECOG performance
status 0 4 (12.9%) 1 27 (87.1%) Histology Adenocarcinoma 11 (35.5%)
Bronchoalveolar carcinoma 1 (3.2%) Squamous cell carcinoma 3 (9.7%)
Non-small cell carcinoma, NOS 11 (35.5%) Small cell carcinoma 5
(16.1%) Prior Therapy Chemotherapy 31 (100%) Radiotherapy 14
(45.2%) Surgery 11 (35.5%) Prior Chemotherapy regimens 1 9 (29%) 2
9 (29%) >2 13 (41.9%) Number of doses received 1 8 (26%) 2 19
(61%) >3 4 (13%) Abbreviations: ECOG, Eastern Cooperative
Oncology Group; NOS, not otherwise specified
[0250] A total of 74 cycles of DOTAP:chol-TUSC2 were administered,
with a median of 2 cycles (range, 1 to 12 cycles) per patient.
Patients were treated at 6 dose levels ranging from 0.01 to 0.09
mg/kg. The dose escalation scheme, including number of patients,
number of cycles, DLTs, and grade 2 toxicities judged to be related
to DOTAP:chol-TUSC2 are listed in Table 3.
TABLE-US-00004 TABLE 3 Dose-Escalation Scheme Dose No. No. Co-
level of of No. Grade 2 hort (mg/ Pa- cy- patients toxicity No. kg)
tients cles with DLT (No. patients) 1 0.02 3 9 0 Fever (1) 2 0.03 3
6 0 0 3.sup.1 0.01 3 4 2; G3 fever Fever (1) (n = 2), G3
hypotension (n = 1) 4* 0.01 3 9 0 0 5* 0.02 3 6 0 0 6* 0.04 3 6 0 0
7* 0.06 3 6 0 ALT (1), neuropathy (n = 1) 8* 0.09 3 5 1; G3 Fever
(1) hypophosphatemia 9* 0.06 3 16 0 Hypophosphatemia (1), nausea
(1), myalgia (1) 10* 0.06 3 5 1; G3 Fever (1), hypophosphatemia
myalgia (1), hypophosphatemia (1), 11 0.06 1 2 0 0 Abbreviations:
G3, grade 3; ALT, alanine aminotransferase elevation .sup.1This
cohort did not receive dexamethasone or diphenhydramine
premedications *Cohorts used to determine maximum tolerated dose
(MTD)
[0251] The first patient in cohort 1 (receiving 0.02 mg/kg)
developed grade 2 fever within 3 hours of the DOTAP:chol-TUSC2
infusion. The subsequent patients in cohorts 1 and 2 were given
dexamethasone and diphenhydramine prior to receiving
DOTAP:chol-TUSC2, and no grade 1 or higher toxicites were observed.
However, after discussions with the FDA, it was mandated that the
next patient cohort receive DOTAP:chol-TUSC2 at a lower dose level
of 0.01 mg/kg without dexamethasone or diphenhydramine
premedication. All three patients developed grade 2 or 3 fever and
one patient developed grade 3 hypotension. The FDA then allowed the
protocol to be amended to require dexamethasone and diphenhydramine
premedications beginning with the next cohort (patient 10),
starting at a dose level of 0.01 mg/kg. Due to this amendment, it
was decided not to use the toxicity data from the first nine
patients for MTD determination, and a subgroup of 21 patients
enrolled between Sep. 28, 2006 and Oct. 29, 2009 were used to
determine the final MTD.
[0252] The only subsequent DLTs observed were grade 3
hypophosphatemia in two patients with one at 0.06 mg/kg and another
at 0.09 mg/kg. In both cases the patients had either grade 1 or 2
fevers and the hypophosphatemia was an incidental laboratory
finding. The MTD was determined to be 0.06 mg/kg. As listed in
Table 2, grade 2 toxicities included myalgias, hypophosphatemia,
fever, nausea, and transaminase elevation.
Peripheral Blood Mononuclear Cell (PBMC) Cryo Preservation and
Fluorescent Activated Cell Sorter Analysis (FACS)
[0253] Patient blood samples were collected in capped glass tubes
containing ficoll at room temperature (RT). Blood samples were
centrifuged at 3500 rpm at RT for 30 minutes in a swing-out rotor.
Separated plasma and lymphocytes were collected separately in a
centrifuge tubes. An equal volume of PBS was added to the
lymphocyte-containing tube and centrifuged at 900.times.g at RT for
10 min. After centrifugation, the supernatant was removed. The cell
pellets were washed again with the same volume of PBS as the first
wash. The cell suspension was centrifuged at 700.times.g for 10
minutes, and the supernatant was removed. Cell concentrations were
determined and adjusted to a final concentration of 5.times.106
cells/mL with cell-freezing medium containing 10% of DMSO and 90%
fetal bovine serum. Eight hundred uL aliquots of 5.times.106/mL
lymphocytes were transferred into cryogenic vials. The
PBMC-containing cryogenic vials were stored in a -80.degree. C.
freezer for 48 h and then transferred to a liquid nitrogen
freezer.
[0254] Frozen PBMC were thawed immediately in a 37.degree. C. water
bath, then washed with 10 ml of RPMI1640 with 10% FBS. The cells
were then lysed with 1.times.BD FACS Lysing Solution (BD
Biosciences, San Jose Calif.) for 10 minutes at room temperature.
The cells were centrifuged at 400.times.g for 10 minutes, followed
by treatment with 1.times.FACS Permeabilizing Solution 2 (BD
Biosciences, San Jose, Calif.) for 10 minutes at room temperature.
The cells were then rinsed with PBS containing 1% FBS and
centrifuged for 10 minutes at 400.times.g and re-suspended in 400
.mu.L of PBS with 1% FBS. Aliquots were made in the required number
of BD Falcon 5 mL polystyrene tubes. Antibodies (BD, Franklin
Lakes, N.J.) were then added to each tube according to the table
listed below under the fluorescent dye in bold letters:
TABLE-US-00005 FITC PE PerCP APC IgG1 IgG1 CD14 IgG1 TNF-a IL-6
CD14 IL-15 IL-1b IFN-g CD14 IL-8 CD14
[0255] The cells were incubated with antibodies for 30 minutes at
room temperature protected from light, washed with PBS containing
1% FBS, re-suspended in 250 uL of PBS with 1% paraformaldehyde, and
analyzed by 6-color flow cytometry (LSRII, BD). The cytokine data
was analyzed using FlowJo software (Tree Star, Inc., Ashland,
Oreg.).
[0256] Results of these studies showed that intracellular levels of
TNF-a, IL-15, IL-6, IL1b, IFNg, and IL-8 in peripheral blood
monocytes and lymphocytes remained unchanged 24 hours after
treatment (FIG. 11A-B).
Antibodies to Single and Double Stranded DNA
[0257] Serum antibodies to single and double standed DNA were
determined by an ELISA assay performed at the Mayo Clinic
Department of Laboratory Medicine and Pathology, Rochester, Minn.
For single stranded DNA antibodies a value of <69 U/ml is
considered negative for antibody detection. For double stranded DNA
antibodies a value of <1 is considered negative for antibody
detection.
[0258] Results showed that antibodies to single and double stranded
DNA were not detected 14 months after completion of 12 cycles of
therapy in patient 26.
Example 6
TUSC2 RNA Expression
[0259] All specimens were blinded for patient identity, for
clinical information and for specimen timing (pre- vs
post-treatment) during all studies. Ectopic expression of the TUSC2
gene in patient biopsy samples was analyzed using a TaqMan.TM.
based quantitative real time reverse transcriptase-polymerase chain
reaction (RT-PCR) (Applied Biosystems, Foster City, Calif.) that
enables quantification of gene expression from a limited amount of
starting material as detailed below.
[0260] RNA was isolated using RNeasy.TM. minikit from Qiagen
(Valencia, Calif.) following the manufacturer's instructions. The
fine-needle biopsy tissues that were immediately fixed in RNAlater
(Ambion, Austin, Tex.) were washed once with cold PBS and then the
total RNAs were isolated with (reagent and methods). The quality of
the purified RNA was analyzed using an Agilent 2100 Nano
Bioanalyzer (Agilent Technologies, Santa Clara Calif.). Reverse
transcription was done using a High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, Calif.) with
MultiScribe Reverse Transcriptase for two hours at 37.degree. C. in
a thermal cycler according to the manufacturer's instructions. The
PCR reaction was setup with 10 ml of 2.times. TaqMan.TM. gene
expression master mix containing the polymerase, buffer and dNTPs,
1 ml of 20.times. TaqMan.TM. gene expression assay solution
containing primers, probe, and 5 ml of cDNA template, and 4 ml of
sterile distilled water. The primers and probes used were specific
to the exogenous TUSC2 transcripts expressed through the plasmid
gene expression cassette (Forward primer: 5' GGA CCT GCA GCC CAA
GCT 3' (SEQ ID NO: 3) and Reverse primer: 5' GCC CAT GTC AAG CCG
AAT T 3' (SEQ ID NO: 4), and TaqMan.TM. probe: 6-FAM-CGA GCT CGG
ATC CAC TAG TCC AGT GTG-TAMRA; SEQ ID NO: 5) to avoid detection of
endogenous TUSC2 mRNA. PCR analysis was performed using a 7500
Real-Time PCR System (Applied Biosystems, Foster City, Calif.) and
run with an absolute quantification mode with a standard curve. The
DNA amount values were then used for the calculation of TUSC2 copy
numbers using the University of Rhode Island's website available on
the world wide web at uri.edu/research/gsc/resources/cndna.html
(URI Genomics & Sequencing Center Calculator for determining
the number of copies of a template).
[0261] TUSC2 transgene RNA expression by RT-PCR was not detected in
pre-treatment biopsies (Table 4). Five of six post-treatment
biopsies showed expression of the TUSC2 transgene. In a seventh
patient (Patient No. 31), TUSC2 mRNA was detected by RT-PCR
transgene specific primers included in the qRT Profiler Apoptosis
PCR Array and was detected only in the post-treatment sample (see
below). Expression was not detected in pre- and post-treatment
peripheral blood lymphocytes collected at the time of the
biopsies.
TABLE-US-00006 TABLE 4 Real Time RT-PCR detection of TUSC2 gene
expression in patients. Tumor Lympho- TUSC2 cyte Tumor Copy TUSC2
Site of TUSC2 Gene Number Gene Patient Dose Tumor Expression
(copies/ug Expression Number (mg/kg) Biopsy Treatment (pg/ug
tissue) tissue) (pg/.mu.l) 1 0.02 Lung Pre- 0 0 NA.sup.1 treatment
Lung Post- .sup. 2.0 .times. 10.sup.-5 .+-. 2.20 .times. 10.sup.-10
4.44 NA treatment 7 0.01 Lung Pre- 0 0 NA treatment Lung Post- 3.6
.times. 10.sup.-6 .+-. 9.1 .times. 10.sup.-7 0.89 NA treatment 13
0.02 Lung Pre- 0 0 NA treatment Lung Post- 3.0 .times. 10.sup.-5
.+-. 1.71 .times. 10.sup.-8 6.22 NA treatment 20 0.06 Liver Pre- 0
0 0 treatment Liver Post- 0 0 0 treatment 24 0.09 Subcutaneous Pre-
0 0 0 nodule treatment Subcutaneous Post- 8.0 .times. 10.sup.-6
.+-. 2.33 .times. 10.sup.-8 1.90 0 nodule treatment 25 0.06 Lung
Pre- 0 0 0 treatment Lung Post- 4.0 .times. 10.sup.-5 .+-. 1.66
.times. 10.sup.-9 8.76 0 treatment .sup.1Specimens not
available
Example 7
TUSC2 Protein Expression
[0262] Anti-TUSC2 antibody was used to detect TUSC2 protein
expression in pre- and post-treatment lung tumor biopsies from
patients 13, 26 and 31 (FIG. 2B). Specifically, Duolink kits from
Olink Biosciences (Uppsala, Sweden) were used. These kits are based
on PLA technology and the rolling circle amplification (RCA)
reaction wherein a pair of oligonucleotide labeled secondary
antibodies (PLA probes) generates a signal only when the two PLA
probes have bound in close proximity, either to the same primary
antibody or two primary antibodies that have bound to the sample in
close proximity. The signal from each detected pair of PLA probes
is visualized as an individual fluorescent spot. Signals can be
quantified (counted) and assigned to a specific subcellular
location based on microscopy images.
[0263] The samples were incubated with primary antibodies that bind
to the protein(s) to be detected. Secondary antibodies conjugated
with oligonucleotides (PLA probe MINUS and PLA probe PLUS) were
then added to the reaction and incubated. The ligation solution,
consisting of two oligonucleotides and ligase, is added and the
oligonucleotides hybridize to the two PLA probes and join to form a
closed circle if they are in close proximity. The amplification
solution, consisting of nucleotides and fluorescently labeled
oligonucleotides, was added together with polymerase. The
oligonucleotide arm of one of the PLA probes acts as a primer for a
rolling-circle amplification (RCA) reaction using the ligated
circle as a template, generating a concatemeric (repeated sequence)
product. The fluorescently labeled oligonucleotides then hybridizes
to the RCA product. The signal was visible as a distinct
fluorescent spot that can be analyzed by fluorescence microscopy.
In order to detect posttreatment TUSC2 protein expression, a single
antibody (TUSC2) raised in rabbits and oligo probes (plus and
Minus) with rabbit secondary antibodies were used. In situ PLA was
performed as per the recommendations of the manufacturer with minor
modifications and also including appropriate controls. The
experiments were carried out in a blinded setting. Patient biopsy
tissues preserved in RNAlater.TM. were washed in 50 ml of cold PBS
for 30 minutes at 4.degree. C. before using OCT to prepare frozen
blocks to cut slides. The slides with were then fixed with 4%
paraformaldehyde and permeabilized with methanol for 20 minutes
each. The tissues were blocked for 30 min at 37.degree. C. in a
humidified chamber with the blocking buffer provided in the kit and
later incubated with anti-rabbit TUSC2 primary antibody overnight
at 4.degree. C. The following day, the primary antibodies were
washed and tissues incubated with oligo-linked secondary antibodies
(anti-rabbit PLA probes plus and minus). Hybridization, ligation,
amplification and detection were then performed according to the
manufacturer's instructions. For non-specific control, rabbit HA
tag antibodies were used in the place of TUSC2 antibody. For
competition experiments, the synthetic oligopeptide
(GASGSKARGLWPFASAA; SEQ ID NO: 2) derived from the N-terminal
amino-acid sequence of the TUSC2 protein that was used to develop
anti-TUSC2 polyclonal antibody in rabbits was used (Ito et al.,
2004).
[0264] The number of in situ proximity ligation signals was counted
using the freeware software Blobfinder (available on the world wide
web at cb.uu.se/.about.amin/BlobFinder). Nuclei were visualized by
DAPI staining and used for cell count. The protein expression level
was quantified by counting all signals (fluorescent spots) obtained
from one image divided by the number of cells in the image, to
derive the average signals/cell. Background subtraction was them
applied with the pre-treatment samples.
[0265] Results of these studies are shown in FIG. 2A-B and
demonstrated that both post-treatment biopsies showed a high level
of TUSC2 protein with absence of TUSC2 protein on the paired
pre-treatment biopsies. A non-specific control antibody showed only
background staining Pre-incubation of the TUSC2 antibody with the
specific TUSC2 peptide used to immunize for antibody production,
but not a non-specific peptide, was able to significantly reduce
TUSC2 fluorescence in the post-treatment biopsies
Example 8
Effects on the Apoptosis Pathway
[0266] The expression of major genes in apoptosis signaling
pathways in pretreatment and posttreatment needle biopsy specimens
were quantified using a qRT Profiler Apoptosis PCR Array with RT
Nano PreAmp-mediated cDNA synthesis (SA Biosciences, Frederick,
Md.). The quantitative apoptotic gene expression data were analyzed
as detailed below and through the use of Ingenuity Pathway Analysis
(IPA) Ingenuity Systems, (available on the world wide web at
ingenuity.com).
[0267] For gene expression profiling experiments, the total RNAs
were isolated from patient fine needle biopsies using Trizol
(Invitrogen, Carlsbad, Calif.) reagent and purified using a RT2
qPCR-Grade RNA isolation kit from SA Biosciences (Frederick, Md.)
according to the manufacturer's instructions. The purified RNA was
then used to synthesize cDNA using RT2 Nano PreAmp cDNA Synthesis
Kit from SA Biosciences (Frederick, Md.). This cDNA kit also
involved pre-amplification of the cDNA target templates. The
preamplified cDNA was applied onto a RT2 Profiler Apoptosis PCR
array (SA Biosciences) for qPCR analysis using an ABI 7500
real-time PCR instrument (Applied Biosystems, Foster City, Calif.)
according to the manufacturers' instructions. The expression level
of the mRNA of each gene in the patient after treatment with
DOTAP:chol-TUSC2 was normalized using the expression levels of
housekeeping genes B2M, HPRT1, RPL13A, GAPDH, and ACTB. For data
analysis, the comparative Ct method was used wherein the relative
changes in gene expression were calculated using the
.DELTA..DELTA.Ct (threshold cycle) method. This method first
subtracts the ct (threshold cycle number) of the gene-average ct of
the five housekeeping genes on the array (B2M HPRT1, RPL13A, GAPDH
and ACTB) to normalize to the RNA amount. Finally, the
.DELTA..DELTA.Ct was calculated as the difference between the
normalized average ct of each gene on the array after
DOTAP:chol-TUSC2 treatment and the normalized average ct of the
pre-treatment control sample. This .DELTA..DELTA.Ct was then raised
to the power of 2 to calculate the relative fold-change of gene
expression after-treatment compared to pre-treatment. Genes that
differed from pretreatment controls by more than two fold were
considered significant and changes of gene expression levels larger
than three-fold were shown as a scatter plot.
[0268] The expression of major genes in apoptosis signaling pathway
in tumor fine needle biopsies from human lung cancer patients
before and after systemic treatment with DOTAP:chol-TUSC2
nanoparticles were quantified using a qRT Profiler Apoptosis PCR
Array with RT Nano PreAmp-mediated cDNA synthesis (SA Biosciences,
Frederick, Md.). The quantitative apoptotic gene expression data
were analyzed through the use of Ingenuity Pathway Analysis (IPA)
Ingenuity Systems, (see, e.g., the world wide web at
ingenuity.com). For the network and canonical pathway analysis, the
quantitative PCR data set containing gene identifiers and
corresponding expression fold change values was uploaded into the
application. Each identifier was mapped to its corresponding object
in Ingenuity's Knowledge Base (IKB). An expression fold change
(posttreatment/pretreatment) cutoff of 3 was set to identify
molecules whose expression was significantly differentially
regulated. These molecules, called Network Eligible molecules, were
overlaid onto a global molecular network developed from information
contained in IKB. Networks of Network Eligible Molecules were then
algorithmically generated based on their direct or indirect
connectivity. The Network molecules associated with biological
functions in IKB were considered for analysis. Right-tailed
Fisher's exact test was used to calculate a p-value determining the
probability that each biological function assigned to a given
network is due to chance alone. Molecules from the data set that
met the above gene expression fold changes cutoff were also
considered for the canonical pathway analysis. The significance of
the association between the data set and the canonical pathway was
measured by a ratio of the total number of molecules from the data
set that map to the pathway to the total number of molecules that
map to the canonical pathway in IKB. A Fisher's exact test was used
to calculate a p-value determining the probability that the
association between the genes in the dataset and the canonical
pathway is explained by chance alone.
[0269] Significant differences in gene expression were detected by
an apoptosis multiplex array between a pre and post-treatment
biopsy from patient No. 31 whose tumor biopsies showed high levels
of TUSC2 mRNA and protein post-treatment (FIG. 1B). The changes in
gene expression and canonical apoptosis pathways in TUSC2-mediated
apoptosis are graphically represented by FIG. 1C. Analysis methods
are detailed below.
Example 9
Response and Survival
[0270] Twenty-three patients received two or more doses. Five
patients achieved stable disease (range 2.6 to 10.8 months, median
5.0, 95% CI 2.0-7.6) and all other patients progressed. Two
patients had reductions in primary tumor size of 14% and 26%. One
patient with stable disease (patient 26) had evidence of a durable
metabolic response on positron emission tomography (PET) imaging
(FIG. 3) and received 12 cycles of therapy. The response was
documented with PET scans performed after the second, fourth (FIG.
2), and sixth doses, all showing decreased metabolic activity with
no changes in size or number of metastases by CT imaging. This
patient remains alive on subsequent therapy 14 months after the
final treatment with DOTAP:chol-TUSC2. Median survival for all
patients was 8.3 months (95% CI 6.0-10.5 months,) and mean survival
time was 13.2 months (95% CI 8.9-7.5 months) with a range of 2 to
21+ months).
Example 10
Predicting Clinical Benefit
[0271] Formalin fixed paraffin embedded (FFPE) pretreatment tumor
samples obtained at initial diagnosis were available from 10
patients for assessment of baseline TUSC2 protein expression and
AI. Only FFPE tissue could be used for this assay. All pre and
posttreatment biopsies obtained specifically for this protocol were
preserved in RNAlater (e.g., Patients 13 and 31) and could not be
used for IHC.
[0272] TUSC2 Protein Expression
[0273] Formalin-fixed and paraffin-embedded (FFPE) tissue histology
sections (5 .mu.m thick) were baked overnight at 56.degree.,
deparaffinized, hydrated. Heat-induced epitope retrieval was
performed in a DAKO antigen retrieval bath (10 mmol/L of sodium
citrate, pH 6.0) at 121.degree. C. for 30 seconds and 90.degree. C.
for 10 seconds in a decloaking chamber (Biocare, Concord, Calif.),
followed by a 30-min cool down. Peroxide blocking was done with 3%
H.sub.2O.sub.2 in methanol at room temperature for 15 min, followed
by 10% bovine serum albumin in TBS-t for 30 min. The slides were
incubated with primary antibody at 1:400 dilution for 65 min at
room temperature. After washing with TBS-t, incubation with
biotin-labeled secondary antibody for 30 min followed. The samples
were incubated with a 1:40 solution of streptavidin-peroxidase for
30 min. The staining was then developed with 0.05%
3',3-diaminobenzidine tetrahydrochloride prepared in 0.05 mol/L of
Tris buffer at pH 7.6 containing 0.024% H.sub.2O.sub.2 and then
counterstained with hematoxylin. Formalin-fixed and
paraffin-embedded lung tissues with normal bronchial epithelia were
used as a positive control. For a negative control, the same
specimens used for the positive controls were used, replacing the
primary antibody with TBS-t. TUSC2 immunostaining was detected in
the cytoplasm of normal epithelium and tumor cells.
Immunohistochemical expression was quantified by two independent
pathologists (M. I. Nunez and I. I. Wistuba) using a four-value
intensity score (0, 1+, 2+, and 3+) and the percentage of the
reactivity extent. A consensus value on both intensity and
extension was reached by the two independent observers. A final
consensual score was obtained by multiplying both intensity and
extension values (range, 0-300).
[0274] TdT-Mediated dUTP Nick End Labeling (TUNEL) Assay and
Apoptotic Index
[0275] FFPE tissue sections were stained using the DeadEnd.TM.
Colorimetric TUNEL System (Cat G7130, Promega, Madison, Wis.)
according to technical manufacture recommendation. The negative
controls were performed omitting the rTdT enzyme in the TUNEL
reaction mixture. The positive controls were performed treating the
tissues with DNase I enzyme (Cat # M6101, Promega, Madison, Wis.)
prior to the reaction mixture. 10 high-powered fields (.times.400)
per case were assessed (at least 1000 cells). The apoptotic index
(AI) was the total number of TUNEL positive cells per 1000 cells
counted.
[0276] Results of these studies showed that TUSC2 protein
expression in pretreatment FFPE tumor biopsies was low in most of
the pretreatment biopsies with only two samples exceeding the level
noted in normal bronchial epithelium (FIG. 4). The level of
pretreatment TUSC2 protein expression did not correlate with
clinical benefit. TUNEL staining was also performed in 10
pretreatment biopsies and an apoptotic index (AI) was calculated as
detailed above. High levels of AI (>10%) were associated with
achieving a minor response or stable disease while those with an AI
of .ltoreq.10% all had progressive disease (FIG. 5).
Example 11
TSC2 Therapy in Combination with EGFR-Targeted Therapy
[0277] To assess cooperative effects of tumor growth data between
FUS-1 (FUS1) and Erlotinib (Erlo) a Bayesian Bootstrapping analysis
approach was used. The Pr(min(.mu..sub.F,
.mu..sub.E)<.mu..sub.C|data) was calculated i.e., the posterior
probability that the minimum of the two posterior mean colony
formation for FUS-1 alone, .mu..sub.F, or Erlotinib alone,
.mu..sub.E, is less than the mean posterior colony formation for
the combination .mu..sub.C. This probability calculates the
likelihood that average colony formation in the combination arm
will be less than the minimum of the two single agent arms.
Cooperative effects are shown if this posterior probability is
large. Thus, the probability of cooperative effect ranges from 0.0
to 1.0 where 0 means no chance of a true cooperative effect given
the data observed while 1 means 100% certainty of a cooperative
effect given the data observed. The Statistical software S-PLUS 8.0
were used for all the analyses.
[0278] In the studies presented here all cell lines, including
H1975 cells, which have two EGFR mutations (L858R/T790M), and doses
of erlotinib showed almost near certainty of a cooperative effect
(See, FIGS. 6-10 and Tables 5-8). The probability of cooperative
effectiveness calculated from the results of each of the studies is
provided below:
[0279] For 1299 cells: FUS1+Erlotinib (1.0 .mu.g) (Probability of
Cooperative Effect=1.00); FUS1+Erlotinib (2.3 .mu.g) (Probability
of Cooperative Effect=1.00). See Table 5; FIG. 6.
[0280] For H322 cells: FUS1+Erlotinib (1.0 .mu.g) (Probability of
Cooperative Effect=1.0); FUS1+Erlotinib (2.3 .mu.g) (Probability of
Cooperative Effect=1.0). See Table 6; FIG. 7.
[0281] For A549 cells: FUS1+Erlotinib (1.0 .mu.g) (Probability of
Cooperative Effect=0.9981); FUS1+Erlotinib (2.3 .mu.g) (Probability
of Cooperative Effect=1.00). See Table 7; FIG. 8.
[0282] For H460 cells: FUS1+Erlotinib (1.0 .mu.g) (Probability of
Cooperative Effect=0.9874); FUS1+Erlotinib (2.3 .mu.g) (Probability
of Cooperative Effect=1.0). See Table 8; FIG. 9.
[0283] For H1975 cells: FUS1+Erlotinib (1.0 .mu.g) (Probability of
Cooperative Effect=1.0); FUS1+Erlotinib (2.3 .mu.g) (Probability of
Cooperative Effect=1). See FIG. 10.
TABLE-US-00007 TABLE 5 Fus1 and Erlotinib Combine Treatment Effect
on Colony Formation of H1299 Cells. Group pc301PBS pc301 + 1 pc301
+ 2.3 EV + PBS EV + 1 EV + 2.3 Fus1 + PBS Fus1 + 1 Fus1 + 2.3 1
40355.0 31957.0 24425.0 15796.0 18287.0 9216.0 12651.0 6212.0
4315.0 2 39639.0 25107.0 18192.0 18653.0 15301.0 8082.0 10095.0
5307.0 4633.0 3 47131.0 32817.0 21246.0 17517.0 17010.0 7128.0
9988.0 7396.0 4070.0 average 42375 29960 21288 17322 16866 8142
10911 6305 4339 SD 4134 4225 3117 1438 1498 1045 1508 1048 282 CV %
9.8% 14.1% 14.6% 8.3% 8.9% 12.8% 13.8% 16.6% 6.5% P value of 0.013
0.009 0.406 0.010 0.027 0.009 Ttest erlo diffent dose 0.014 0.004
0.062 EV vs 0.7231 0.0009 0.0060 FUS1 or Erlo Fus1 + 1 vs 0.0006
0.0122 Fus1 or Erlo Fus1 + 2.3 0.0037 0.0018 vs Fus1 or Erlo
normalized 100% 71% 50% 41% 40% 19% 26% 15% 10% on pc301 normalized
100% 97% 47% 63% 36% 25% on EV normalized 8.3% 8.6% 6.0% 8.7% 6.0%
1.6% % SD
TABLE-US-00008 TABLE 6 Fus1 and Erlotinib Combine Treatment Effect
on Colony Formation of H322 Cells Group pc301PBS pc301 + 1 pc301 +
2.3 EV + PBS EV + 1 EV + 2.3 Fus1 + PBS Fus1 + 1 Fus1 + 2.3 1 57216
44761 28610 46711 43621 20293 23947 11162 3203 2 65399 37701 28943
46887 36971 16469 19851 11259 3653 3 55676 35079 27080 53930 35119
17511 18660 8947 2782 average 59430 39180 28211 49176 38570 18091
20819 10456 3213 SD 5226 5008 994 4118 4471 1977 2773 1308 436 CV %
8.8% 12.8% 3.5% 8.4% 11.6% 10.9% 13.3% 12.5% 13.6% P value of 0.022
0.003 0.073 0.004 0.007 0.004 Ttest erlo diffent dose 0.026 0.003
0.003 EV vs 0.0391 0.0003 0.0006 FUS1 or Erlo Fus1 + 1 vs 0.0005
0.0042 Fus1 or Erlo Fus1 + 2.3 0.0002 0.0004 vs Fus1 or Erlo
normalized 100% 66% 47% 83% 65% 30% 35% 18% 5% on pc301 normalized
100% 78% 37% 42% 21% 7% on EV normalized 8.4% 9.1% 4.0% 5.6% 2.7%
0.9% % SD
TABLE-US-00009 TABLE 7 Fus1 and Erlotinib Combine Treatment Effect
on Colony Formation of A549 Cells. Group pc301PBS pc301 + 1 pc301 +
2.3 EV + PBS EV + 1 EV + 2.3 Fus1 + PBS Fus1 + 1 Fus1 + 2.3 1 4147
3779 1625 2714 3083 2484 1358 932 511 2 6208 2436 1803 3147 2538
1910 1732 1279 435 3 6586 3393 1651 2716 2334 1738 1308 1358 460
average 5647 3203 1693 2859 2652 2044 1466 1190 469 SD 1313 691 96
249 387 391 232 227 39 CV % 23.2% 21.6% 5.7% 8.7% 14.6% 19.1% 15.8%
19.0% 8.3% P value of 0.073 0.016 0.278 0.057 0.116 0.011 Ttest
erlo diffent dose 0.040 0.000 0.021 EV vs 0.4791 0.0382 0.0021 FUS1
or Erlo Fus1 + 1 vs 0.0049 0.2138 Fus1 or Erlo Fus1 + 2.3 0.0023
0.0018 vs Fus1 or Erlo normalized 100% 57% 30% 51% 47% 36% 26% 21%
8% on pc301 normalized 100% 93% 71% 51% 42% 16% on EV normalized
8.7% 13.5% 13.7% 8.1% 7.9% 1.4% % SD
TABLE-US-00010 TABLE 8 Fus1 and Erlotinib Combine Treatment Effect
on Colony Formation of H460 Cells. Group EV + PBS EV + 1 EV + 2.3
Fus1 + PBS Fus1 + 1 Fus1 + 2.3 1 2117 1216 750 1564 1158 330 2 2179
1393 968 1751 1322 261 3 2106 1470 858 2018 1094 289 average 2134
1360 859 1778 1191 293 SD 39 130 109 228 118 35 CV % 1.8% 9.6%
12.7% 12.8% 9.9% 11.8% P value of 0.005 0.001 0.037 0.005 T-test on
erlo diffent dose 0.006 0.004 EV vs 0.0006 0.00004 0.056 FUS1 or
Erlo Fus1 + 1 vs 0.1719 0.0167 Fus1 or Erlo Fus1 + 2.3 0.0010
0.0004 vs Fus1 or Erlo normalized 100% 64% 40% 83% 56% 14% on EV
normalized 1.8% 6.1% 5.1% 10.7% 5.5% 1.6% % SD
[0284] The use of FUS1 expression to enhance the effectiveness of
gefitinib and overcome gefitinib resistance was also explored in
human NSCLC. Re-expression of wild-type FUS1 by
FUS1-nanoparticle-mediated gene transfer into FUS1-deficient and
gefitinib-resistant NSCLC cell lines H1299, H322, H358, and H460
cells that have a wild-type EGFR significantly (P<0.001)
sensitized their response to gefitinib treatment and
synergistically induced apoptosis in vitro and in an H322
orthotopic lung cancer mouse model (FIG. 12, Note that these
studies included the K-ras mutant cell line H460 which is
significant in that patients with K-ras mutant tumors are in
general unresponsive to EGFR TKIs). Supra-additive induction of
apoptosis was seen with the combination of nanoparticle FUS1 and
concentrations of gefitinib similar to steady-state serum
concentrations achievable with oral dosing. To understand the
mechanism of gefitinib-induced resistance, a gefitinib-resistant
HCC827GR NSCLC cell line (IC.sub.50=16 .mu.M) was established by
selecting against gefitinib from the parental HCC827 cells that
contain an activating deletion mutation of the EGFR gene and are
extremely sensitive to gefitinib treatment (IC.sub.50=0.016 uM). No
secondary mutations in the EGFR gene in the HCC827GR cells was
found, but these cells registered a significantly elevated level of
phosphorylated AKT protein. Combination treatment with
FUS1-nanoparticles and gefitinib at a dose level of IC.sub.10
significantly re-sensitized the cells to gefitinib, as demonstrated
by synergistically enhanced growth inhibition and apoptosis. FUS1
nanoparticle treatment alone or with gefitinib markedly inactivated
EGFR and AKT, as shown by decreased phosphorylation levels of both
proteins on Western blots, compared with either agent alone (FIG.
12D). Cleavage of caspase-3, caspase-9, and PARP was also
significantly induced by the combination of FUS1 and gefitinib in
HCC872GR and other gefitinib-resistant NSCLC cells. The combination
of FUS1 and erlotinib induced similar levels of tumor cell growth
inhibition, apoptosis induction, and inactivation of oncogenic PTKs
as those observed in NSCLC cells treated by a combination of FUS1
and gefitinib (FIG. 12A-D).
Example 12
In Vivo Assessment of TSC2 Therapy in Combination with
EGFR-Targeted Therapy
[0285] The cooperative interaction between erlotinib and FUS1
nanoparticles was confirmed in vivo using a lung colony formation
metastases model in nu/nu mice with A549 human lung cancer cells
injected in the tail vein. Following injection mice were treated
with FUS1 nanoparticles and erlitinib and various controls (FIG.
13). The greatest reduction in lung colonies occurred with the FUS1
nanoparticle/erlotinib combination (90% reduction) which was
significantly reduced compared to all control groups
(p<0.0005).
[0286] These studies along with those in Example 11 showed that a
combination treatment of FUS1 nanoparticles and gefitinib or
erlotinib can promote a synergistic tumor cell killing and overcome
drug-induced resistance by simultaneously inactivating the EGFR and
the AKT signaling pathways and by inducing apoptosis in resistant
cells with wild-type EGFR.
Example 13
Bystander Effect of FUS1-Nanoparticle in NSCLC
[0287] Many currently available gene transfer protocols and
techniques are capable of transducing only a fraction of tumor
cells in vivo, and thus, relying on a bystander effect (killing of
non-transduced cells by products of transduced cells) to achieve
clinically-meaningful therapeutic efficacy. For example, bystander
effects have been observed in cancer gene therapy by adenoviral or
retroviral vector-mediated gene transfer of tumor suppressor genes
such as p53 and TRAIL and for suicide gene HSV-TK in cancer cells.
These bystander effects are induced through various mechanisms
including intercellular communication, interaction of cell surface
receptors and ligands, secretion of cytotoxic or apoptotic
metabolites and peptides, and activation of anticancer cytokine
cascades and the immune response. To test whether ectopic
expression of FUS1 in tumor cells can cause neighboring cell
killing by triggering the release of cytotoxic soluble factors,
conditioned medium (CM) was collected from FUS1-transduced H1299
cells. CM was collected after 48 h in cell culture either
containing or free of bovine fetal serum (BFS) and concentrated 2-5
fold by lyophilization. The CMs from untransduced (PBS), or empty
vector (EV), and myristoylation-deficient mutant FUS1
(mt-FUS1)-transduced cells were used as controls. A marked
inhibition of tumor cell growth (FIG. 14A) and induction of
apoptosis (FIG. 14B) were detected in H1299 cells treated by
concentrated CMs from wt-FUS1-transduced cells compared with those
of controls. In addition, distinct soluble protein/peptide species
were clearly detected in the serum-free wt-FUS1-CM on protein mass
spectra by a ProteinChip array-based SELDI-TOF-MS analysis (FIG.
14C), compared to those of control CMs, suggesting release of
specific soluble peptides. To further test the potential bystander
effects of FUS1 on lung cancer cells, the
wt-FUS1-nanoparticles-transfected H1299 cells were used as effector
cells and mixed them with the Ad-GFP-transduced H1299 target cells,
which do not express FUS1, at a ratio of 1:1. The empty vector
(EV)-nanoparticle-transfected H1299 effectors were used as the
control. The mixed cells were then seeded into a 6-well plate and
cultured for 48 hr. The dead/apoptotic cells were labeled by PI
staining and analyzed by flow cytometry to determine the extent of
cell death and apoptosis in both effector and target (GFP) cells.
An increased population of dead/apoptotic cells was detected in the
GFP-expressing target cells mixed with wt-FUS1-transfected H1299
effecter cells, compared with that of target cells mixed with
EV-transfected effectors (FIG. 15). This effect is comparable to
that seen with a secreted protein such as TRAIL. These preliminary
data support the presence of bystander effects induced by
FUS1-nanoparticle-mediated gene transfer in lung cancer cells.
Example 14
Preclinical Animal Studies with FUS1-Nanoparticles
[0288] Murine Studies
[0289] The mouse LD.sub.10 for a single intravenous dose of
DOTAP:Cholesterol-Fus1 liposome complex was determined from a
series of experiments. For each experiment, C3H strain mice (4 to 6
weeks old, estimated total blood volume 1 ml) were injected over a
period of approximately 3 minutes. The doses ranged from 50 to 150
mcg of DOTAP:Cholesterol-Fus1 liposome complex, and the total
injection volume ranged from 100 to 300 microliters. The results of
the dose-escalation study in mice are summarized below in Table
9.
TABLE-US-00011 TABLE 9 DOTAP:Chol-Fus1 dose escalation in mice
DOTAP:Chol-Fus1 Total number Injection Number of dose (mcg) of mice
volumn (ml) deaths (%) 50 8 100 0 60 8 120 0 70 8 140 0 80 8 160 0
90 8 180 0 100 23 200 2 (8.6%) 110 18 220 0 120 18 240 1 (5.6%) 130
18 260 7 (39%) 150 23 300 7 (30%)
[0290] The LD.sub.10 for a single intravenous injection in mice was
conservatively estimated to be 100 micrograms. Of significance, the
drug was infused over approximately 3 minutes, and the injection
volumes ranged from 100 to 300 microliters, or the equivalent of 10
to 30% of the animals' total blood volume. This rapid rate of
infusion would never be used in humans, and the relationship of the
rapid infusion rate to the observed animal toxicity remains
unclear.
[0291] Autopsies were obtained on all animals that died secondary
to acute toxicity. Pathological examination of the brain, heart,
lungs, spleen, liver, gastrointestinal tract, and kidneys were
performed by an attending veterinary pathologist. The pathology
findings are summarized below in Table 10.
TABLE-US-00012 TABLE 10 Pathology in DOTAP:Chol-Fus1 treated mice
Dose Number of (mcg) autopsies Pathology findings (number of
animals) 100 2 Lymphoid tissue and spleen, necrosis, apoptosis, and
atrophy, moderate (2) Multifocal liver degeneration and necrosis,
mild (1) Acute liver necrosis, mild (1) 120 1 Lymphoid tissue,
spleen, and GALT necrosis, apoptosis, and atrophy, moderate (1)
Acute liver necrosis, moderate (1) Malignant lymphoma, kidney (1)
Glomerulonephritis (1) 130 7 Lymphoid tissue and spleen, necrosis,
apoptosis, and atrophy, mild (1), moderate (6) Acute liver
necrosis, mild (3), moderate (3), severe (1) Multifocal myocardial
degeneration, necrosis, and mineralization, moderate (2), severe
(1) Acute tubular necrosis, kidney, minimal (1) Lung
granuloma/foreign bodies (1) Intestinal crypt epithelial acute
necrosis, mild (1) 150 7 Lymphoid tissue and spleen, necrosis,
apoptosis, and atrophy, mild (3), moderate (4) Acute liver
necrosis, mild (4), moderate (1), severe (2) Multifocal myocardial
degeneration, necrosis, and mineralization, mild (1), moderate (2)
Acute tubular necrosis, kidney, mild (1) Multiple subacute to
chronic kidney infarcts (1) Spleen red pulp myeloid hyperplasia (1)
Spleen sinus histiocyte marked hyperplasia (1) Intestinal crypt
epithelial acute necrosis, mild (1) Note: Multifocal myocardial
degeneration, necrosis, and mineralization are most likely
incidental findings observed in control C3H mice (ref. Vargas, K J,
Stephens, L C, Clifford, C B, et al. Dystrophic Cardiac Calcinosis
in C3H/HeN Mice. Lab Anim Sci, 46:572-575, 1996.) Minimal = 1+,
5-10%; Mild = 2+, 10-20%; Moderate = 3+, 20-50%: Severe = 4+,
>50%.
[0292] GLP Toxicology Studies
[0293] The objective of this study was to determine single dose
toxicology of DOTAP:Chol/fus1 in preparation for Phase I studies.
The non-toxic dose and dose-limiting toxicity for C3H/HeNCR mice
were determined. The study contained three control groups: D5W
(vehicle), 4 mM DOTAP:Chol (highest dose of lipid), and 70 .mu.g
DNA (highest dose of fus1 plasmid). The study also contained three
experimental groups: 70 .mu.g DNA, DOTAP:Chol, 40 .mu.g DNA,
DOTAP:Chol and 10 .mu.g DNA, DOTAP:Chol. Each group contained 15
mice (8 female and 7 male). Acute (0-72 hours), subacute (14 days)
and chronic (6 weeks) toxicity were evaluated. At 3 and 14 days and
at 6 weeks, five mice per group were euthanized. For each mouse, an
attempt was made to collect urine for analysis for CBC and serum
chemistries. Necropsies were performed and histopathological
analysis done on all mice, including those that died during the
study. This study was conducted in an AAALAC accredited facility
(2000).
[0294] All mice in the three control groups (D5W, 4 mM DOTAP:Chol,
and 70 micrograms DNA alone) and in the experimental group
receiving 10 micrograms DNA, DOTAP:Chol were observed to be normal
at all observation time points.
[0295] Mice in the experimental group receiving 40 micrograms DNA,
DOTAP:Chol appeared normal at the end of the 4 hours post-injection
observation period. When observed later that day at approximately 7
hours post injection 14/15 mice were squinting and appeared to be
lethargic. One female mouse was very weak, trembling and sat
hunched with her eyes closed. She was euthanized and sent to
necropsy at that time. On day one post injection (PI), all mice had
decreased activity levels and the eyes appeared to be swollen. On
day two PI, all mice appeared to have returned to normal activity
levels and general appearance. One female mouse had an area of
necrosis involving approximately 20% of one pinna at this time
point, but otherwise appeared normal. The damaged pinna was
interpreted to be the result of trauma. All mice were thereafter
normal at all observation time points. In summary, one female mouse
became moribund on day zero and was euthanized.
[0296] Mice in the experimental group receiving 70 micrograms DNA,
DOTAP:Chol appeared normal at the end of the 4 hours post-injection
observation period. When observed later that day at approximately 7
hours post injection, all mice were squinting and appeared to be
lethargic. On day one PI, one female mouse died. Three male mice
and one female mouse were found to be moribund and were euthanized
and necropsied. One female mouse was reported to have a swollen
face. This mouse and the remaining mice in the group all appeared
to have decreased activity levels and abnormal appearance at day
one PI. On day two PI, the female mouse that had the swollen face
on day one PI was found to be moribund and was euthanized and
necropsied. Another female mouse was found dead on day two PI. The
remaining mice had decreased or slightly decreased activity levels
and some were squinting. On day three PI, 2/8 remaining mice
appeared normal, while 6/8 still had decreased activity levels and
abnormal general appearance. From day four PI and thereafter, all
mice appeared normal at all observation time points. In summary,
two female mice died. Three male and two female mice were found
moribund and were euthanized.
[0297] Non-Human Primate Toxicology
[0298] Ten (10) cynomolgus monkeys (Macaca fascicularis) were used
in the study. Six experimental animals (three male and three
female) were injected with DOTAP:Chol/Fus1 complex on Day 1 and Day
21 of the study. Four control animals (two male and two female)
were injected with DOTAP:Cholesterol alone on Day 1 and Day 21 of
the study. At days 46-52 the animals were necropsied, blood was
collected for hematology and chemistries, and organs were collected
for histopathological analysis.
[0299] Significant gross and microscopic lesions were found in 1/10
monkeys on protocol. This animal received 1 dose of 0.6 mg/kg DNA,
DOTAP: Chol (high dose) and died within 18-20 hours. Lesions in
this monkey were most likely treatment related. A second monkey
that received the high dose of DNA, DOTAP: Chol had changes in a
lymph node. The significance of these minimal changes is not known.
Equivocal lesions were found in the femoral bone marrow of two low
dose (0.2 mg/kg DNA, DOTAP: Chol) monkeys. The latter may be
incidental findings, but were not seen in other protocol animals.
No significant gross or microscopic lesions were found in the
remaining six animals that received either DOTAP: Chol only or 0.2
mg/kg DNA, DOTAP: Chol.
Example 15
Enhancement of Anti-Tumor Activity of MK2206 in Human Lung Cancer
Cells by Tumor Suppressor Gene FUS1
[0300] Studies were undertaken to investigate whether FUS1
nanoparticles can sensitize lung cancer cells to chemotherapeutic
agents such as MK2206. First preliminary studies were performed to
determine the DC transfection efficiency in various lung cancer
cell lines. Results of these studies are shown below in Tables 11
and 12.
TABLE-US-00013 TABLE 11 Cell lines with high DC transfection
efficiency Cell line GFP (%) H2882 53.9 H1395 52.9 H2450 51.4 H358
46.1 H1299 40.2 H1171 37.8 H2887 34.6 H661 33.1 H522 30.8 Calu-1 25
H1650 24.8 H322 24.7 HCC827 23.6 HCC366 22.4
TABLE-US-00014 TABLE 12 Cell lines with low DC transfection
efficiency Cell line GFP (%) H196 17 H460 11.46 H1944 11.4 H1703
11.3 H1355 9.03 H1648 8.9 Calu-6 8.6 H1993 8.4 H1975 7.97 Calu-3
7.74 HCC193 7.53 H2052 6.01 H515 6 H2009 3.51 H838 2.88 H2935 1.83
H1792 1.67 H157 1 H3122 0.88 H226 0.56 H1437 0.44 H125 0.36
[0301] Next the effect of FUS1 nanoparticle treatment alone was
assessed in an array of lung cancer cell lines. As shown in FIG.
16, FUS1 was effectively expressed in the HCC366, H322, A549 and
H2887 cell lines. FUS1 expression resulted in a consistent (but not
significant) decrease in cell viability in all cell lines (bottom
panel).
[0302] Single drug treatment with AKT inhibitor MK2206 was also
assessed in a wide range of lung cancer cell lines. The effective
IC.sub.50 on the various cells are shown in FIG. 17. Cell lines
indicated by arrows (H322, A549, H2887 and HCC386) were subjected
to further analysis. First, each of the cell lines was treated with
FUS1 nanoparticles or empty vector at increasing concentrations of
MK2206. Results shown in FIG. 18 show synergistic cell killing
mediated by the combination of FUS1 and the kinase inhibitor. The
effect of the combined therapy was especially evident in H2887,
H322 and HCC366 cells. Next, the ability of combined FUS1/MK2206
treatment to inhibit colony formation was studied in the cell
lines. Graphs shown in FIG. 19 demonstrate that the combination of
MK2206 and FUS1 was significantly more effective than either
treatment alone at inhibiting colony formation. Thus, FUS1
treatment is able to sensitize cancer cells to the effects of
kinase inhibitors such as the AKT inhibitor MK2206. This effect was
quantified relative to each studied cell line below in Table
13.
[0303] Additional colony formation assays in both H322 and H1299
cells demonstrated that TUSC2 nanoparticles synergistically
inhibited colony formation in the cancer cells when combined with
the EGFR-targeted therapeutic afatinib (FIG. 26A-B). In these
studies, afatinib showed even greater effect in combination with
TUSC2 than similar concentrations of erlotinib combined with TUSC2.
Still further studies indicated that dasatinib has enhanced
anti-cancer activity when used in conjunction with TUSC2
nanoparticles.
TABLE-US-00015 TABLE 13 Fold decrease in IC.sub.50 of MK2206 when
combined with FUS1-nanoparticles and gene mutation status IC.sub.50
IC.sub.50 Fold Cell line (MK2206 alone) (MK2206 + FUS1) reduction
kras Braf EGFC PIK3CA LKB1 H322 20.39 1.24 16.4 wt wt wt wt mutant
HCC366 18.4 2.17 8.5 -- -- -- -- mutant H2887 16.53 1.28 12.9 -- --
-- -- -- A549 2.86 0.56 5.1 mutant wt wt wt mutant
[0304] Further studies were undertaken to evaluate the ability of
FUS1 and MK2206 treatment to induce apoptosis. Cells were treated
with the two agents, or each individually, stained by propidium
iodide (PI) and analyzed by flow cytometry. Results of these
studies are shown in the histograms of FIG. 20. In the case of each
cell line, combined FUS1 and MK2206 treatment resulted in
significantly more apoptotic cells as compared to either agent
alone (indicated by the horizontal bar in the histograms).
[0305] To better determine the mechanism for synergistic
FUS1/MK2206 effect, treated cells were subjected to an immunoblots
to assess the phosphorylation status of cell signaling molecules.
As shown in FIG. 21, FUS1 alone resulted in an increased in
phosphorylated AMPK (p-AMPK), but had little effect on the level of
phosphorylated AKT (p-AKT). On the other hand, the addition of
MK2206 significantly reduced phosphorylated AKT levels, while a
robust increase in phosphorylated AMPK (mediated by FUS1) was
maintained. Indeed, the role of AMPK signaling in FUS1-mediated
cell killing was confirmed by the fact that treatment of cells with
a AMPK-targeted siRNA partially protected the cells from the
effects of FUS1/MK2206 treatment (see, e.g., FIG. 22). Likewise, an
inhibitor of AMPK activity (Compound C) was also able to partially
protect cancer cells from FUS1/MK2206-mediated killing (FIG. 23).
Thus, FUS1-increased sensitivity to MK2206 is associated with the
down-regulation of AKT and mTOR phosphorylation and up-regulation
of AMPK phosphorylation. In view of these studies a proposed
FUS/MK2206 signaling pathway is provided as FIG. 25.
[0306] The in vivo effectiveness of combination FUS1 and MK2206
treatment was further assessed using a mouse xenograft model. For
these studies H322 cells were transplanted into mice and the
explanted cells allowed to grow in vivo. Tumor mass was assessed at
various time points in the presence of FUS1 therapy, MK2206 therapy
or the combination of the two. In all cases expression of FUS1 and
activity of MK2206 (as evidenced by reduced p-AKT expression) was
histologically evaluated in samples from the mice. As shown in FIG.
24, results of these studies showed that combined FUS1 and MK2206
therapy was far more effective than either treatment alone at
inhibiting tumor growth in the animals.
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Sequence CWU 1
1
517936DNAArtificial SequencePlasmid vector 1cgaaatcatc aataatatac
cttattttgg attgaagcca atatgataat gctttagtag 60ttattatatg gaataaaacc
taacttcggt tatactatta gagggggtgg agtttgtgac 120gtggcgcggg
gcgtgggaac ggggcgggtg ctcccccacc tcaaacactg caccgcgccc
180cgcacccttg ccccgcccac acgtagtagt gtggcggaag tgtgatgttg
caagtgtggc 240ggaacacatg tgcatcatca caccgccttc acactacaac
gttcacaccg ccttgtgtac 300taagcgacgg atgtggcaaa agtgacgttt
ttggtgtgcg ccggtgtata attcgctgcc 360tacaccgttt tcactgcaaa
aaccacacgc ggccacatat cgggaagtga caattttcgc 420gcggttttag
gcggatgttg tagtaaattt gcccttcact gttaaaagcg cgccaaaatc
480cgcctacaac atcatttaaa gggcgtaacc aagtaatatt tggccatttt
cgcgggaaaa 540ctgaataaga cccgcattgg ttcattataa accggtaaaa
gcgccctttt gacttattct 600ggaagtgaaa tctgaataat tctgtgttac
tcatagcgcg taatatttgt ccttcacttt 660agacttatta agacacaatg
agtatcgcgc attataaaca ctagggccgc ggggactttg 720accgtttacg
tggagactcg cccaggtgtt gatcccggcg cccctgaaac tggcaaatgc
780acctctgagc gggtccacaa tttctcaggt gttttccgcg ttccgggtca
aagttggcgt 840tttattatta aaagagtcca caaaaggcgc aaggcccagt
ttcaaccgca aaataataat 900tagtcagctc tagagcccga cattgattat
tgactagtta ttaatagtaa atcagtcgag 960atctcgggct gtaactaata
actgatcaat aattatcatt tcaattacgg ggtcattagt 1020tcatagccca
tatatggagt tccgcgttac agttaatgcc ccagtaatca agtatcgggt
1080atatacctca aggcgcaatg ataacttacg gtaaatggcc cgcctggctg
accgcccaac 1140gacccccgcc tattgaatgc catttaccgg gcggaccgac
tggcgggttg ctgggggcgg 1200cattgacgtc aataatgacg tatgttccca
tagtaacgcc aatagggact gtaactgcag 1260ttattactgc atacaagggt
atcattgcgg ttatccctga ttccattgac gtcaatgggt 1320ggagtattta
cggtaaactg cccacttggc aaggtaactg cagttaccca cctcataaat
1380gccatttgac gggtgaaccg agtacatcaa gtgtatcata tgccaagtac
gccccctatt 1440gacgtcaatg tcatgtagtt cacatagtat acggttcatg
cgggggataa ctgcagttac 1500acggtaaatg gcccgcctgg cattatgccc
agtacatgac cttatgggac tgccatttac 1560cgggcggacc gtaatacggg
tcatgtactg gaataccctg tttcctactt ggcagtacat 1620ctacgtatta
gtcatcgcta ttaccatggt aaaggatgaa ccgtcatgta gatgcataat
1680cagtagcgat aatggtacca gatgcggttt tggcagtaca tcaatgggcg
tggatagcgg 1740tttgactcac ctacgccaaa accgtcatgt agttacccgc
acctatcgcc aaactgagtg 1800ggggatttcc aagtctccac cccattgacg
tcaatgggag tttgttttgg cccctaaagg 1860ttcagaggtg gggtaactgc
agttaccctc aaacaaaacc caccaaaatc aacgggactt 1920tccaaaatgt
cgtaacaact ccgccccatt gtggttttag ttgccctgaa aggttttaca
1980gcattgttga ggcggggtaa gacgcaaatg ggcggtaggc gtgtacggtg
ggaggtctat 2040ataagcagag ctgcgtttac ccgccatccg cacatgccac
cctccagata tattcgtctc 2100ctcgtttagt gaaccgtcag atcgcctgga
gacgccatcc acgctgtttt gagcaaatca 2160cttggcagtc tagcggacct
ctgcggtagg tgcgacaaaa gacctccata gaagacaccg 2220ggaccgatcc
agcctccgcg gccgggaacg ctggaggtat cttctgtggc cctggctagg
2280tcggaggcgc cggcccttgc gtgcattgga acggacctgc agcccaagct
tggtaccgag 2340ctcggatcca cacgtaacct tgcctggacg tcgggttcga
accatggctc gagcctaggt 2400ctagtccagt gtggtggaat tcggcttgac
atgggcgcca gcgggtccaa gatcaggtca 2460caccacctta agccgaactg
tacccgcggt cgcccaggtt agctcggggc ctgtggccct 2520tcgcctcggc
ggccggaggc ggcggctcag tcgagccccg gacaccggga agcggagccg
2580ccggcctccg ccgccgagtc aggcagcagg agctgagcaa gctttggtgc
ggcctcgggg 2640ccgagctgtg tccgtcgtcc tcgactcgtt cgaaaccacg
ccggagcccc ggctcgacac 2700ccccccttcg tattcacgcg ccgcggctct
atgttctatg atgaggatgg ggggggaagc 2760ataagtgcgc ggcgccgaga
tacaagatac tactcctacc ggatctggct cacgagttct 2820atgaggagac
aatcgtcacc aagaacgggc cctagaccga gtgctcaaga tactcctctg
2880ttagcagtgg ttcttgcccg agaagcgggc caagctgagg cgagtgcata
agaatctgat 2940tcctcagggc tcttcgcccg gttcgactcc gctcacgtat
tcttagacta aggagtcccg 3000atcgtgaagc tggatcaccc ccgcatccac
gtggatttcc ctgtgatcct tagcacttcg 3060acctagtggg ggcgtaggtg
cacctaaagg gacactagga ctatgaggtg tgaccctgga 3120agccgaattc
tgcagatatc cagcacaagt gatactccac actgggacct tcggcttaag
3180acgtctatag gtcgtgttca ggcggccgct cgagtctaga gggcccgttt
aaacccgctg 3240atcagcctcg ccgccggcga gctcagatct cccgggcaaa
tttgggcgac tagtcggagc 3300actgtgcctt ctagttgcca gccatctgtt
gtttgcccct cccccgtgcc tgacacggaa 3360gatcaacggt cggtagacaa
caaacgggga gggggcacgg ttccttgacc ctggaaggtg 3420ccactcccac
tgtcctttcc taataaaatg aaggaactgg gaccttccac ggtgagggtg
3480acaggaaagg attattttac aggaaattgc atcgcattgt ctgagtaggt
gtcattctat 3540tctggggggt tcctttaacg tagcgtaaca gactcatcca
cagtaagata agacccccca 3600ggggtggggc aggacagcaa gggggaggat
tgggaagaca atagcaggca ccccaccccg 3660tcctgtcgtt ccccctccta
acccttctgt tatcgtccgt tgctggggat gcggtgggct 3720ctatggcttc
tactgggcgg ttttatggac acgaccccta cgccacccga gataccgaag
3780atgacccgcc aaaatacctg agcaagcgaa ccggaattgc cagctggggc
gccctctggt 3840aaggttggga tcgttcgctt ggccttaacg gtcgaccccg
cgggagacca ttccaaccct 3900agccctgcaa agtaaactgg atggctttct
cgccgccaag gatctgatgg tcgggacgtt 3960tcatttgacc taccgaaaga
gcggcggttc ctagactacc cgcaggggat caagctctga 4020tcaagagaca
ggatgaggat cgtttcgcat gcgtccccta gttcgagact agttctctgt
4080cctactccta gcaaagcgta gattgaacaa gatggattgc acgcaggttc
tccggccgct 4140tgggtggaga ctaacttgtt ctacctaacg tgcgtccaag
aggccggcga acccacctct 4200ggctattcgg ctatgactgg gcacaacaga
caatcggctg ctctgatgcc ccgataagcc 4260gatactgacc cgtgttgtct
gttagccgac gagactacgg gccgtgttcc ggctgtcagc 4320gcaggggcgc
ccggttcttt ttgtcaagac cggcacaagg ccgacagtcg cgtccccgcg
4380ggccaagaaa aacagttctg cgacctgtcc ggtgccctga atgaactgca
agacgaggca 4440gcgcggctat gctggacagg ccacgggact tacttgacgt
tctgctccgt cgcgccgata 4500cgtggctggc cacgacgggc gttccttgcg
cagctgtgct cgacgttgtc gcaccgaccg 4560gtgctgcccg caaggaacgc
gtcgacacga gctgcaacag actgaagcgg gaagggactg 4620gctgctattg
ggcgaagtgc cggggcagga tgacttcgcc cttccctgac cgacgataac
4680ccgcttcacg gccccgtcct tctcctgtca tctcaccttg ctcctgccga
gaaagtatcc 4740atcatggctg agaggacagt agagtggaac gaggacggct
ctttcatagg tagtaccgac 4800atgcaatgcg gcggctgcat acgcttgatc
cggctacctg cccattcgac tacgttacgc 4860cgccgacgta tgcgaactag
gccgatggac gggtaagctg caccaagcga aacatcgcat 4920cgagcgagca
cgtactcgga tggaagccgg gtggttcgct ttgtagcgta gctcgctcgt
4980gcatgagcct accttcggcc tcttgtcgat caggatgatc tggacgaaga
gcatcagggg 5040ctcgcgccag agaacagcta gtcctactag acctgcttct
cgtagtcccc gagcgcggtc 5100ccgaactgtt cgccaggctc aaggcgagca
tgcccgacgg cgaggatctc ggcttgacaa 5160gcggtccgag ttccgctcgt
acgggctgcc gctcctagag gtcgtgaccc atggcgatgc 5220ctgcttgccg
aatatcatgg tggaaaatgg cagcactggg taccgctacg gacgaacggc
5280ttatagtacc accttttacc ccgcttttct ggattcatcg actgtggccg
gctgggtgtg 5340gcggaccgct ggcgaaaaga cctaagtagc tgacaccggc
cgacccacac cgcctggcga 5400atcaggacat agcgttggct acccgtgata
ttgctgaaga gcttggcggc tagtcctgta 5460tcgcaaccga tgggcactat
aacgacttct cgaaccgccg gaatgggctg accgcttcct 5520cgtgctttac
ggtatcgccg ctcccgattc cttacccgac tggcgaagga gcacgaaatg
5580ccatagcggc gagggctaag gcagcgcatc gccttctatc gccttcttga
cgagttcttc 5640tgaattatta cgtcgcgtag cggaagatag cggaagaact
gctcaagaag acttaataat 5700acgcttacaa tttcctgatg cggtattttc
tccttacgca tctgtgcggt tgcgaatgtt 5760aaaggactac gccataaaag
aggaatgcgt agacacgcca atttcacacc gcatacaggt 5820ggcacttttc
ggggaaatgt gcgcggaacc taaagtgtgg cgtatgtcca ccgtgaaaag
5880cccctttaca cgcgccttgg cctatttgtt tatttttcta aatacattca
aatatgtatc 5940cgcttaagaa ggataaacaa ataaaaagat ttatgtaagt
ttatacatag gcgaattctt 6000catgtgagca aaaggccagc aaaaggccag
gaaccgtaaa aaggccgcgt gtacactcgt 6060tttccggtcg ttttccggtc
cttggcattt ttccggcgca tgctggcgtt tttccatagg 6120ctccgccccc
ctgacgagca tcacaaaaat acgaccgcaa aaaggtatcc gaggcggggg
6180gactgctcgt agtgttttta cgacgctcaa gtcagaggtg gcgaaacccg
acaggactat 6240aaagatacca gctgcgagtt cagtctccac cgctttgggc
tgtcctgata tttctatggt 6300ggcgtttccc cctggaagct ccctcgtgcg
ctctcctgtt ccgaccctgc ccgcaaaggg 6360ggaccttcga gggagcacgc
gagaggacaa ggctgggacg cgcttaccgg atacctgtcc 6420gcctttctcc
cttcgggaag cgtggcgctt gcgaatggcc tatggacagg cggaaagagg
6480gaagcccttc gcaccgcgaa tctcatagct cacgctgtag gtatctcagt
tcggtgtagg 6540tcgttcgctc agagtatcga gtgcgacatc catagagtca
agccacatcc agcaagcgag 6600caagctgggc tgtgtgcacg aaccccccgt
tcagcccgac cgctgcgcct gttcgacccg 6660acacacgtgc ttggggggca
agtcgggctg gcgacgcgga tatccggtaa ctatcgtctt 6720gagtccaacc
cggtaagaca cgacttatcg ataggccatt gatagcagaa ctcaggttgg
6780gccattctgt gctgaatagc ccactggcag cagccactgg taacaggatt
agcagagcga 6840ggtatgtagg ggtgaccgtc gtcggtgacc attgtcctaa
tcgtctcgct ccatacatcc 6900cggtgctaca gagttcttga agtggtggcc
taactacggc tacactagaa gccacgatgt 6960ctcaagaact tcaccaccgg
attgatgccg atgtgatctt gaacagtatt tggtatctgc 7020gctctgctga
agccagttac cttcggaaaa cttgtcataa accatagacg cgagacgact
7080tcggtcaatg gaagcctttt agagttggta gctcttgatc cggcaaacaa
accaccgctg 7140gtagcggtgg tctcaaccat cgagaactag gccgtttgtt
tggtggcgac catcgccacc 7200tttttttgtt tgcaagcagc agattacgcg
cagaaaaaaa ggatctcaag aaaaaaacaa 7260acgttcgtcg tctaatgcgc
gtcttttttt cctagagttc aagatccttt gatcttttct 7320acggggtctg
acgctcagtg gaacgaaaac ttctaggaaa ctagaaaaga tgccccagac
7380tgcgagtcac cttgcttttg tcacgttaag ggattttggt catggctagt
taatcatgag 7440attatcaaaa agtgcaattc cctaaaacca gtaccgatca
attagtactc taatagtttt 7500aggatcttca cctagatcct tttaaattaa
aaatgaagtt ttaaatcaat tcctagaagt 7560ggatctagga aaatttaatt
tttacttcaa aatttagtta ctaaagtata tatgagtaaa 7620cttggtctga
cagttaccaa tgcttaatca gatttcatat atactcattt gaaccagact
7680gtcaatggtt acgaattagt gtgaggcacc tatctcagcg atctgtctat
ttcgttcatc 7740catagttgcc cactccgtgg atagagtcgc tagacagata
aagcaagtag gtatcaacgg 7800tgactccccg tcgtgtagat aactacgata
cgggagggct taccatctgg actgaggggc 7860agcacatcta ttgatgctat
gccctcccga atggtagacc ccccagtgct gcaatgatgg 7920ggtcacgacg ttacta
7936217PRTHomo sapiens 2Gly Ala Ser Gly Ser Lys Ala Arg Gly Leu Trp
Pro Phe Ala Ser Ala 1 5 10 15 Ala 318DNAArtificial
SequenceSynthetic primer 3ggacctgcag cccaagct 18419DNAArtificial
SequenceSynthetic primer 4gcccatgtca agccgaatt 19527DNAArtificial
SequenceSynthetic primer 5cgagctcgga tccactagtc cagtgtg 27
* * * * *
References