U.S. patent application number 16/773769 was filed with the patent office on 2020-05-21 for polymalic acid-based nanobiopolymer compositions.
The applicant listed for this patent is CEDARS-SINAI MEDICAL CENTER. Invention is credited to Keith L. Black, Hui Ding, Eggehard Holler, Satoshi Inoue, Julia Y. Ljubimova.
Application Number | 20200155593 16/773769 |
Document ID | / |
Family ID | 51061113 |
Filed Date | 2020-05-21 |
United States Patent
Application |
20200155593 |
Kind Code |
A1 |
Inoue; Satoshi ; et
al. |
May 21, 2020 |
POLYMALIC ACID-BASED NANOBIOPOLYMER COMPOSITIONS
Abstract
Nanobiopolymeric conjugates based on biodegradable, non-toxic
and non-immunogenic poly (.beta.-L-malic acid) PMLA covalently
linked to molecular modules that include morpholino antisense
oligonucleotides (AONa), an siRNA or an antibody specific for an
oncogenic protein in a cancer cell, and an antibody specific for a
transferrin receptor protein, are provided. Methods for treating a
cancer in subject with nanobiopolymeric conjugates are
described.
Inventors: |
Inoue; Satoshi; (Beverly
Hills, CA) ; Ding; Hui; (Los Angeles, CA) ;
Holler; Eggehard; (Los Angeles, CA) ; Black; Keith
L.; (Los Angeles, CA) ; Ljubimova; Julia Y.;
(Studio City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CEDARS-SINAI MEDICAL CENTER |
LOS ANGELES |
CA |
US |
|
|
Family ID: |
51061113 |
Appl. No.: |
16/773769 |
Filed: |
January 27, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15447439 |
Mar 2, 2017 |
10583151 |
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16773769 |
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13930533 |
Jun 28, 2013 |
9623041 |
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15447439 |
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PCT/US2010/062515 |
Dec 30, 2010 |
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13930533 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/713 20130101;
C07K 16/2881 20130101; A61K 2039/505 20130101; C07K 16/32
20130101 |
International
Class: |
A61K 31/713 20060101
A61K031/713; C07K 16/32 20060101 C07K016/32; C07K 16/28 20060101
C07K016/28 |
Goverment Interests
GOVERNMENT SUPPORT
[0003] The invention was made in part with support from grants
RO1CA123495 and RO1CACA1136841 from the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A composition comprising: a polymalic acid-based scaffold; a
tyrosine kinase inhibitor covalently attached to the polymalic
acid-based scaffold; and a laminin-411 inhibitor covalently
attached to the polymalic acid-based scaffold.
2. The composition of claim 1, wherein the polymalic acid-based
scaffold comprises poly(.beta.-L-malic acid).
3. The composition of claim 1, further comprising an endosomal
escape unit.
4. The composition of claim 1, further comprising an anti-TfR
antibody.
5. The composition of claim 1, further comprising polyethylene
glycol.
6. The composition of claim 1, wherein the tyrosine kinase
inhibitor and the laminin-411 inhibitor are covalently attached to
the polymalic acid-based scaffold via a glutathione-cleavable bond
or via a disulfide bond.
7. The composition of claim 1, wherein the laminin-411 inhibitor
targets an .alpha.4 or .beta.1 subunit of laminin-411.
8. The composition of claim 1, wherein the laminin-411 inhibitor
targets an .alpha.4 subunit of laminin-411, and wherein the
composition further comprises a second laminin-411 inhibitor that
targets a .beta.1 subunit of laminin-411 and is covalently attached
to the polymalic acid-based scaffold.
9. The composition of claim 1, wherein the tyrosine kinase
inhibitor targets HER2 or EGFR.
10. The composition of claim 9, wherein the tyrosine kinase
inhibitor comprises an antibody.
11. The composition of claim 1, wherein the tyrosine kinase
inhibitor comprises gefitinib.
12. The composition of claim 9, wherein the tyrosine kinase
inhibitor or the laminin-411 inhibitor comprises an
oligonucleotide.
13. The composition of claim 12, wherein the oligonucleotide
comprises a morpholino antisense oligonucleotide or an siRNA.
14. The composition of claim 1, wherein the tyrosine kinase
inhibitor comprises a nucleotide sequence comprising
5'-AGGGAGCCGCAGCTTCATGTCTGTG-3' (SEQ ID NO: 1),
5'-CATGGTGCTCACTGCGGCTCCGGC-3' (SEQ ID NO: 2),
5'-TCGCTCCGGCTCTCCCGATCAATAC-3' (SEQ ID NO: 3),
5'-CCUAUAAUGCUACGAAUAUtt-3' (SEQ ID NO: 6),
5'-AUAUUCGUAGCAUUUAUGGag-3' (SEQ ID NO: 7),
5'-GUUGGAUGAUUGACUCUGAtt-3' (SEQ ID NO: 8), or
5'-UCAGAGUCAAUCAUCCAACat-3' (SEQ ID NO: 9).
15. The composition of claim 1, wherein the laminin-411 inhibitor
comprises a nucleotide sequence comprising
5'-AGCTCAAAGCCATTTCTCCGCTGAC-3' (SEQ ID NO:4) or
5'-CTAGCAACTGGAGAAGCCCCATGCC-3' (SEQ ID NO:5).
16. A pharmaceutical composition comprising a therapeutically
effective amount of the polymalic acid-based scaffold of claim 1
and a pharmaceutically acceptable carrier.
17. The pharmaceutical composition of claim 16, formulated for
intravenous administration.
18. A method of treating a cancer in a subject, by inhibiting the
synthesis or activity of laminin-411 and HER2 or EGFR, the method
comprising administering to a subject in need thereof the
pharmaceutical composition of claim 16.
19. The method of claim 18, wherein the cancer is breast
cancer.
20. The method of claim 18, wherein the administration inhibits a
cancer stem cell marker comprising CD133 protein, c-myc protein,
CD44 protein, Notch1 protein, or nestin protein.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation application which claims
the benefit of U.S. application Ser. No. 15/447,439 filed Mar. 2,
2017, which is a continuation of U.S. application Ser. No.
13/930,533 filed Jun. 28, 2013, now issued as U.S. Pat No.
9,623,041 on Apr. 18, 2017, which claims the benefit and is a
continuation-in-part of PCT Patent Application Serial No.
PCT/US2010/062515, filed Dec. 30, 2010, which is incorporated by
reference as if fully set forth.
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 27, 2020, is named 50585-707_302SL.txt and is 2,710 bytes
in size.
FIELD OF INVENTION
[0004] The present invention generally relates to compositions and
methods for treating patients having cell proliferative disorders
with polymalic acid-based nanobiopolymeric compositions that
inhibit synthesis and activity of an oncogenic protein.
BACKGROUND
[0005] Breast cancer is a disease affecting a significant
population of women around the world. About 1 in 8 women in the
United States (between 12 and 13%) will develop invasive breast
cancer over the course of her lifetime. Prognosis and survival rate
varies greatly depending on cancer type and staging. Breast cancers
expressing genetic characteristics such as human epidermal growth
factor receptor-2 (HER2) are associated with a poor prognosis.
[0006] Research has focused on the use of recombinant humanized
monoclonal antibodies for the treatment of cancers with cells that
overexpress protein p185HER2. This 185-kDa growth factor receptor
is encoded by the her-2 proto-oncogene, also referred to as neu and
c-erbB-2 (Slamon et al. 1987 Science 235:177). The her-2 gene is
closely related to the gene encoding epidermal growth factor
receptor (EGFR). Amplification of the her-2 gene has been linked to
neoplastic transformation in human breast cancer cells (Slamon et
al. 1987 Science 235:177). Overexpression of the HER2 protein has
been identified in 20-30% of breast cancer patients, and has been
correlated with regionally advanced disease, increased probability
of tumor recurrence, and reduced patient survival. As many as
30-40% of patients having gastric, endometrial, salivary gland,
non-small cell lung, pancreatic, ovarian, peritoneal, prostate, or
colorectal cancers may also exhibit overexpression of this
protein.
[0007] A more difficult-to-treat form of HER2-negative breast
cancer known as "triple-negative," affects some patients. This form
tests negative for three primary receptors: HER2, estrogen receptor
and progesterone receptor. However, it is positive for epidermal
growth factor receptor (EGFR, HER1).
[0008] Humanized anti-HER2/neu monoclonal antibody trastuzumab
(Herceptin.RTM., Genentech Inc., San Francisco, Calif.) is used
alone or combined with chemotherapy for treatment of patients with
advanced breast cancer overexpressing HER2/neu (Baselga J. 2006
Science 312:1175; Baselga J et al. 1999 Semin Oncol 26:78; Slamon D
J et al. 2009 J Natl Cancer Inst 101:615), with significant
anti-tumor effect. However, serious adverse effects on normal
organs have been reported (Keef D L. 2002 Cancer 95:1592; Vahid B
et al, 2008 Chest 133:528). Moreover, many patients develop
resistance to Herceptin.RTM. within one year of treatment, which
renders this treatment ineffective (Tseng PH et al. 2006 Mol
Pharmacol 70:1534). Therefore, new drugs with minimal side effects
for non-tumor tissues are urgently needed to improve
HER2/neu-positive tumor therapy.
SUMMARY
[0009] In an aspect, the invention relates to a drug delivery
composition for treating a cancer in a subject. The drug delivery
composition includes a plurality of biologically active molecular
modules comprising at least one module that targets a tumorigenic
cell or a cancer cell, at least one module that inhibits synthesis
or activity of a human epidermal growth factor receptor (HER)
protein in the cell, and at least one module for cytoplasmic
delivery. The drug delivery composition also includes a polymalic
acid-based molecular scaffold. The molecular modules are covalently
linked to the scaffold.
[0010] In an aspect, the invention relates to a kit for treating a
patient having a cancer. The kit includes a drug delivery
composition comprising a nanobiopolymeric conjugate of a scaffold
that includes a PMLA and molecular modules. The molecular modules
includes an antisense molecule that substantially inhibits
synthesis or activity of a HER protein, a molecular module to
facilitate delivery of the antisense molecule to cytoplasm, at
least one targeting antibody specific for the HER protein, at least
one antibody specific for a tumor vasculature protein, and a
molecular module that prolongs circulation of the composition. The
PMLA is covalently linked to the molecular modules, in a
container.
[0011] In an aspect, the invention relates to a method for treating
a cancer in a subject. The method includes contacting the subject
with a drug delivery composition. The drug delivery composition
includes a PMLA covalently linked to a plurality of molecular
modules. The molecular modules include at least one module that
targets a tumorigenic cell or a cancer cell, at least one module
that inhibits synthesis or activity of a HER protein in the cell,
and at least one module for cytoplasmic delivery. The drug delivery
composition is effective for inhibiting at least one of tumor
growth, tumor regression and eliminating of cancer in a
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The following detailed description of the preferred
embodiments will be better understood when read in conjunction with
the appended drawings. For the purpose of illustration, there are
shown in the drawings embodiments which are presently preferred. It
is understood, however, that the invention is not limited to the
precise arrangements and instrumentalities shown. In the
drawings:
[0013] FIG. 1 illustrates a chemical structure and schematic
drawings of a nanobiopolymeric conjugate designed to inhibit
HER2/neu expression by antisense oligonucleotides (AON) and to
attenuate HER2/neu-mediated cell signaling by Herceptin.RTM..
[0014] FIG. 2 illustrates data obtained from an in vitro cell
viability assay.
[0015] FIGS. 3A-3C illustrate photographs of immunoblots showing
changes observed in HER2/neu expression, Akt phosphorylation, and
apoptosis resulting from various treatments of breast cancer cells
in vitro. FIG. 3A illustrates a comparison of HER2/neu and TfR
expression in various cell lines. FIG. 3B illustrates expression
analysis of various markers in cell line SKBR-3. FIG. 3C
illustrates expression of the markers in cell line BT-474. HER2/neu
overexpressing breast cancer cells shown in FIG. 3A treated with
various compounds.
[0016] FIG. 4 illustrates distribution of various compounds herein
labeled with Alexa Fluor 680 in live mice with BT-474 breast tumors
and in tumors in isolated organs.
[0017] FIG. 5 illustrates distribution of various compounds in
BT-474 breast tumor cells.
[0018] FIGS. 6A-6C illustrate mouse tumor inhibition, pathology,
signaling and apoptosis marker expression. FIG. 6A illustrates data
of histopathological analysis of respective tumors from two
representative animals for each group administered with different
drugs. FIG. 6B illustrates extent of tumor growth inhibition in
mice. FIG. 6C illustrates expression of select markers after
treatment of HER2/neu positive tumors in vivo.
[0019] FIG. 7 illustrates extent of tumor growth inhibition by
compositions herein in subjects bearing triple-negative breast
tumors.
[0020] FIGS. 8A-8B illustrate distribution of two cancer stem cell
markers, CD44 and c-Myc, in human BT-474 breast tumor cells grown
in the brain of nude mice as a model of breast cancer metastasis to
the brain, and their inhibition by compositions herein. FIG. 8A
illustrates treatment with PBS as a negative control. FIG. 8B
illustrates treatment with
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] Certain terminology is used in the following description for
convenience only and is not limiting. The words "right," "left,"
"top," and "bottom" designate directions in the drawings to which
reference is made. The words "a" and "one," as used in the claims
and in the corresponding portions of the specification, are defined
as including one or more of the referenced item unless specifically
stated otherwise. This terminology includes the words above
specifically mentioned, derivatives thereof, and words of similar
import. The phrase "at least one" followed by a list of two or more
items, such as "A, B, or C," means any individual one of A, B or C
as well as any combination thereof.
[0022] As used herein the term "molecular scaffold" refers to a
molecule having at least two or more modules that transport a
covalently conjugated drug to a targeted tissue; bind to cell
surface receptors of the tissue; internalize into endosomes; escape
the endosomes into the cytoplasm; and release reactive free drug in
the cytoplasm by chemical reaction with glutathione and other
sulfhydryl groups of the cytoplasmic content. The specificity of
high molecular mass drug vehicles and particles rests primarily on
the tumor tissue targeting by tumor-specific conjugated targeting
molecules and their enhanced permeability and retention in tumors
that originates from high molecular mass such as greater than 20000
(Duncan R. 1999 Research Focus 2:441; Seymour L W et al., 1995 Eur
J Cancer Res 31A:766).
[0023] The term "polymalic acid" or PMLA as used herein refers to a
polymer, e.g., a homopolymer that contains a main chain ester
linkage, is biodegradable and of a high molecular flexibility,
soluble in water (when ionized) and organic solvents (in its acid
form), non-toxic, and non-immunogenic (Lee B et al., Water-soluble
aliphatic polyesters: poly(malic acid)s, in: Biopolymers, vol. 3a
(Doi Y, Steinbuchel A eds., pp 75-103, Wiley-VCH, New York 2002).
Drug carrying PMLA is synthesized by ring-opening polymerization of
derivatized malic acid lactones. Doxorubicin-poly-malic acid has
been synthesized from synthetic poly-.beta.-D, L-malic acid
(Abdellaoui K et al., 1998 Eur J Pharmaceutical Sciences 6:61). The
carrier consists of poly(.beta.-L-malic acid), herein referred to
as poly-.beta.-L-malic acid or PMLA, representing the molecular
backbone or scaffold that is chemically conjugated at its
carboxylic groups at defined ratios with a variety of modules each
of which performs at least one of the following functions: delivery
of a pro-drug via a releasable functional module that becomes
effective in the cytoplasm; directing the carrier towards a
specific tissue by binding to the surfaces of cells, e.g., a
monoclonal antibody (mAB); internalization into the targeted cell
through endosomes (usually via internalization of a targeted
surface receptor); promoting escape from endosomes into the
cytoplasm by virtue of hydrophobic functional units that integrate
into and finally disrupt endosomal membranes; increasing
effectiveness during acidification of endosomes en route to
lysosomes; and protection by polyethylene glycol (PEG) against
degradative enzyme activities, e.g., peptidases, proteases,
etc.
[0024] The term "module" as used herein refers to a biologically
active molecular structure that forms a part of a composition
herein, for example, a small drug molecule or a chromophore
molecule; a protein molecule such as an antibody or lectin; or a
portion thereof that are covalently joined to PMLA in constructing
the composition. In the examples herein a biologically active
module is exemplified by morpholino antisense oligonucleotides
(AON) that are specific to HER2/neu receptor protein. Tissue
targeting is exemplified by use of a monoclonal antibody (mAB)
module that specifically recognizes and binds a transferrin
receptor protein.
[0025] The term "transferrin receptor protein" as used herein
refers to the receptor expressed on endothelium cell surfaces, and
at elevated levels on certain tumors (Lee J H et al. 2001 Eur J
Biochem 268:2004; Kovar M K et al., 2003 J Drug Targeting 10:23).
Transferrin receptors are used as a target for a drug delivery
system in compositions herein, to chemically bind to transferring,
for example using a monoclonal antibody that binds the transferrin
receptor and thereby achieves transcytosis through endothelium
associated with blood brain barrier. Antibody binding to
transferrin receptor and internalization into endosomes has been
demonstrated (Broadwell R D et al., 1996 Exp Neurol 142:47). It
will be appreciated that in the case of the transferrin receptor
any appropriate antibody monoclonal antibody, for example, a
humanized or chimeric antibody, or a lectin or another ligand
specific to the transferrin receptor can be used. Other appropriate
ligands to any number of cell surface receptors or antigens can be
used as targets in the compositions herein and transferrin receptor
is merely examplary.
[0026] The phrase "endosomal escape unit" as used herein refers to
a carrier module attached to the PMLA scaffold that becomes active
by acidification during maturation of the endosomal vesicles
towards lysosomes (Bulmus V et al., 2001 Cancer Research 61:5601;
Lackey C A et al., 2002 Bioconjugate Chem 13:996). The carrier
module includes a plurality of leucine or valine residues, or a
leucine ethylester linked to the PMLA scaffold by amide bonds.
During acidification of the endosomes en route to lysosomes, these
stretches of the carrier molecule become charge-neutralized and
hydrophobic, and capable of disrupting membranes. Other molecules
that become charge neutralized at lysomal pH's may be used in place
of leucine or valine residues, or a leucine ethylester in
construction of the compositions containing PMLA and an endosomal
escape unit module.
[0027] PEGylation is generally used in drug design to increase the
in vivo half-life of conjugated proteins, to prolong the
circulation time, and enhance extravasation into targeted solid
tumors (Arpicco S et al. 2002 Bioconjugate Chem 13:757; Maruyama K
et al., 1997 FEBS Letters 413:1771). Other molecules known to
increase half-life may be used in design of compositions
herein.
[0028] As used herein, the terms "cancer" and "cancerous" refer to
the physiological condition in mammals in which a population of
cells are characterized by unregulated cell growth. Examples of
cancers include, without limitation, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia. More particular examples of such cancers
include squamous cell cancer, small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of
the lung, cancer of the peritoneum, hepatocellular cancer,
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney cancer, liver
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma and various types of head and neck cancers.
[0029] The terms "proliferative disorder" and "proliferative
disease" refer to disorders associated with abnormal cell
proliferation such as cancer.
[0030] The terms "tumor" and "neoplasm" as used herein refer to any
mass of tissue that result from excessive cell growth or
proliferation, either benign (noncancerous) or malignant
(cancerous) including pre-cancerous lesions.
[0031] The term "primary cancer" refers to the original site at
which a cancer originates. For example, a cancer originating in the
breast is called a primary breast cancer. If it metastasizes, i.e.,
spreads to the brain, the cancer is referred to as a primary breast
cancer metastatic to the brain.
[0032] The term "metastasis" as used herein refers to the process
by which a cancer spreads or transfers from the site of origin to
other regions of the body with the development of a similar
cancerous lesion, i.e., having the same or substantially the same
biochemical markers at the new location. A "metastatic" or
"metastasizing" cell is one that has a reduced activity for
adhesive contacts with neighboring cells and migrates by the
bloodstream or within lymph from the primary site of disease to
additional distal sites, for example, to invade neighboring body
structures or distal structures.
[0033] The terms "cancer cell", "tumor cell" and grammatical
equivalents refer to a cell derived from a tumor or a pre-cancerous
lesion including both a non-tumorigenic cell and a tumorigenic
cell, i.e., cancer stem cell.
[0034] As used herein "tumorigenic" refers to the functional
features of a solid tumor stem cell including the properties of
self-renewal i.e., giving rise to additional tumorigenic cancer
cells, and proliferation to generate other tumor cells i.e., giving
rise to differentiated and thus non-tumorigenic tumor cells, such
that cancer cells form a tumor.
[0035] The phrase "target a tumorigenic cell or a cancer cell" as
used herein refers to delivery of a composition to a population of
tumor-forming cells within tumors, i.e., tumorigenic cells. The
preferential delivery of the composition to the tumorigenic
population of cancer cells in comparison to other populations of
cells within tumors is referred herein as targeting to eliminate
cancer cells, a property that improves specificity and efficacy of
the composition.
[0036] The term "antibody" is used herein to mean an immunoglobulin
molecule that is a functional module included in compositions
herein for ability to recognize and specifically bind to a target,
such as a protein, polypeptide, peptide, carbohydrate,
polynucleotide, lipid, or combinations of the foregoing through at
least one antigen recognition site within the variable region of
the immunoglobulin molecule. In certain embodiments, antibodies
included as functional modules of compositions herein include a
class described as antagonist antibodies, which specifically bind
to a cancer stem cell marker protein and interfere with, for
example, ligand binding, receptor dimerization, expression of a
cancer stem cell marker protein, and/or downstream signaling of a
cancer stem cell marker protein. In alternative embodiments,
antibodies as functional modules in compositions herein include
agonist antibodies that specifically bind to a cancer stem cell
marker protein and promote, for example, ligand binding, receptor
dimerization, and/or signaling by a cancer stem cell marker
protein. In alternative embodiments, antibodies that do not
interfere with or promote the biological activity of a cancer stem
cell marker protein instead function to inhibit tumor growth by,
for example, antibody internalization and/or recognition by the
immune system.
[0037] As used herein, the term "antibody" encompasses intact
polyclonal antibodies, intact monoclonal antibodies, antibody
fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single
chain Fv (scFv) mutants, multispecific antibodies such as
bispecific antibodies generated from at least two intact
antibodies, chimeric antibodies, humanized antibodies, human
antibodies, fusion proteins comprising an antigen determination
portion of an antibody, and any other modified immunoglobulin
molecule comprising an antigen recognition site so long as the
antibodies exhibit the desired biological activity. An antibody
includes any the five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their
heavy-chain constant domains referred to as alpha, delta, epsilon,
gamma, and mu, respectively. Antibodies can be naked or conjugated
to other molecules such as toxins, radioisotopes, etc. In other
embodiments an antibody is a fusion antibody.
[0038] As used herein, the term "antibody fragment" refers to a
portion of an intact antibody and refers to the antigenic
determining variable regions of an intact antibody. Examples of
antibody fragments include, but are not limited to Fab, Fab',
F(ab')2, and Fv fragments, linear antibodies, single chain
antibodies, and multispecific antibodies formed from antibody
fragments.
[0039] An "Fv antibody" refers to the minimal antibody fragment
that contains a complete antigen-recognition and -binding site
either as two-chains, in which one heavy and one light chain
variable domain form a non-covalent dimer, or as a single-chain
(scFv), in which one heavy and one light chain variable domain are
covalently linked by a flexible peptide linker so that the two
chains associate in a similar dimeric structure. In this
configuration the complementarity determining regions (CDRs) of
each variable domain interact to define the antigen-binding
specificity of the Fv dimer. Alternatively a single variable domain
(or half of an Fv) can be used to recognize and bind antigen,
although generally with lower affinity.
[0040] A "monoclonal antibody" as used herein refers to homogenous
antibody population involved in specific recognition and binding of
a single antigenic determinant, or epitope. Polyclonal antibodies
include a population of antibody species each directed to a
different antigenic determinant. The term "monoclonal antibody"
encompasses both and full-length monoclonal antibodies and antibody
fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv)
mutants, fusion proteins comprising an antibody portion, and any
other modified immunoglobulin molecule comprising an antigen
recognition site. Furthermore, "monoclonal antibody" refers to
those obtained without limitation by methods including and not
limited to hybridoma expression, phage selection, recombinant
expression, and by transgenic animals.
[0041] In an embodiment, a drug delivery composition for treating a
cancer in a subject is provided. The drug delivery composition may
include a plurality of biologically active molecular modules. The
plurality of the biologically active molecular modules may include
at least one module that targets a tumorigenic cell or a cancer
cell. The drug delivery composition may include at least one module
that inhibits synthesis or activity of a human epidermal growth
factor receptor (HER) protein in the cell. The drug delivery
composition may include at least one module for cytoplasmic
delivery. The drug delivery composition may include a polymalic
acid-based molecular scaffold. The molecular modules may be
covalently linked to the polymalic acid-based molecular scaffold.
The HER protein may be at least one protein selected from the group
consisting of: HER1, HER2, HER3 and HER4. The at least one module
that inhibits synthesis or activity of the protein may be selected
from the group consisting of: an antisense oligonucleotide (AON),
an siRNA oligonucleotide, an antibody, a polypeptide, an
oligopeptide and a low molecular weight drug. The scaffold in a
related embodiment includes a poly-.beta.-L-malic acid (PMLA). The
PMLA may be also denoted as poly(-.beta.-L-malic acid).
[0042] In an embodiment, the AON may be a Morpholino AON. The
Morpholino AON may include a sequence complementary to a sequence
contained in an mRNA transcript of HER2/neu protein. For example,
the AON may be selected from: 5'-AGGGAGCCGCAGCTTCATGTCTGTG-3' (SEQ
ID NO: 1), and 5'-CATGGTGCTCACTGCGGCTCCGGC-3' (SEQ ID NO:2).
[0043] In an embodiment, the at least one module that targets the
cell may include an antibody that binds specifically to a
vasculature protein in the cell. The vasculature protein may
include a transferrin receptor protein. The antibody may be
selected from at least one of: anti-human, rat anti-mouse, rat
anti-human, rabbit anti-human and goat anti-human.
[0044] In an embodiment, the at least one module that inhibits
activity of the protein includes an antibody binding specifically
to a HER2/neu protein. The antibody may be Herceptin.RTM..
[0045] In an embodiment, the drug delivery composition may include
a Morpholino AON that include sequence complementary to a sequence
contained in an mRNA transcript of an epidermal growth factor
receptor (EGFR) or HER1 protein. The sequence of the Morpholino AON
may include 5'-TCGCTCCGGCTCTCCCGATCAATAC-3' (SEQ ID NO:3).
[0046] In an embodiment, the drug delivery composition may include
a Morpholino AON that includes a sequence complementary to a
sequence contained in an mRNA transcript of at least one subunit of
laminin-411. The subunit may be at least one of an .alpha.4 subunit
and a .beta.1 subunit. The sequence complimentary to the transcript
of the .alpha.4 subunit may include the following sequence:
[0047] 5'-AGCTCAAAGCCATTTCTCCGCTGAC-3' (SEQ ID NO:4). The sequence
complimentary to the transcript of the .beta.1 subunit may include
the following sequence: 5'-CTAGCAACTGGAGAAGCCCCATGCC-3' (SEQ ID
NO:5).
[0048] In an embodiment, the drug delivery composition may include
the siRNA oligonucleotide. The siRNA oligonucleotide may include a
sequence complementary to a gene encoding an EGFR/HER1 protein. The
sequence may include a sense sequence as follows:
5'-CCUAUAAUGCUACGAAUAUtt-3' (SEQ ID NO:6). The sequence may include
an antisense sequence as follows: 5'-AUAUUCGUAGCAUUUAUGGag-3' (SEQ
ID NO:7).
[0049] In an embodiment, the siRNA oligonucleotide may include a
sequence complementary to a gene encoding a HER2 protein. The
sequence may include a sense sequence as follows:
5'-GUUGGAUGAUUGACUCUGAtt-3' (SEQ ID NO:8). The sequence may include
an antisense sequence as follows: 5'-UCAGAGUCAAUCAUCCAACat-3' (SEQ
ID NO:9).
[0050] In an embodiment, the at least one module for cytoplasmic
delivery may include an endosome escape unit. The endosome escape
unit may be but is not limited to leucine residues, valine
residues, or a leucine ethylester. The endosome escape unit may be
a plurality of leucine or valine residues, or a single or a
plurality of leucine residues, or mixture of any of these. The
leucine ethylester may be included in the drug delivery composition
in a concentration of about 40% of the drug delivery
composition.
[0051] In an embodiment, the plurality of modules of the drug
delivery composition may further include a polyethylene glycol
(PEG). The PEG may have a molecular weight of about 1,000 Da, about
5,000 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da, about
25,000 Da, or about 30,000 Da.
[0052] In an embodiment, the drug delivery composition may be
provided in a unit dose effective for treatment of the cancer in
the patient. The unit dose may be at least one selected from: 1
.mu.g/kg, 50 .mu.g/kg, 100 .mu.g/kg, 500 .mu.g/kg, 1 mg/kg, 5
mg/kg, 10 mg/kg, 50 mg/kg, and 100 mg/kg. The unit dose may be at
least 1 mg/kg. The unit dose may be less than about 10 mg/kg.
[0053] In an embodiment, the cancer is at least one selected from
the list of: gastric, endometrial, salivary gland, lung, non-small
cell lung, pancreatic, ovarian, peritoneal, prostate, colorectal,
breast, cervical, uterine, ovarian, brain, head and neck,
testicular and teratoma cancers. The breast cancer may be a
triple-negative breast cancer. The cancer may be either a primary
cancer or a metastatic cancer, or both. The cancer may include
cells overexpressing a HER2/neu receptor protein.
[0054] In an embodiment, a drug delivery composition for treating a
cancer in a subject may include: a polymerized carboxylic acid
molecular scaffold and a plurality of biologically active molecular
modules. The polymerized carboxylic acid molecular scaffold may
include a poly-.beta.-L-malic acid (PMLA). The plurality of
biologically active molecular modules may include an antisense
molecule that substantially inhibits synthesis of a HER2/neu
receptor protein, a molecular module to facilitate delivery of the
antisense molecule to cytoplasm, at least one antibody specific for
the receptor protein that inhibits activity of the receptor
protein, at least one antibody targeting a tumor vasculature
protein, and a molecular module that prolongs circulation of the
composition. The molecular modules may be covalently linked to the
scaffold.
[0055] In an embodiment, a drug delivery composition for treating a
cancer in a subject including: a polymerized carboxylic acid
molecular scaffold and a plurality of biologically active molecular
modules. The polymerized carboxylic acid molecular scaffold may be
a poly-.beta.-L-malic acid (PMLA). The plurality of biologically
active molecular modules may include an antisense molecule that
substantially inhibits synthesis of an epidermal growth factor
receptor (EGFR/HER1) protein, an antisense molecule that
substantially inhibits at least one subunit of laminin-411, a
molecular module to facilitate delivery of the antisense molecule
to cytoplasm, at least one antibody targeting a tumor vasculature
protein, and a molecular module that prolongs circulation of the
composition. The molecular modules may be covalently linked to the
scaffold.
[0056] In an embodiment, a pharmaceutical composition is provided
that includes a nanobiopolymeric conjugate of poly(.beta.-L-malic
acid) referred to as poly-.beta.-L-malic acid or PMLA herein. PMLA
may be covalently linked to an antisense molecule. The antisense
molecule may be a functional module that inhibits expression of an
oncogenic protein. The PMLA may be covalently linked to at least
one module that is an antibody specific for the protein. The PMLA
may optionally further comprise a module that is an antibody
specific for an oncogenic vascular protein. The pharmaceutical
composition may include a pharmaceutically acceptable carrier.
[0057] In an embodiment, the pharmaceutical composition may
optionally further include one or more additional modules that are
additional therapeutic agents. The additional therapeutic agent or
agents may be selected from the group consisting of growth factors,
anti-inflammatory agents, vasopressor agents, collagenase
inhibitors, topical steroids, matrix metalloproteinase inhibitors,
ascorbates, angiotensin II, angiotensin III, calreticulin,
tetracyclines, fibronectin, collagen, thrombospondin, transforming
growth factors (TGF), keratinocyte growth factor (KGF), fibroblast
growth factor (FGF), insulin-like growth factors (IGF), epidermal
growth factor (EGF), platelet derived growth factor (PDGF), neu
differentiation factor (NDF), hepatocyte growth factor (HGF), and
hyaluronic acid.
[0058] As used herein, the term "pharmaceutically acceptable
carrier" includes any and all solvents, diluents, or other liquid
vehicle, dispersion or suspension aids, surface active agents,
isotonic agents, thickening or emulsifying agents, preservatives,
solid binders, lubricants and the like, as suited to the particular
dosage form desired. Remington's Pharmaceutical Sciences Ed. by
Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various
carriers used in formulating pharmaceutical compositions and known
techniques for the preparation thereof. Materials which can serve
as pharmaceutically acceptable carriers may include, but are not
limited to, sugars, lactose, glucose, and sucrose; starches, corn
starch and potato starch; cellulose and its derivatives, sodium
carboxymethyl cellulose, ethyl cellulose, and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, cocoa butter
and suppository waxes; oils, peanut oil, cottonseed oil, safflower
oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, a
propylene glycol; esters, ethyl oleate and ethyl laurate; agar;
buffering agents, magnesium hydroxide and aluminum hydroxide;
alginic acid; pyrogen-free water; isotonic saline; Ringer's
solution; ethyl alcohol, or phosphate buffer solutions.
Pharmaceutically acceptable carriers may include non-toxic
compatible lubricants, sodium lauryl sulfate and magnesium
stearate. Pharmaceutically acceptable carriers may include coloring
agents, releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants.
[0059] In an embodiment, a kit for treating a patient having a
cancer is provided. The kit may include a drug delivery
composition. The drug delivery composition may include a
nanobiopolymeric conjugate of a scaffold and molecular modules. The
scaffold may be a poly-.beta.-L-malic acid (PMLA). The molecular
modules may include an antisense molecule that substantially
inhibits synthesis or activity of a human epidermal growth factor
receptor (HER) protein. The molecular modules may include a
molecular module to facilitate delivery of the antisense molecule
to cytoplasm. The molecular modules may include at least one
targeting antibody specific for the HER protein. The molecular
modules may include at least one antibody specific for a tumor
vasculature protein. The molecular modules may include a molecular
module that prolongs circulation of the composition. The PMLA may
be covalently linked to the molecular modules. The drug delivery
composition may be included in a container.
[0060] In an embodiment, the kit may further include a
pharmaceutically acceptable buffer and instructions for use.
[0061] In an embodiment, a method for treating a cancer in a
subject is provided. The method may include contacting the subject
with a drug delivery composition. The drug delivery composition may
include a poly-.beta.-L-malic acid (PMLA) covalently linked to a
plurality of molecular modules. The plurality of molecular modules
may include at least one module that targets a tumorigenic cell or
a cancer cell. The plurality of molecular modules may include at
least one module that inhibits synthesis or activity of a human
epidermal growth factor receptor (HER) protein in the cell. The HER
protein may be selected from a group consisting of: HER1, HER2,
HER3, and HER4. The plurality of molecular modules may include at
least one module for cytoplasmic delivery. The drug delivery
composition may be effective for inhibiting at least one of tumor
growth, tumor regression and eliminating of cancer in a
subject.
[0062] In an embodiment, the drug delivery composition may be
further effective for inhibiting expression of cancer stem cell
markers in the subject. The cancer stem cell markers may include at
least one marker selected from the group consisting of: CD133
protein, c-myc protein, CD44 protein, Notch1 protein, and nestin
protein. The inhibition of expression of cancer stem cell markers
may indicate inhibition of growth of drug resistant tumors.
[0063] The method may also include analyzing at least one of:
inhibition of tumor growth, tumor regression and elimination of
cancer in the subject, thereby treating the cancer in the
subject.
[0064] In an embodiment, the module that inhibits synthesis or
activity of the HER protein may be at least one selected from the
group consisting of: an antisense oligonucleotide (AON), an siRNA
oligonucleotide, an antibody, a polypeptide, an oligopeptide and a
low molecular weight drug. The AON may include a sequence
complementary to an mRNA transcript of at least one protein
selected from the group consisting of: HER2, an epidermal growth
factor receptor (EGFR/HER1) protein, and a subunit of laminin-411.
The sequence complementary to the HER2 mRNA transcript may include
the following sequence: 5'AGGGAGCCGCAGCTTCATGTCTGTG-3' (SEQ ID NO:
1). The sequence complementary to the HER2 mRNA transcript may
include the following sequence 5'-CATGGTGCTCACTGCGGCTCCGGC-3' (SEQ
ID NO:2). The sequence complementary to the EGFR/HER1 mRNA
transcript may include the following sequence:
5'-TCGCTCCGGCTCTCCCGATCAATAC-3' (SEQ ID NO:3).
[0065] In an embodiment, the subunit of laminin-411 may be selected
at least one of .alpha.4 and .beta.1 subunits. The .alpha.4
transcript sequence may include the following sequence:
5'-AGCTCAAAGCCATTTCTCCGCTGAC-3' (SEQ ID NO:4). The .beta.1
transcript sequence may include the following sequence:
5'-CTAGCAACTGGAGAAGCCCCATGCC-3' (SEQ ID NO:5).
[0066] In an embodiment, the siRNA oligonucleotide may include a
sequence complementary to a gene encoding at least one of an
epidermal growth factor receptor (EGFR/HER1) protein and HER2. The
sequence complementary to a gene encoding EGFR/HER1 sequence may be
selected from the group consisting of: 5'-CCUAUAAUGCUACGAAUAUtt-3'
(SEQ ID NO:6), and 5' -AUAUUCGUAGCAUUUAUGGag-3' (SEQ ID NO:7). The
sequence complementary to a gene encoding HER2 may be selected
from: 5'-GUUGGAUGAUUGACUCUGAtt-3' (SEQ ID NO:8), and
5'-UCAGAGUCAAUCAUCCAACat-3' (SEQ ID NO:9).
[0067] In an embodiment, the antibody may bind specifically to
HER2/neu protein. The antibody may be Trastuzumab Herceptin.RTM..
The at least one module that targets the cell may include an
antibody that binds specifically to a transferrin receptor protein.
The antibody may be selected from at least one of: anti-human, rat
anti-mouse, rat anti-human, rabbit anti-human and goat anti-human.
The at least one module for cytoplasmic delivery may include an
endosome escape unit. The endosome escape unit may be a leucine
ethylester.
[0068] In an embodiment, the plurality of modules may further
include a polyethylene glycol (PEG). The PEG may have a molecular
weight of about 1,000 Da. The PEG may have a molecular weight of
about 5,000 Da.
[0069] In an embodiment, the method may include analyzing
inhibition of tumor growth. The step of analyzing may include
observing more than about 60%, 70%, 80% or about 90% inhibition of
tumor growth in the subject. The step of analyzing may include
observing the inhibition of HER2/neu receptor signaling by
suppression of Akt phosphorylation.
[0070] In an embodiment, the subject may be a mammal. The may be
but is not limited to a human, a simian, an equine, a bovine, or a
high value agricultural or zoo animal. The mammal may be a rodent.
The rodent may be an experimental human-breast tumor-bearing nude
mouse.
[0071] In an embodiment, the step of contacting may include
administering the drug delivery to the subject. The drug delivery
compositions may be formulated with an appropriate pharmaceutically
acceptable carrier in a desired dosage. The drug delivery
compositions may be administered to humans and other mammals
topically. Topical administration may include drug delivery
compositions formulated as powders, ointments, or drops. The drug
delivery compositions may be administered orally, rectally,
parenterally, intracisternally, intravaginally, intraperitoneally,
or intravenously, depending on the severity and location of the
cancer or other condition being treated. Intravenous administration
may include injection as a bolus, or as a drip.
[0072] In an embodiment, dosage forms for topical or transdermal
administration of the drug delivery compositions may include
ointments, pastes, creams, lotions, gels, powders, solutions,
sprays, inhalants, or patches. The drug delivery composition may be
admixed under sterile conditions with a pharmaceutically acceptable
carrier and any needed preservatives or buffers as may be required.
Administration may be therapeutic or it may be prophylactic.
Prophylactic formulations may be present or applied to the site of
potential tumors, or to sources of tumors. The ointments, pastes,
creams, and gels may contain, in addition to the drug delivery
compositions, excipients. Excipients may be but are not limited to
animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc, zinc oxide, or mixtures thereof.
Powders and sprays may contain, in addition to the drug delivery
compositions, excipients. Excipients may include lactose, talc,
silicic acid, aluminum hydroxide, calcium silicates, polyamide
powder, or mixtures of these substances. Sprays may additionally
contain customary propellants. Customary propellants may include
chlorofluorohydrocarbons.
[0073] In an embodiment, the drug delivery composition may be
administered using transdermal patches. The transdermal patches may
have the added advantage of providing controlled delivery of the
active ingredients to the body. Controlled delivery may be achieved
using dosage forms. Dosage forms may be made by dissolving or
dispensing the compound in the proper medium. Absorption enhancers
may also be used to increase the flux of the drug delivery
composition across the skin. The rate of delivery may be controlled
by either providing a rate controlling membrane or by dispersing
the compound in a polymer matrix or gel.
[0074] In an embodiment, the step of administering may include
administering injectable preparations. The injectable preparations
may include sterile injectable aqueous solutions or oleaginous
suspensions formulated according to the known art using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable preparation may be formulated as a sterile injectable
solution, suspension or emulsion in a nontoxic parenterally
acceptable diluent or solvent. The sterile injectable preparation
may be formulated as a solution in 1,3-butanediol. The acceptable
vehicles and solvents may include water, Ringer's solution, U.S.P.
or isotonic sodium chloride solution. In addition, sterile, fixed
oils may be employed as a solvent or suspending medium. Any bland
fixed oil may be employed including synthetic mono- or
diglycerides. In addition, fatty acids such as oleic acid may be
used in the preparation of injectables. The injectable formulations
may be sterilized. The injectable preparations may be sterilized by
filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use. To prolong the
effect of a drug delivery composition, the absorption of the drug
from subcutaneous or intramuscular injection may be slowed. Delayed
absorption of a parenterally administered active agent may be
accomplished by dissolving or suspending the drug delivery
composition in an oil vehicle. Injectable depot forms may be made
by forming microencapsule matrices of the drugs in biodegradable
polymers such as polylactide-polyglycolide as described herein, and
in Ljubimova et al., U.S. Pat. No. 7,547,511 issued Jun. 16, 2009,
Ljubimova et al., U.S. patent application Ser. No. 12/473,992
published Oct. 22, 2009, Ljubimova et al., U.S. patent application
Ser. No. 10/580,999 published Nov. 8, 2007, and Ding et al.,
International patent application PCT/US2009/40252 filed Apr. 10,
2009. The rate of active agent release is controlled by the ratio
of active agent to polymer and the nature of the particular polymer
employed. Examples of other biodegradable polymers include
poly(orthoesters) and poly(anhydrides). Depot injectable
formulations may also be prepared by entrapping the agent in
liposomes or microemulsions which are compatible with body
tissues.
[0075] In an embodiment, the drug delivery compositions may be used
for rectal or vaginal administration. The drug delivery
compositions may be administered as suppositories. Suppositories
may be prepared by mixing the drug delivery compositions with
suitable non-irritating excipients or carriers. The non-irritating
excipients or carriers may include cocoa butter, polyethylene
glycol or a suppository wax which are solid at ambient temperature
but liquid at body temperature and therefore melt in the rectum or
vaginal cavity and release the drug delivery compositions.
[0076] In an embodiment, the drug delivery composition may be
administered for the treatment of a cancer associated with a
particular receptor. The drug deliver composition may be
administered in a therapeutically effective amount. The
therapeutically effective amount may inhibit expression of at least
one ligand of the receptor to a subject in need thereof. It will be
appreciated that this encompasses administering an inventive
pharmaceutical as a therapeutic measure to promote regression of a
cancer or prevent further development or metastasis, or as a
prophylactic measure to minimize complications associated with
development of a tumor or cancer. As used herein, the
"therapeutically effective amount" of the pharmaceutical
composition is that amount effective for preventing further
development of a cancer or transformed growth, and even to effect
regression of the cancer. The drug delivery compositions may be
administered using any amount and any route of administration
effective for prevention of development of a cancer. Thus, the
expression "amount effective for inhibiting expression or activity
of the oncogenic protein", as used herein, refers to a sufficient
amount of composition to prevent or retard development of a cancer,
and even cause regression of a cancer or solid tumor. The cancer
need not be limited to a solid tumor, and includes various types of
lymphomas and leukemias.
[0077] In an embodiment, the exact dosage may be chosen by the
individual physician with regard to the need of the patient to be
treated. Dosage and administration may be adjusted to provide
sufficient levels of the active agent(s) or to maintain the desired
effect. Additional factors which may be taken into account include
the severity of the disease state, e.g., cancer size and location;
age, weight and gender of the patient; diet, time and frequency of
administration; drug combinations; reaction sensitivities; and
tolerance/response to therapy. Long acting pharmaceutical
compositions might be administered every 3 to 4 days, every week,
or once every two weeks depending on half-life and clearance rate
of the particular composition.
[0078] In an embodiment, the drug delivery compositions may be
formulated in dosage unit form for ease of administration and
uniformity of dosage. The expression "dosage unit form" as used
herein refers to a physically discrete unit of active agent
appropriate for the patient to be treated. The total daily usage of
the compositions of the present invention may be decided by the
attending physician within the scope of sound medical judgment. For
any drug delivery composition described herein, the therapeutically
effective dose may be estimated initially either in cell culture
assays or in animal models. Animal models may be mice, rabbits,
dogs, or pigs as shown in Examples herein. The animal model may
also be used to achieve a desirable concentration range and route
of administration. Such information may then be used to determine
useful doses and routes for administration in humans. A
therapeutically effective dose refers to that amount of active
agent, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity of active agents may be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., ED50 (the dose is therapeutically effective in 50%
of the population) and LD50 (the dose is lethal to 50% of the
population). The dose ratio of toxic to therapeutic effects is the
therapeutic index, and it can be expressed as the ratio, LD50/ED50.
Pharmaceutical compositions herein exhibit large therapeutic
indices. The data obtained from the animal studies herein is used
in formulating a range of dosage for human use.
[0079] In an embodiment, an initial dose of Herceptin.RTM. for
human treatment accepted by the FDA may be 4 mg/kg followed by 2
mg/kg weekly for a total of 52 doses. An efficient dose of the
composition herein for treatment of a mouse was 100 .mu.l of
observed 40 .mu.g/ml, which may be equivalent to about 3.2 mg/kg
for human use.
[0080] In an embodiment, the method may further include
administering an additional therapeutic agent. The additional
therapeutic agent may be selected from the group consisting of: an
antibody, an enzyme inhibitor, an antibacterial agent, an antiviral
agent, a steroid, a non-steroid-inflammatory agent, an
antimetabolite, a cytokine, a cytokine blocking agent, an adhesion
molecule blocking agent, and a soluble cytokine receptor.
[0081] In an embodiment, the method may include further
administering antineoplastic agents. The antineoplastic agents may
include agents for overcoming trastuzumab resistance. A variety of
agents including monoclonal antibodies, recombinant proteins, and
drugs, are known to have activity in treating breast cancer, and
are here contemplated to be useful agents in combination with
compositions described herein.
[0082] In an embodiment, the step of administering drug delivery
composition including Herceptin.RTM. may include combining the drug
delivery composition with other agents. The drug delivery
composition may be administered with paclitaxel (taxol,
Bristol-Myers Squibb) and docetaxel (taxotere, Sanofi-Aventis). The
method may yield increases in response rates, time to disease
recurrence, and overall survival (Esteva F J et al. 2002 J Clin
Oncol. 20:1800; Slamon D J et al. 2001 N Engl J Med. 344:783;
Wardley A M et al. 2009. J Clin Oncol 49:976).
[0083] In an embodiment, the step of administering may include
combining targeting of HER2 and other tyrosine kinases. Tyrosine
kinases are associated with breast cancer tumorigenesis and are of
substantial interest as potential drug targets (Ocana A et al. 2008
Clin Cancer Res 14:961). The insulinlike growth factor 1 receptor
(IGF-1R), a receptor tyrosine kinase (RTK), has been shown to
increase the growth of breast cancer cells and is also implicated
in developing resistance to trastuzumab (Nahta R et al. 2006 Nat
Clin Pract Oiled 3:269). Cotargeting or simultaneous targeting of
IGF-1R and 1-HER2 may offer an advantage compared to targeting of
the individual RTKs in breast cancer cells (Esparis-Ogando A et al.
2008 Ann Oncol 19:1860). The v-kit Hardy-Zuckerman 4 feline sarcoma
viral oncogene homolog (c-KIT) RTK is overexpressed in
triple-negative breast cancers (those that do not express estrogen
receptor, progesterone receptor; and HER2) (Nielsen T O et al. 2004
Clin Res 10:5367). The activation of two nonreceptor cytosolic
tyrosine kinases, c-abl oncogene 1 (ABL1) and c-SRC tyrosine kinase
(CSK), is associated with the aggressiveness of breast cancer (Finn
R S. 2008 Ann Oncol 19:1379) and proliferation of triple-negative
breast cancers (Finn R S. 2008 Ann Oncol 19:1379; Finn R S et al.
2007 Breast Cancer Res Treat 105:319), respectively. Moreover,
c-SRC has also been associated with antiestrogen resistance in
estrogen receptor-positive breast tumors (van Agthoven T et al.
2009 J Clin Oncol 27:542). The step of administering may include
combining the drug delivery composition with Dasatinib,
(Sprycel.RTM., Bristol-Myers Squibb) a small-molecule tyrosine
kinase inhibitor. Dasatinib targets the cytosolic c-SRC and ABL1
kinases, and RTKs c-KIT and platelet-derived growth factor
receptors alpha and beta (Finn R S et al. 2007 Breast Cancer Res
Treat 105:319; Rix U et al. 2007 Blood 110:4055; Huang F. et al.
2007 Cancer Res 67: 2226; Huang F. et al. 2007 Cancer Res 67:2226).
The activity of Dasatinib for treatment of triple-negative breast
cancer not expressing estrogen receptor, progesterone receptor, or
HER2/neu (Finn R S et al. 2007 Breast Cancer Res Treat 105:319;
Huang F. et al. 2007 Cancer Res 67: 2226), and favorable
antitumoral activity in head and neck cancer in combination with
gefitinib (Koppikar P et al. 2008 Clin Cancer Res 14:4284), led to
combining trastuzumab and dasatinib for treatment of HER2 -positive
breast cancers. This combination was found to be highly effective
against breast cancer cells overexpressing HER2 receptors. Both
drugs individually inhibited cell proliferation in vitro and
exhibited antitumoral action, and the combination resulted in a
more potent effect on HER2-overexpressing cells.
[0084] In an embodiment, the drug delivery composition may be
administered in combination with other drugs and may lead to
decreased levels of phosphorylated HER2 and phosphorylated. HER3,
and a decrease observed in the total amount of these receptors. The
combined treatment may affect downstream signaling routes, such as
the ERK1 or AKT pathways that regulate cell proliferation and
survival (Garcia-Echeverria C et al. 2008 Oncogene 27:5511; Roberts
P J et al. 2007 Oncogene 26:3291). Dasatinib alone was as
inhibitory for phosphorylated levels of ERK1 as the combined drug
treatment. Treatment with Dasatinib also inhibited SRC or FAK
phosphorylation to the same degree as the combined drug treatment.
These two kinases are known targets of Dasatinib (Huang F. et al.
2007 Cancer Res 67: 2226) and participate in several oncogenic
processes (Kim L C et al. 2009 Nat Rev Clin Oncol 6:587). Combined
treatment and not the individual drugs was observed to decrease the
level of phosphorylated AKT. Downstream targets of AKT such as
p70S6K and BAD were also affected by the combined drug treatment,
and not by the individual drugs, as the resting phosphorylated
levels of these proteins were reduced by treatment with trastuzumab
and dasatinib.
[0085] In an embodiment, the step of administering may include
providing drug combination that may also induce caspase-independent
apoptosis as determined by the lack of an effect of caspase
inhibitors on apoptosis induced by the drug combination. One of the
possible mediators in caspase-independent apoptosis is NAIF1 a
protein that may be released from the mitochondrial intermembrane
space by certain apoptotic stimuli. The release of NAIF1 from
mitochondria to the cytosol, by treatment with the drug
combination, may indicate that this mechanism could be responsible
for caspase-independent apoptosis.
[0086] In an embodiment, the drug combination may also affect DNA
repair machinery and lead to accumulation of double-stranded breaks
(DSBs) which indicate control of DNA repair machinery by tyrosine
kinases and potential clinical implications.
[0087] In an embodiment, the chug delivery composition may be
administered in combination with Erlotinib (Tarceva, Roche), an
inhibitor of EGFR. Erlotinib may block homologous recombination
repair of the DSBs in breast cancer cells through reduction of
RAD51 foci formation (Li L et al 2008 Cancer Res 68:9141). Previous
studies have indicated that RTKs may regulate DNA repair (Tanaka T
et al, 2008 Clin Cancer Res 14:1266; Ganapathipillai S S et al.
2008 Cancer Res 68:5769).
[0088] In an embodiment, the drug delivery composition may be
administered in combination with Gefitinib (Iressa, Astra Zeneca
and Teva) is an EGFR inhibitor. Gefitinib may impede DNA repair in
response to ionizing radiations in macrocytic lung cancer cells
(Tanaka T et al, 2008 Clin Cancer Res 14:1266). Mutated forms of
MET protein, an RTK implicated in several oncogenic processes such
as invasion and metastasis (Benvenuti S et al. 2007 J Cell Physiol
213:316) or drug resistance (Engelman J A et al. 2007 Science
316:1039), have been reported to bind to and phosphorylate RAD51,
facilitating DNA repair in tumor cells (Ganapathipillai S S et al.
2008 Cancer Res 68:5769).
[0089] In an embodiment, a drug delivery composition may be
administered with other drugs or agents. The agents may affect a
transcription factor associated with Williams-Beuren syndrome
(WSTF; also known as BAZ1B), a tyrosine kinase component of the
WICH complex (WSTF-ISWI ATP-dependent chromatin-remodeling
complex), that regulates the DNA damage response through
phosphorylation of Tyr142 of H2AX (Xiao A et al. 2009 Nature
457:57).
[0090] It is here envisioned that drugs such as dasatinib in
combination with other antineoplastic agents such as gefitinib and
erlotinib (Koppikar P et al. 2008 Clin Cancer Res 14:4284), are
further combined with drug delivery compositions described
herein.
[0091] In an embodiment, the drug delivery composition may be
administered in combination with Lapatinib (Tyverb.RTM., GSK) is a
dual EGFR/HER2 tyrosine kinase inhibitor (Rusnak D W et al. 2001
Mol Cancer Ther 1:85) which is highly selective to EGFR and HER2
(Karaman M W et al. 2008 Nat Biotechnol 26:127). In preclinical
models of trastuzumab resistance, lapatinib inhibited
phosphorylation of HER2 and overall growth in HER2 overexpressing
breast cancer cell lines specifically chosen for extent of in vitro
resistance to trastuzumab (Konechny G E et al. 2006 Cancer Res
66:1630). Further, treatment with lapatinib may be combined with
trastuzumab and may result in a greater degree of survival and
greater apoptosis induction than either agent alone (Xia et al.
2005 Oncogene 24: 6213). A substantial number of HER2-positive
metastatic breast cancer patients treated with trastuzumab
experience symptomatic central nervous system (CNS) metastasis,
which unlike visceral diseases, are not well controlled by
trastuzumab. Lapatinib and not trasuzumab has been shown to cross
the blood-brain barrier, providing rationale for testing lapatinib
in patients with CNS metastases (Nielsen D L et al. 2009 Cancer
Treat Rev 35:121). Trastuzumab in combination with lapatinib may be
to be superior to lapatinib alone in HER2-positive metastatic
breast cancer patients (Blackwell K L et al. 2010 J Clin Oncol
28:1124).
[0092] In an embodiment, the drug delivery composition may be
administered in combination with Pertuzumab (2c4, omnitarg,
Genentech). Pertuzumab is a monoclonal antibody specific for the
extracellular domain of HER2 protein. Pertuzumab may attach to a
different epitope of HER2 compared to trastuzumab. Pertuzumab was
observed to inhibit heterodimer formation between HER2 and EGFR or
HER3 (Agus D B et al. 2002 Cancer Cell 2:127). Although the
HER2/HER3 heterodimer may be important in HER2-driven cell
signaling, the heregulin-dependent HER2/HER3 heterodimer may be
disrupted by pertuzumab and may not be disrupted by trastuzumab
(Jitunttila et al. 2009 Cancer Cell 15:429). In a phase II clinical
trial involving combination treatment with pertuzumab and
trastuzumab in HER2-positive breast cancer patients, treatment
produced a response rate of 24.2%, and disease control rate of 50%
(Baselga J et al. 2010 J Clin Oncol 28: 1138).
[0093] In an embodiment, the drug delivery composition may be
administered in combination with Trastuzumab-DM1 comprised of
trastuzumab and DM1, an agent that is an inhibitor of tubulin
polymerization derived from maytansine. A stable MCC linker
conjugates the DM1 to the trastuzumab. The compound may be designed
to deliver DM1 to HER2-overexpressing cancer cells. Preclinical
studies have indicated the growth-inhibitory effect of
trastuzumab-DM1 in HER2-overexpressing and trastuzumab resistant
cells (Lewis Phillips G D et al. 2008 Cancer Res 68:9280). In a
phase II clinical trial involving HER2-positive metastatic breast
cancer patients with disease progression despite trastuzumab-based
therapy, trastuzumab-DM1 yielded an independently reviewed response
rate and progression-free survival of 26.9% and 4.6 months,
respectively (Vogel C L et al. 2009 J Clin Oncol 27: 15s (suppl;
abstr 1017). Trastuzumab-DM1 had similar antitumor activity and an
independently reviewed response rate of 24.2% even in patients
previously treated with lapatinib and trastuzumab (n=66).
[0094] In an embodiment, the drug delivery compositions may be
administered in combination with PI3K pathway inhibitors. The PI3K
pathway inhibitors may be used for treating HER2 expressing tumors.
HER2-overexpressing breast cancer cells are believed to be
dependent on the PI3K signaling pathway, and a number of genetic or
epigenetic alterations in PI3K signaling molecules have been shown
to cause resistance to trastuzumab or small-molecule HER2 kinase
inhibitors. HER2-overexpression and PIK3CA mutations frequently
occur simultaneously in breast cancer cells (Oda K et al. 2008
Cancer Res 68:8127), and cell lines with either HER2 amplification
or PIK3CA mutation are equally Akt-dependent (She Q B et al. 2008
PLoS ONE 3:e3065). PI3K pathway inhibitors may therefore be useful
in overcoming resistance to anti-HER2 agents. PI3K/mTOR dual
inhibitor and Akt inhibitor were shown to effectively inhibit
cellular growth in trastuzumab-and lapatinib resistant cells. At
present, many classes of PI3K pathway inhibitors are in clinical
development, and their roles in overcoming trastuzumab resistance
will be tested in the future. These inhibitors may be used in
combination with the drug delivery compositions herein.
[0095] In an embodiment, the drug delivery compositions may be
administered in combination with inhibitors of alternative
signaling molecules. The inhibitors of alternative signaling
molecules may be used to treat trasuzumab resistant cancer cells.
Alternative signaling from IGF-1R or MET may be associated with
trastuzumab resistance. Small-molecular weight inhibitors of IGF-1R
or MET receptor tyrosine kinase, and anti-IGF-1 antibody and
anti-HGF antibody are in clinical development at present.
Monotherapy or combination therapy with these agents and the drug
delivery composition that includes trastuzumab may be therefore an
attractive therapeutic strategy.
[0096] In an embodiment, the drug delivery compositions may be
administered in combination with HER2 vaccines and adoptive
immunotherapy targeting the HER2 extracellular domain tested in
clinical trials. Results of these tests showed that significant
levels of durable T-cell HER2 immunity may be generated with active
immunization without significant consequences with regard to
autoimmunity against normal tissues (Bernhard H et al 2002 Endoctr
Relat Cancer 9:33). Early data from clinical trials testing the
potential use of HER2-specific vaccines in adjuvant therapy for
high-risk breast cancer patients show promising results (Peoples G
E et al. 2008 Clin Cancer Res 14:797).
[0097] In an embodiment, the drug delivery composition may be
administered in combination with Ertumaxomab (Rexomum, Fresenius
Biotech GmbH, phase II study). Ertumaxomab is an intact bispecific
antibody targeting HER2 and CD3 on T cells with preferential
binding to activating Fcc type I/III receptors and redirecting T
cells, macrophages, dendritic cells, and natural killer cells to
HER2 expressing tumor sites (Kiewe P et al. 2008 Expert Opin
Investig Drugs 17: 1553). In a phase I trial, ertumaxomab treatment
was associated with one complete response, two partial responses,
and two stable diseases in patients with metastatic breast cancer
who had received extensive prior treatment (Kiewe P et al. 2006
Clin Cancer Res 12:3085). The effects of ertumaxomab are being
evaluated in phase II studies.
[0098] In an embodiment, the drug delivery compositions may be
administered using defucosylated trastuzumab. Defucosylated
trastuzumab may be used to treat trastuzumab resistant cancer
cells. Removal of fucose from antibody oligosaccharides attached to
the heavy chain of Asn.sup.297 (defucosylation) has been shown to
significantly enhance antibody-dependent cellular cytotoxicity
(ADCC) compared to the activity of regular antibodies. In addition,
defucosylation of trastuzumab was also found to enhance ADCC in an
in vitro assay as compared to regular trastuzumab (Suzuki E et al.
2007 Clin Cancer Res 13:1875). Defucosylated trastuzumab more than
doubled the median progression-free survival compared with
conventional trastuzumab in preclinical models of HER2-amplified
breast cancer (Juntilla et al. 2010 Cancer Res 70:4481).
[0099] Any of the above agents including paclitaxel, docetaxel,
dasatinib, erlotinib, gefitinib, lapatinib, pertuzumab,
trastuzumab, ertumaxomab, trasuzumab-DM1, defucosylated
trastuzumab, PI3K pathway inhibitors and HER2 vaccines are here
envisioned to be useful in combination with nanobiopolymer
conjugate compositions herein to treat breast cancers by methods
described herein.
[0100] In an embodiment, the drug delivery composition may include
at least one module that targets a tumorigenic or a cancer cell to
be selected from the group of agents consisting of: paclitaxel,
docetaxel, dasatinib, erlotinib, gefitinib, lapatinib, pertuzumab,
trastuzumab, ertumaxomab, trasuzumab-DM1, defucosylated
trastuzumab, PI3K pathway inhibitors and HER2 vaccines.
[0101] In an embodiment, the step of contacting the subject with
the composition may further include providing the composition in a
unit dose effective for treatment the cancer in the subject. For
example, the effective dose may be at least one dose selected from
the group consisting of: 1 .mu.g/kg, 50 .mu.g/kg, 100 .mu.g/kg, 200
.mu.g/kg, 300 .mu.g/kg, 400 .mu.g/kg, 500 .mu.g/kg, 600 .mu.g/kg,
700 .mu.g/kg, 800 .mu.g/kg, 900 .mu.g/kg, 1 mg/kg, 50 mg/kg, 100
mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700
mg/kg, 800 mg/kg, 900 mg/kg, and 1 g/kg
[0102] In an embodiment, of the cancer may be selected from the
list consisting of: gastric, endometrial, salivary gland, lung,
non-small cell lung, pancreatic, ovarian, peritoneal, prostate,
colorectal, breast, cervical, uterine, ovarian, brain, head and
neck, testicular and teratoma cancers.
[0103] The cancer may be either a primary cancer, or a metastatic
cancer, or both.
[0104] As discussed above and described in greater detail in the
Examples, inhibition of expression or activity of an oncogenic
protein may be useful to prevent development or metastasis of a
cancer conditions. These inhibitors may be clinically useful in
preventing further growth of a particular cancer type, including
but not limited to the breast cancer; skin cancer; ovarian cancer;
cervical cancer; the retinoblastoma; colon cancer and other
conditions, e.g., those arising from the lining of the
gastrointestinal tract; lung cancer and cancers of the respiratory
tract; renal carcinoma and other tumors arising from the inner
surface of kidney tubules; leukemias and lymphomas and disorder of
blood; and other types of genital cancer including those associated
with various strains of papilloma virus; brain tumors; and cancers
of the uterus, of the vagina, of the urethra.
[0105] In an embodiment, the diagnostic, prognostic and therapeutic
methods described herein may not be limited to treating conditions
in humans, but may be used to treat similar conditions in any
mammal. The mammal may be but not limited to bovine, canine,
feline, caprine, ovine, porcine, murine, or equine species. When
treating tumors in a given species, it is preferred, but not
required, that the antisense oligonucleotides have a nucleotide
sequence that is substantially identical in base sequence to that
as it occurs naturally in the species.
[0106] The invention having been fully described is it further
exemplified in a research paper by Satoshi Inoue et al. entitled
"Polymalic acid-based nanobiopolymer provides efficient systemic
breast cancer treatment by inhibiting both HER2/neu receptor
synthesis and activity", which was published Feb. 15, 2011 in
Cancer Research 71(4): 1454-1464, and is incorporated herein by
reference as if fully set forth. A skilled person will recognize
that many suitable variations of the methods may be substituted for
or used in addition to those described above and in the claims. It
should be understood that the implementation of other variations
and modifications of the embodiments of the invention and its
various aspects will be apparent to one skilled in the art, and
that the invention is not limited by the specific embodiments
described herein and in the claims. The present application
mentions various patents, scientific articles, and other
publications, each of which is hereby incorporated in its entirety
by reference.
[0107] Further embodiments herein may be formed by supplementing an
embodiment with one or more element from any one or more other
embodiment herein, and/or substituting one or more element from one
embodiment with one or more element from one or more other
embodiment herein. Further embodiments herein may be described by
reference to any one of the appended claims following claim 1 and
reading the chosen claim to depend from any one or more preceding
claim.
EXAMPLES
[0108] The following non-limiting examples are provided to
illustrate particular embodiments. The embodiments throughout may
be supplemented with one or more detail from one or more example
below, and/or one or more element from an embodiment may be
substituted with one or more detail from one or more example
below
Example 1. Experimental Design
[0109] Compositions and methods of the present invention provide a
nanobiopolymeric drugs based on poly-.beta.-L-malic acid (PMLA)
platform specifically designed for delivery into HER2/neu-positive
tumors. Targeted nanobiopolymeric conjugates based on
poly-.beta.-L-malic acid (PMLA) are biodegradable, non-toxic, and
non-immunogenic. The PMLA nanoplatform was synthesized for
repetitive systemic treatments of HER2/neu-positive human breast
tumors in a xenogeneic mouse model. Various moieties were
covalently attached to PMLA, including a combination of morpholino
antisense oligonucleotides (AON) directed against HER2/neu mRNA, to
block HER2/neu synthesis; anti-HER2/neu antibody trastuzumab
(Herceptin.RTM.), to target breast cancer cells and inhibit
receptor activity simultaneously; and transferrin receptor
antibody, to target the tumor vasculature and mediate delivery of
the nanobiopolymer through the host endothelial system.
[0110] The Examples herein include tests of the lead compound, and
data show that this compound significantly inhibited growth of
HER2/neu-positive breast cancer cells in vitro and in vivo, and
enhanced apoptosis and inhibition of HER2/neu receptor signaling
with suppression of Akt phosphorylation was observed in treated
cells and animals. In vivo imaging analysis and confocal microscopy
demonstrated selective accumulation of the nanodrug in tumor cells
as a result of an active delivery mechanism resulting from design
of the lead compound. Systemic treatment of human breast
tumor-bearing nude mice resulted in more than 90% inhibition of
tumor growth and tumor regression, compared to partial (50%) tumor
growth inhibition in mice treated with control trastuzumab alone or
control AON alone, either free or attached to PMLA. Data from
Examples herein offer a preclinical demonstration of use of the
PMLA nanoplatform for combination cancer therapy.
[0111] The epidermal growth factor receptor or ErbB family of
receptor tyrosine kinases is exemplified by an epidermal growth
factor receptor (also called HER1 or ErbB1), HER2 (ErbB2 or neu),
HER3 (ErbB3), and HER4 (ErbB4). Upon ligand binding, ErbB family
members form homodimers and heterodimers followed by the
phosphorylation within intracellular kinase domains (Yarden et al.
2001 Nat Rev Mol Cel Biol 2:127). Upon ErbB1 and ErbB2 activation,
phosphotyrosylated sites in Src-homology 2 (SH2) domains in these
proteins serve as docking sites for adaptor proteins such as Shc,
Grb2, and Sos resulting in the activation of the of
Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK
and PI3K/protein kinase B (PKB) pathways and promotion of
proliferation and mitogenesis (Yarden et al. 2001 Nat Rev Mol Cel
Biol 2:127).
[0112] The HER2/neu proto-oncogene, also known as erbB-2, encodes a
185-kDa type I transmembrane receptor tyrosine kinase that is
member of the epidermal growth factor receptor family (Hynes N E et
al., 2005 Nat Rev Cancer 5:341; Bargmann C I et al. 1986. Nature
319:226; Coussens L et al. 1985 Science 230:1132). Early studies
have identified HER2/neu protein overexpression in several human
carcinomas, including subsets of ovarian and breast cancers (Hynes
N E et al. 1994. Biochim Biophys Acta 1198:165; D'Emilia J et al.
1989 Oncogene 4:1233; Slamon D J et al. 1989 Science 244:707).
HER2/neu overexpression has been linked to a short relapse time and
poor survival of breast cancer patients (Slamon D J et al. 1987
Science 235:177), as this protein plays a role in the molecular
mechanisms of human cancers.
[0113] The ErbB2 gene is amplified and overexpressed in up to 30%
of primary breast cancers and this is associated with poor patient
prognosis (Slamon D J et al., 1989 Science 244:707). ErbB1 is also
overexpressed in up to 30% of primary invasive breast cancers and
this is correlated with reduced overall survival, proliferation,
and higher metastatic potential (Tsutsui S et al. 2002 Breast
Cancer Res Treat 71:67). Inhibition of ErbB1 signaling reduces both
ErbB1 and ErbB2 activity and delays tumorigenesis in MMTV/Neu mice
(Lenferink A E G et al. 2000 Proc Natl Acad Sci 97:9609). The
cooperative activation of proliferative pathways by these two
receptors has stimulated the development of a number of small
molecule inhibitors of members of the ErbB family for use as
anticancer agents.
[0114] Newly diagnosed estrogen positive breast cancers are
commonly treated with the antiestrogen agent tamoxifen. In
estrogen-positive breast cancers, overexpression of both Erb1 and
Erb 2 is associated with resistance to tamoxifen therapy. It was
shown that administration of such anticancer agents as lapatinib
(GW572016) and tamoxifen together was advantageous and restored
tamoxifen-mediated cell cycle arrest and inhivited
tamoxifen-resistant breast tumor growth (Chu I et al. 2005 Cancer
Res 65: 18).
[0115] Characteristics such as extracellular accessibility, high
expression, and association with poor prognosis make HER2/neu an
attractive candidate for antibody therapy. Metastatic breast cancer
patients are currently being treated with Trastuzumab (also known
as Herceptin; Genentech, Inc., San Francisco, Calif.), a Food and
Drug Administration-approved humanized monoclonal anti-HER2/neu
(Kaptain S et al. 2001 Diagn Mol Pathol 10:139). Breast cancer
clinical trials for patients with advanced disease expressing high
levels of HER2/neu showed that use of Trastuzumab as a single
immunotherapeutic agent resulted in an objective response rate of
12% to 26% (Cobleigh M A et al. 1999 J Clin Oncol 17:2639; Baselga
J et al. 1996 J Clin Oncol 14:737; Vogel C L et al. 2002 J Clin
Oncol 20:719). Subsequent clinical trials in patients with advanced
disease have also shown that targeting metastatic breast cancer
with Trastuzumab in combination with chemotherapy resulted in a 50%
objective response, but disease relapse still affected most cases
(Slamon D J et al. 2001 N Engl J Med 344:783). In addition,
Trastuzumab lacks considerable activity against tumors expressing
HER2/neu that are not of breast origin (Burstein H J 2005 N Engl J
Med 353:1652). Furthermore, resistance to Trastuzumab is a growing
problem in patients with breast tumors. Novel treatments for
patients with HER2/neu-expressing tumors are still needed.
[0116] In 66% to 88% of cases, HER2/neu-overexpressing tumors
demonstrate primary resistance to Herceptin.RTM. (Baselga J et al.
1999 Semin Oncol 26:78; Nahta R et al. 2004 Cancer Res. 64:398).
This resistance may be due to epitope masking by overexpressed
mucins, loss of receptor ability to influence pro-survival
signaling through PI3K-Akt axis, or loss of protein phosphatase
PTEN leading to the activation of PI3K-Akt signaling (Tseng P H et
al. 2006 Mol Pharmacol. 70:1534-41; Nagy P et al 1998 Cytometry
32:120; Tanner M et al. 2004 Cancer Ther. 3:1585-92).
Nanobiopolymers as a Platform for Carrying Multiple Drugs for
Treatment of HER2/neu Cancers
[0117] Advantages of drug combinations can be offered in a single
molecular entity such as a nanobiopolymeric conjugate. These
compounds offer enhanced cancer cell specificity because of the
presence of tumor targeting antibodies, bypass drug resistance by
delivering polymer-bound drugs into cancer cell cytoplasm, and can
carry multiple drugs on a single platform (Wu K et al. 2010 Angew
Chem Int Ed Engl. 9:1451). Efficient delivery of
nanobiopolymer-attached drugs to tumors is increased by passive
targeting through enhanced permeability and retention (EPR) effect
typical for tumors (Maeda H et al. 2009 Eur J Pharm Biopharm
71:409), and additionally, by active targeting using antibodies,
such as anti-TfR (Maeda H et al. 2009 Eur J Pharm Biopharm 71:409;
Liu X, et al. 2008 Cancer Gene Ther. 15:126; Peterson C M et al.
2003 Adv Exp Med Biol. 519:101). Table 1 shows the size (smaller
than 30 nm) of conjugates used in Examples herein.
[0118] The slightly negative .zeta. potentials promote interaction
of the conjugate with the cell membrane and enhance intracellular
internalization (Wilhelm C et al. 2003 Biomaterials
24:1001-11).
[0119] A general problem with anti-cancer drugs is lack of specific
tumor targeting, resulting in an extent of random tissue
accumulation and significant side effects for normal tissues
(Shukla R et al. 2008 Nanotech 19:1; Shukla R et al. 2006 Bioconjug
Chem 17:1109). To circumvent this drawback, tumor-targeting
antibodies have been used as drug carriers or directly as
therapeutics (e.g., Herceptin.RTM.). Dendrimer nanoconjugates with
attached Herceptin.RTM. displayed enhanced accumulation in breast
cancer cells in animal models (Shukla R et al. 2006 Bioconjug Chem
17:1109). Methotrexate-loaded dendrimers produced a cytotoxic
effect in tumor cells in vitro resulting from
Herceptin.RTM.-mediated complex internalization (Shukla R et al.
2008 Nanotech 19:1). However, the efficacy of these nanodrugs was
limited because of lack of efficient endosome release unit (Shukla
R et al. 2008 Nanotech 19:1). Drugs were specifically delivered to
cancer cells and tumor growth was inhibited as was angiogenesis in
brain glioma-bearing animals (Fujita M et al. 2006 Angiogenesis
9:183; Ljubimova J Y et al. 2008 Chem Biol Interact. 171:195). The
efficiency of the polymers was associated with properties of tumor
targeting, use of AON drugs to more than one tumor marker at the
same time, and the presence of endosome disruption moiety ensuring
drug release inside the target cell (Gasslmaier B et al. 2000 Eur J
Biochem267: 5101).
[0120] Table 1 summarizes nanobiopolymer drugs synthesized for use
in Examples herein.
TABLE-US-00001 TABLE 1 Nanobiopolymer drugs and controls for
treatment of cancers overexpressing HER2/neu, molecular sizes, and
.zeta. potentials .zeta. potential Nanobiopolymer variant Version
Size (nm) (mV) P/mPEG/LOEt/AON/ Lead drug 22.1 (.+-.2.3) -5.2 .+-.
(0.4) .sup. Herceptin .RTM./TfR(M) with AON, Herceptin .RTM. and
TfR(M) P/mPEG/LOEt/AON/ with AON and 20.1 (.+-.2.4) -5.7 (.+-.0.6)
TfR(H/M) TfR (Human/ Mouse) P/mPEG/LOEt/ with 15.1 (.+-.1.2) -4.1
(.+-.0.4) Herceptin .RTM. Herceptin .RTM. alone P/mPEG/LOEt/IgG
Control version N/A N/A for imaging study with IgG
[0121] PMLA is a natural polymer obtained from the slime mold
Physarum polycephalum (Lee B S et al. 2006 Bioconjug Chem 17:317;
Lee B S et al. 2002 Water-soluble aliphatic polyesters:poly(malic
acid)s, in: Doi YSA, eds, Biopolymers, Weinheim: Wiley-VCH, 2002
pp. 75-103). PMLA is non-toxic, non-immunogenic, and biodegradable
in vitro and in vivo, stable in the bloodstream, and highly
water-soluble (Gasslmaier B et al. 1997 Eur J Biochem 250:308;
Gsslmeier B et al. 2000 Eur J Biochem 267:5101). Systemic delivery
of morpholino AONs having nucleotide sequences specific to .alpha.4
and .beta.1 chains of a tumor vasculature-specific protein,
laminin-411 (formerly, laminin-8), to intracranial glioblastoma was
shown to result in marked inhibition of tumor angiogenesis and
growth (Ljubimova J Y et al. 2008 Nanomedicine 3:247; Ding H et al.
2010 Proc Natl Acad Sci online publication). Further, to target
tumor vasculature, a mAb to transferrin receptor (TfR) was attached
to the same nanoplatform. The nanobiopolymer composition carrying
each of anti-HER2/neu antibody (Herceptin.RTM.), anti-TfR antibody,
and AON to HER2/neu is shown herein to enhance the specificity and
anti-tumor effect towards HER2/neu positive breast cancer. Without
being limited by any specific theory or molecular mechanism, the
lead compound tested herein is a nanoplatform designed to work on
several molecular levels, to inhibit the synthesis of new HER2/neu
receptors with AON, and to block the activity of existing HER2/neu
on the tumor cell membrane with Herceptin.RTM..
[0122] Antisense oligonucleotides (AONs) that bind specifically to
mRNA and block protein synthesis are tools specific for silencing
gene expression. Efficient delivery of AONs and siRNAs in systemic
treatment of tumors however still presents significant problems
(Patil S D et al. 2005 AAPS 7:E61; Thierry et al., 2003 Curr Opin
Mol Ther 5:133). Preclinical studies of AON for cancer treatment
showed promising results, and stability of AON in plasma renders
these molecules feasible for systemic treatment (Busch R K et al.
1994 Cancer Lett 86:151; Sekhon H S et al. 2008 Lung Cancer 60:347;
Garbuzenko O B et al. 2010 Proc Natl Acad Sci 107:10737). Further,
Morpholino AONs specific for dystrophin have been delivered to
dystrophic muscle cells in vivo in a Duchenne muscular dystrophy
mouse model and to patients (Wu B et al. 2010 Gene Ther 17:132;
Kinali M et al 2009 Lancet Neurol 8:918). An AON specific for
HER2/neu was observed to be more potent for inhibiting neoplastic
cell proliferation in vitro than mAb inhibition of HER2/neu
receptor (Roh H et al. 2000 Cancer Res 60:560). Combination
treatment of HER2/neu-positive breast cancer cells in vitro with
HER2/neu AON and conventional chemotherapeutic agents results in
synergistic inhibition of tumor cell growth by activation of
apoptosis (Rait A S et al. 2001 Cancer Gene Ther 8:728; Lewis P G D
2008 Cancer Res 68:9280).
[0123] Nanoparticles are used in drug delivery as carriers for
small and large molecules. Nanoparticles are defined as particulate
dispersions or solid particles with a size in the range of 10-1000
nm. The drug is dissolved, entrapped, encapsulated or attached to a
nanoparticle matrix (Langer R. 2000 Acc Chem Res 33:94).
Nanobiopolymers of the present invention differ from nanoparticles
in that nanoparticles have no covalent bonds between the particle
and drug cargo, generally merely leak the drug, and accordingly
cannot directly transport cargo to and release the cargo inside
tumor cells.
[0124] Contrary to nanoparticles, nanobiopolymer compositions
provided herein comprise a single unitary molecular entity having
functional modules including a plurality of the following: tumor
cell-targeting antibodies, two or more anti-tumor drugs, an
endosomal disruption moiety, and a glutathione-cleavable bond to
release the drug inside tumor cell cytoplasm, covalently attached.
Such a construct functions to eliminate leakiness, suppresses
non-tumor accumulation thereby minimizing side effects, and
increase drug half-life dwell time of the composition in plasma. As
a result, tumor uptake and drug specificity were observed in
examples herein to be enhanced, leading to a significant reduction
of tumor growth and volume. Moreover, the combined drug action
through inhibiting Akt activation and increase of tumor cell
apoptosis was also observed in examples herein.
[0125] Nanobiopolymers of the present invention offer a great
potential in cancer therapy.
Example 2. Reagents
[0126] Morpholino.sup..TM.-3'-NH2 antisense oligonucleotides (AONs)
used in Examples herein were custom made by Gene Tools (Philomath,
Oreg.).
[0127] AONs specific for HER2/neu included two sequences:
TABLE-US-00002 version 1: (SEQ ID NO: 1)
5'-AGGGAGCCGCAGCTTCATGTCTGTG-3', version 2: (SEQ ID NO: 2)
5'-CATGGTGCTCACTGCGGCTCCGGC-3'.
and
[0128] AONs specific for an epidermal growth factor receptor (EGFR)
included:
TABLE-US-00003 (SEQ ID NO: 3) 5'-TCGCTCCGGCTCTCCCGATCAATAC-3'.
[0129] AONs specific for .alpha.4 and .beta.1 subunits of
laminin-411 included:
TABLE-US-00004 .alpha.4 subunit: (SEQ ID NO: 4)
5'-AGCTCAAAGCCATTTCTCCGCTGAC-3', .beta.1 subunit: (SEQ ID NO: 5)
5'-CTAGCAACTGGAGAAGCCCCATGCC-3'.
and
[0130] siRNA specific for EGFR included sequences as follows:
TABLE-US-00005 sense: (SEQ ID NO: 6) 5'-CCUAUAAUGCUACGAAUAUtt-3',
and antisense: (SEQ ID NO: 7) 5'-AUAUUCGUAGCAUUUAUGGag-3'.
[0131] siRNA specific for HER2 receptor protein included:
TABLE-US-00006 sense: (SEQ ID NO: 8) 5'-GUUGGAUGAUUGACUCUGAtt-3',
and antisense: (SEQ ID NO: 9) 5'-UCAGAGUCAAUCAUCCAACat-3'.
[0132] Small letters "tt", "ag" and "at" at the 3'-terminus of the
siRNA sequence denote DNA oligonucleotides that are synthesized to
anneal siRNA to a DNA molecule.
[0133] Highly purified, endotoxin-free poly-.beta.-L-malic acid, Mw
(weight-averaged) =100 kDa, polydispersity=1.1, was obtained from
the culture broth of Physarum polycephalum. Rat anti-mouse TfR mAb
R17217 (mTfR) was purchased from Southern Biotech (Birmingham,
Ala.). Cysteamine (2 -mercaptoethyl-1-amine hydrochloride),
N-hydroxysuccinimide, other reagents and solvents were of highest
available purity and purchased from Sigma-Aldrich (St. Louis,
Mo.).
Example 3. Synthesis of Polymalic Acid Nanobiopolymers
[0134] The nanobiopolymers contain five to six components: PMLA as
the backbone; functional modules include: morpholino AON to inhibit
HER2/neu protein synthesis; targeting anti-TfR mAb; anti-tumor
Herceptin.RTM.; 40% leucine ethyl ester (LOEt) as endosome escape
unit to achieve cytoplasmic AON delivery, and 5% PEG.sub.5000 to
increase stability in the bloodstream. FIG. 1 illustrates a
chemical structure and schematic drawings showing a
nanobiopolymeric conjugate designed to inhibit HER2/neu expression
by antisense oligonucleotides (AON) and to attenuate
HER2/neu-mediated cell signaling by Herceptin.RTM.. The modules are
HER2/neu morpholino AON (indicated 1 in Figure) conjugated to the
PMLA scaffold by disulfide bonds (S--S) that are cleaved by
cytoplasmic glutathione to release the free drugs; targeting and/or
effector antibodies that include antibody specific to a transferrin
receptor protein (TfR) either alone or in combination with
monoclonal antibodies (mAbs) to mouse TfR (indicated 2a in Figure),
human TfR (indicated 2b) and Herceptin.RTM. (indicated 2c) for
tumor endothelial and cancer cell targeting, receptor-mediated
endocytosis, and anti-tumor effect, polyethylene glycol (PEG) for
drug protection (indicated 3), stretches of conjugated L-leucine
ethyl ester (LOEt) for endosomal escape of the drug (indicated 4),
and optional fluorescent reporter dye (Alexa Fluor 680) for imaging
(indicated 5). The nanopolymer also contained free unsubstituted
pendant carboxyl groups for enhancing solubility and nonfunctional
disulfides originating from chemical masking of excess sulfhydryls
with 3-(2-pyridyldithio)-propionates.
[0135] Referring to FIG. 1, anti-mouse TfR mAb on
Herceptin.RTM.-containing conjugate was used to target tumor
vasculature. The conjugate with AON without Herceptin.RTM. included
an anti-human TfR mAb attached to it to promote drug binding to
human tumor cells and its internalization. The preconjugate
containing 40% LOEt, 5% PEG.sub.5000 and 10% of cysteamine (%
referring to the total amount of pendant carboxyl groups in
polymalic acid) was synthesized by the methods described previously
(Lee B S et al. 2006 Bioconjug Chem 17:317). The antibodies
conjugated with the preconjugate were qualitatively and
quantitatively assayed by size exclusion HPLC. ELISA with purified
TfR and HER2/neu was used to assess functional reactivity of
attached antibodies as described (Fujita M et al. 2007 J Control
Release. 122:356).
[0136] Conjugates for imaging were fluorescently labeled with Alexa
Fluor.RTM. 680 C2-maleimide (Invitrogen, Carlsbad, Calif.) by
forming thioether with sulfhydryl groups. Antibody conjugates were
then reacted with HER2/neu AON (FIG. 1). A control conjugate
contained Herceptin.RTM. (FIG. 1) and not HER2/neu-specific
AON.
Example 4. The Nanobiopolymer Characterization
[0137] Chemical and physical characterization of polymeric
nanobioconjugate was performed by various methods including
L-malate dehydrogenase assay after nanobiopolymer hydrolysis at
100.degree. C. in the presence of 6 M HCl, PEG colorimetric
determination and protein quantification, size and .zeta.
potential, HPLC, and ELISA. HPLC was performed on a Hitachi
analytical Elite LaChrom HPLC-UV system (Hitachi, Pleasanton,
Calif.) and size exclusion, on a BioSep-SEC-S 3000 column
(Phenomenex, Torrance, Calif.). The nanobiopolymer variants were
characterized by their size (hydrodynamic diameter) on the basis of
noninvasive back-scattering (NIBS), and .zeta. potential from
electrophoretic mobility based on the Helmholtz-Smoluchowski
formula, using electrophoresis M3-PALS (Gasslaier B et a. 1997 Eur
J Biochem 250:308). Both measurements were performed in a Zetasizer
Nano System ZS90 (Malvern Instruments, Malvern, UK). Data on
molecular size and .zeta. potential represent mean.+-.standard
deviation obtained from three independent measurements.
Example 5. Cell Lines and Culture Conditions
[0138] Human breast cancer cell lines BT-474, SKBR-3, MDA-MB-231,
MDA-MB-435, MDA-MB-468, and MCF-7 were obtained from American Type
Culture Collection (Manassas, Va.). BT-474, MDA-MB-231, MDA-MB-435,
MDA-MB-468, and MCF-7 were cultured in DMEM with 10% fetal bovine
serum and antibiotics. SKBR-3 was cultured in McCoy's 5A medium
with 10% fetal bovine serum and antibiotics.
Example 6. Nomenclature
[0139] The term "nanobiopolymer" denotes a drug delivery system
with PMLA as a nanoplatform and functional module groups covalently
attached to the PMLA, including an AON, a rat anti-mouse or a mouse
anti-human targeting TfR mAbs (M and H, respectively), and LOEt as
the endosomal escape unit module. The nanobiopolymer drugs (FIG. 1
and Table 1) described herein to treat HER2/neu-positive breast
cancer contained either a drug HER2/neu AON or drug Herceptin.RTM.
or both HER2/neu AON+Herceptin.RTM..
Example 7. Cell Proliferation Assay
[0140] HER2/neu-overexpressing breast cancer cells each of BT-474
or SKBR-3 were seeded into six-well plates at 3.times.10.sup.5
cells/well. The next day, cells were treated with one of Endoporter
(4 .mu.M; control); Herceptin (40 .mu.g/ml);
P/mPEG/LOEt/Herceptin.RTM. (40 .mu.g/ml); Endoporter (4 .mu.M) and
AON (4 .mu.M); P/mPEG/LOEt/AON/TfR(H/M);
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M); and PBS control. Seventy-two
hours after treatment, the cells were stained with Trypan Blue.
Cell viability was determined by calculating the mean of cell
counts for each treatment group (in triplicate) and was expressed
as a percentage of the total number of cells treated normalized to
the number of cells treated with PBS.
Example 8. Western Blotting
[0141] BT-474 and SKBR-3 breast cancer cells were treated with
Herceptin.RTM. (40 .mu.g/ml P/mPEG/LOEt/Herceptin.RTM. (40 .mu.g/ml
equivalent to Herceptin.RTM.); Endoporter (4 .mu.M) and AON (4
.mu.M); P/mPEG/LOEt/AON/TfR(H/M);
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M); PBS control, or 4 .mu.M
Endoporter. Cell lysates were collected after 72 hours and were
analyzed by western blotting as described previously (Inoue S et
al. 2005 Mol Ther. 12:707-15). Lysates of excised breast tumors
after various treatments were analyzed by these methods. The
following anti-human primary antibodies were used: HER2/neu, Akt,
phosphorylated Akt (p-Akt), glyceraldehyde 3-phosphate
dehydrogenase (GAPDH, to normalize gel load) (all from Cell
Signaling Technology, Beverly, Mass.), and poly(ADP ribose)
polymerase (PARP; BD Biosciences, San Jose, Calif.).
Example 9. Tumor Xenografts in Nude Mice
[0142] Animal experiments were performed in accordance with the
protocols approved by the Cedars-Sinai Medical Center Institutional
Animal Care and Use Committee. Athymic mice (CrTac: NCr-Foxn1nu
Homozygous; Taconic, Hudson, N.Y.) were used. A 0.72-mg, 90-day
release, 17.beta.-estradiol pellet (Innovative Research of America,
Sarasota, Fla.) was inserted subcutaneously into the back of each
mouse seven days prior to injection. An amount of 1.times.10.sup.7
BT-474 cells suspended in 150 .mu.l of Matrigel (BD Biosciences,
Bedford, Mass.) were injected into the right flank of each of 35
mice (5 mice per group), and treatment was initiated when tumors
achieved an average size of >120 mm.sup.3 (21 days after
injection). Mice were divided into five treatment groups and each
group was administered either of: sterile PBS (control);
Herceptin.RTM. (40 .mu.g/ml); P/mPEG/LOEt/Herceptin.RTM. (40
.mu.g/ml equivalent to Herceptin.RTM.); P/mPEG/LOEt/AON/TfR(H/M);
or P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M); into the tail vein twice
a week. Treatments were performed six times during a period of
three weeks.
[0143] Tumor xenografts were measured with calipers twice a week,
and tumor volumes were determined using the formula:
(length.times.width.sup.2).times.(.pi./6).
[0144] Eighteen days after the last treatment, the animals were
anesthetized with 3% isoflurane-air mixture and were euthanized.
Tumor samples were stained with hematoxylin and eosin for
morphological observation. The data are the average of two
independent examples.
Example 10. Confocal Microscopy
[0145] Alexa Fluor 680-labeled nanobiopolymers (P/mPEG/LOEt/IgG,
control); P/mPEG/LOEt/Herceptin.RTM., 40 .mu.g/ml; or
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M)) was each injected into the
tail vein of mice. Twenty-four hours after drug administration,
mice were euthanized; the tumors were harvested to detect the
fluorescent signal, snap-frozen in liquid nitrogen and embedded in
OCT compound for confocal microscopy (TCS SP5.times. microscope;
Leica Microsystems, Mannheim, Germany).
Example 11. In Vivo Imaging
[0146] BT-474 human breast cancer cells were implanted into the
right thigh of mice as described. When tumor size attained 120
mm.sup.3, 160 .mu.l of Alexa Fluor 680 labeled nanobiopolymers was
injected intravenously (4 .mu.M). P/mPEG/LOEt/IgG was used as a
negative control. Drug distribution and localization was assessed
in tumor-bearing mice using Xenogen IVIS 200 imager (Caliper Life
Sciences, Hopkinton, Mass.), at different time points before drug
administration, 1 h, 3 h, 6 h, and 24 h after the drug injection).
Twenty-four hours after drug administration, mice were euthanized
and the circulating drugs eliminated by intraarterial PBS
perfusion. The tumor and major organs were harvested to detect the
fluorescent signal.
Example 12. Statistical Analysis
[0147] Student's t-test (for two groups) and analysis of variance
(ANOVA, for three and more groups) were used to calculate
significance of the experimental results. GraphPad Prism4 program
(GraphPad Software, statistical San Diego, Calif.) was utilized for
all calculations. Data are presented as mean.+-.standard error of
mean (SEM). The significance level was set at P<0.05.
Example 13. Synthesis of Polymer Conjugates
[0148] Of the HER2/neu-specific AON sequences, a version that did
not inhibit HER2/neu expression well in comparison with another
version was observed; therefore, only the effective version was
conjugated to the polymer platform. The absolute molecular weight
of the lead version of nanobiopolymer (FIG. 1) was 1,300 kDa by
light scattering and close to the calculated value based on design.
Hydrodynamic diameters (nano sizes) and .zeta. potentials of the
nanobiopolymers in FIG. 1 are summarized in Table 1. Parameters for
.zeta. potentials in the range of -4.1 to -5.7 mV have been
reported for other nanoparticles as compatible with cell membrane
attachment and nanoparticle internalization (Lorenz M R et al.
2006. Biomaterials 27:2820; Wilhelm C et al. 2003 Biomaterials.
24:1001).
Example 14. The Lead Nanobiopolymer Carrying Both Herceptin.RTM.
and HER2/neu AON (P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M)) Inhibited
Growth of Breast Cancer Cells In Vitro
[0149] Breast cancer cell growth inhibition following
administration of anti-HER2/neu AON and Herceptin.RTM. was first
examined. Based on optimization experiments, each of AON at 4 .mu.M
with 4 .mu.M Endoporter (in vitro AON delivery agent, GeneTools),
and Herceptin.RTM., at 40 .mu.g/ml was analyzed. Results in FIG. 2
are shown for HER2/neu high-expressing cells BT474 and SKBR-3, as
well as for low-expressing cells, MDA-MB-231 and MDA-MB-435.
Referring to this figure, HER2/neu overexpressing breast cancer
cells (BT-474 and SKBR-3; also shown in FIG. 3A) were treated with
various drugs as indicated (top row). After 72 hours, cell
viability was determined using a Trypan Blue exclusion assay.
Percentage of cell growth was calculated as average cell counts for
each group and expressed relative to parallel samples treated with
PBS (control) set to 100%. Growth of tumor cells treated with lead
compound P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) was observed to be
significantly inhibited compared with other treatments in both cell
lines. In cell lines expressing low amounts of HER2/neu (FIG. 3A),
the data showed that the lead compound had greatest ability to
inhibit cell growth (bottom row). One asterisk indicates that
P<0.05; two asterisks indicate that P<0.01; three asterisks
indicate that P<0.003 compared to PBS control treatment. The
lead compound also showed significant differences at P<0.005
when compared to all treatment groups (top row), and at P<0.02
when compared to Herceptin.RTM. (bottom row). At the concentrations
used, it was observed that each of free AON and Herceptin.RTM.
resulted in some growth inhibition in HER2/neu high-expressing
cells. Low-expressing cell lines were observed to be significantly
less responsive to these treatments.
[0150] These nanobiopolymeric conjugates (a two-drug compound and
single-drug compounds shown in FIG. 1) were then tested for tumor
cell growth inhibitory effect. The nanobiopolymers, Herceptin.RTM.,
and free AON caused significant growth inhibition compared to PBS
control in HER2/neu high-expressing cells (FIG. 2 top, P<0.01).
The lead two-drug compound produced the strongest inhibitory effect
that was significantly higher than that of the other
nanobiopolymers tested and higher than Herceptin.RTM. (P<0.005
compared to all groups). In HER2/neu low-expressing cells, only the
lead compound with AON, Herceptin.RTM. and TfR(M) was able to
induce statistically significant inhibition of tumor growth
compared to PBS (FIG. 2 bottom, P<0.02).
Example 15. The Lead Compound Inhibits HER2/neu and p-Akt
Expression and Induces Apoptosis of HER2/neu-Overexpressing Breast
Cancer Cells In Vitro
[0151] A phosphatidylinositol-3 kinase (PI3K) and its downstream
target, the serine/threonine kinase Akt, play an important role in
HER2/neu positive breast cancer cell growth and proliferation, as
well as in anti-tumor effect of Herceptin.RTM. (Tseng P H et al,
2006 Mol Pharmacol. 70:1534; Yakes F M et al. 2002 Cancer Res.
62:4132). HER2/neu signaling can activate the PI3K/Akt/mTOR
cascade, and activated Akt stimulates increases in cell size,
metabolism and survival (Plas D R et al. 2005 Oncogene
24:7435).
[0152] Therefore, to examine the mechanism responsible for the
enhanced growth inhibitory effect of the lead nanobiopolymer, drug
effects on the expression and phosphorylation of pertinent
signaling markers HER2/neu, Akt, and p-Akt were assessed.
[0153] HER2/neu high-expressing cell lines BT-474 and SKBR-3 were
used (FIG. 3A). To determine whether the nanobiopolymer carrying
both HER2/neu AON and Herceptin.RTM. induces apoptosis, PARP
cleavage was examined by western blot analysis. Breast cancer cell
lines used in Examples herein were observed to express high levels
of TfR.
[0154] In HER2/neu high-expressing cell lines, HER2/neu expression
was inhibited to different extents by each of Herceptin.RTM., AON,
and the single-drug versions of the nanobiopolymer
[P/mPEG/LOEt/Herceptin and P/mPEG/LOEt/AON/TfR(H/M)] in comparison
with controls. The strongest inhibition of HER2/neu expression was
observed upon treatment with the lead nanobiopolymer having AON and
Herceptin.RTM. attached to the PMLA carrier molecule.
[0155] Expression of p-Akt, a key downstream mediator of HER2/neu
signaling (Tseng P H et al, 2006 Mol Pharmacol. 70:1534), was
inhibited to different extents in tumor cells treated with
Herceptin.RTM., AON, or single-drug versions of nanobiopolymer
compared to control cells treated with PBS or AON transduction
reagent Endoporter. The p-Akt signal upon treatment of both breast
cancer cell lines with the lead drug carrying both Herceptin.RTM.
and HER2/neu AON was observed to be markedly lower in comparison to
treatment with any other agent (FIG. 3B). The amount of total Akt
on western blots remained unchanged by each of the treatment.
[0156] Apoptosis assessed by PARP cleavage was induced to some
extent by each of Herceptin.RTM., AON, and single-drug
nanobiopolymers in HER2/neu high-expressing cells, for example in
BT-474 cell line. Significantly, the lead compound,
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M), triggered apoptosis to a
greater extent than the other agents in both cell lines, as shown
by increased PARP cleavage compared to the other agents (FIG.
3B).
[0157] Western blot analyses showed decreased HER2/neu and
phosphorylated Akt after treatment with each of Herceptin.RTM.,
P/mPEG/LOEt/Herceptin.RTM., AON or P/mPEG/LOEt/AON/TfR(H/M)-treated
tumor cells, and not with control treatment PBS or Endoporter in
both cell lines. Treatment with lead compound
P/mPEG/LOEVAON/Herceptin.RTM./TfR(M) further reduced both HER2/neu
and p-Akt. Assay of generation of cleaved poly(ADP-ribose)
polymerase (PARP) as a measure of apoptosis was observed at highest
levels in P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M)-treated cells.
Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) was used as an
internal loading control.
Example 16. The Lead Compound P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M)
Specifically Accumulates in HER2/neu-Overexpressing Breast Tumors
In Vivo
[0158] Imaging studies in vivo showed that the lead compound
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) having anti-mouse TfR and
anti-human HER2/neu combined on the same PMLA molecule provided
tumor-specific drug delivery through host endothelial system into
subcutaneous human breast tumors. Twenty-four hours after injection
of drugs, the compounds were observed to accumulate mostly in the
tumor and draining organs, kidney and liver (FIG. 4). FIG. 4 shows
distribution of various compounds herein labeled with Alexa Fluor
680 in live mice with BT-474 breast tumors and in tumors in
isolated organs. Referring to this figure, major organ analysis
compared breast tumors and organs before injection (left panel)
with those twenty-four hours after intravenous injection (right
panels). Live mice herein were injected with each of the lead drug
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) (bottom row), positive
control P/mPEG/LOEt with Herceptin.RTM. (middle row) and control
conjugate P/mPEG/LOEt/IgG (top row). Control mice (top row) had
little BT-474 tumor accumulation, and most of the control polymer
accumulated in drug clearing organs, liver and kidneys. Polymer
P/mPEG/LOEt with Herceptin.RTM. alone had a moderate tumor
accumulation (middle row). The highest accumulation in breast tumor
cells was observed in mice treated with the lead compound
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M). Arrows mark tumor
implantation site.
[0159] The nanobiopolymer with only Herceptin.RTM. accumulated to a
lesser extent in tumors than the version with Herceptin.RTM., AON
and anti-TfR mAb (the lead drug). These data show the enhanced
targeting of tumor vasculature with anti-TfR mAb compared to
Herceptin.RTM.. Control nanobiopolymer with IgG showed only a small
amount of tumor accumulation (FIG. 4).
[0160] Confocal microscopy was performed on sections of brain
tumors removed 24 hours after intravenous injection of Alexa Fluor
680-labeled drugs. A significantly stronger signal in tumor cells
for P/mPEG/LOEt/Herceptin.RTM. was observed than for the control
conjugate P/mPEG/LOEt/IgG, and the highest tumor accumulation was
observed with the lead compound compared to other nanobiopolymers
(FIG. 5). FIG. 5 shows distribution of various compounds in BT-474
breast tumor cells. Referring to FIG. 5, animals were administered
compounds intravenously as shown in FIG. 4, were sacrificed 24
hours after drug injection, tumors were excised, and sections were
analyzed by confocal microscopy. Nuclei were counterstained with
DAPI (grey area). Animals injected with control conjugate
P/mPEG/LOEt/IgG with attached Alexa Fluor 680 tracking dye (grey)
showed little if any tumor cell accumulation (top row). Animals
injected with P/mPEG/LOEt/Herceptin.RTM. displayed considerable
accumulation in tumor cells, and the highest accumulation was
observed in animals injected with the lead drug
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M), consistent with live animal
imaging data shown in FIG. 4. Scale bar=50 .mu.m.
Example 17. The Lead Compound P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M)
Significantly Inhibits HER2/neu Positive Breast Tumor Growth In
Vivo
[0161] The therapeutic effect of compositions herein following
intravenous administration in subcutaneous mouse models of human
breast tumor xenografts was investigated. Cell line BT-474 was
selected for in vivo analysis because of its high HER2/neu
expression and tumorigenicity. Treatment of BT-474 tumor-bearing
mice with Herceptin.RTM., single-drug nanobiopolymers and the lead
compound P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) was performed and
compared to negative control PBS. No decreases in body weight or
morbidity, or death was observed, indicating that each treatment
was well tolerated.
[0162] FIG. 6A-6C show mouse tumor inhibition, pathology, signaling
and apoptosis marker expression.
[0163] FIG. 6A shows data obtained and histopathological analysis
of respective tumors from two representative animals for each group
administered with different drugs. Variable amounts of dead tissue
were observed to be present in all treated groups. Tumor size
reduction data and pronounced disappearance of tumor cells were
observed following treatment with the lead drug
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M), and mostly necrotic areas
were observed to be present.
[0164] FIG. 6B shows extent of tumor growth inhibition in mice.
Referring to this figure, animals treated with each of unconjugated
Herceptin.RTM. (squares) and with positive control
P/mPEG/LOEt/Herceptin.RTM. (triangles), or with
P/mPEG/LOEt/AON/TfR(H/M) (circles) showed significant inhibition
compared with PBS control (diamonds) (P<0.03).
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) treatment (large squares) was
observed to produce the greatest inhibition of tumor growth
compared to other treatment groups, resulting in 80 to 95% tumor
regression observed during the follow-up period (P<0.02 vs.
Herceptin.RTM. and other drugs; P<0.001 vs. PBS). Error bars
denote standard error of the mean (SEM).
[0165] FIG. 6C shows expression of select markers after treatment
of HER2/neu positive tumors in vivo. Referring to this figure,
Western blot analysis data showed a decrease in HER2/neu and p-Akt
(but not total Akt) expression in each of Herceptin.RTM.-,
P/mPEG/LOEt/Herceptin.RTM.-, or P/mPEG/LOEt/AON/TfR(H/M)-treated
mice and not in control PBS-treated ones.
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) further inhibited HER2/neu
expression, with near disappearance of a p-Akt band. PARP cleavage
as a measure of apoptosis was observed also to be substantially
greater in P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M)-treated mice than
that in other groups. GAPDH was an internal control to normalize
gel loading.
[0166] Each the compounds inhibited tumor growth after six
treatments (from days 21-38 post tumor implantation) and during
follow-up to 56 days (FIG. 6B). Control unconjugated Herceptin.RTM.
showed a similar tumor growth inhibition as a function of time as
PMLA-bound Herceptin.RTM.. Both these drugs produced a somewhat
stronger effect than HER2/neu AON bound to PMLA (FIG. 6B). This
effect was significant for all three of these single drug compounds
(P<0.03 vs. PBS). The compound having both Herceptin.RTM. and
HER2/neu AON combined on one nanobiopolymer showed the highest
degree of inhibition of tumor growth, with a clear synergistic
effect compared to single-drug treatments (FIG. 6B; P<0.001 vs.
PBS; P<0.03 vs. other treatment groups). The observed tumor
regression following treatment with
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) was 80% at the start of
follow-up to 95% at the end of this period (day 56; FIG. 6B).
Moreover, tumors in the group treated with this lead compound
started to regress within the two weeks after the initial
treatment, and tumors in this group remained suppressed for an
additional 20 days, at which time the treatment was terminated.
[0167] Hematoxylin and eosin staining revealed that the tumors
treated with each of Herceptin.RTM., P/mPEG/LOEt/Herceptin.RTM., or
P/mPEG/LOEt/AON/TfR(H/M) showed some areas of cell death compared
with PBS (control) treated tumor. Significantly, treatment with the
lead compound led to the appearance of massive morphologically
necrotic areas with little unaffected tumor tissue remaining (FIG.
6A).
[0168] The mechanism of this antitumor effect was further
investigated by western blot analysis using lysates of subcutaneous
BT-474 breast tumors after different treatments. Tumor HER2/neu
expression was partially inhibited by each of Herceptin.RTM., AON,
and single-drug versions of the PMLA nanobiopolymer
[P/mPEG/LOEt/Herceptin.RTM. and P/mPEG/LOEt/AON/TfR(H/M)] in
comparison with PBS controls (FIG. 6C). The lead compound
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) produced the highest
inhibition of HER2/neu tumor expression, consistent with the in
vitro western blot analysis. Phosphorylated Akt was also reduced
after drug treatments. Again, lead drug
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) resulted in the most
pronounced decrease, with little p-Akt signal observed remaining
(FIG. 6C). Total Akt remained unchanged upon treatments, as in the
in vitro experiments.
[0169] Apoptosis assessed by PARP cleavage was induced to some
extent by each of the compounds in HER2/neu high-expressing tumors
compared to PBS treatment. Significantly, lead
P/mPEG/LOEt/AON/Herceptin.RTM./TfR(M) markedly increased PARP
cleavage compared to the other treatments indicating that this
nanobiopolymer induced apoptosis to a greater extent than the other
used drugs (FIG. 6C).
Example 18. Nanobiopolymer Conjugates Significantly Inhibited
Triple Negative Breast Cancer Growth In Vivo
[0170] Potential therapeutic effects of each of the compounds in
Table 2 following intravenous administration using subcutaneous
mouse models of human triple-negative breast cancer (TNBC)
xenografts were investigated. Cell line MDA-MB-468 was selected for
in vivo analysis because it lacked expression of estrogen and
progesterone, and the HER2 protein in these cells is expressed
normally. Treatment of TNBC tumor-bearing mice was performed with a
single-drug nanobiopolymer containing AONs specific for .alpha.4
and .beta.1 subunits of laminin-411; or with a single-drug
nanobiopolymer containing AONs specific for an epidermal growth
factor receptor (EGFR) protein; or with a two-drug nanobiopolymer
conjugate combing AONs specific for EGFR protein with AONs specific
for .alpha.4 and .beta.1 subunits of laminin-411, in comparison
with negative control PBS using the treatment protocol schedule
shown in Table 2.
TABLE-US-00007 TABLE 2 Nanobiopolymer drugs and controls for
treatment of triple-negative breast cancers. Group 1 Group 2 Group
3 Group 4 (n = 6) (n = 6) (n = 6) (n = 6) PBS P/PEG(5%)/ P/PEG(5%)/
P/PEG(5%)/ LOEt(40%)/ LOEt(40%)/ LOEt(40%)/ EGFR(2.1%)/
.alpha..sub.4.beta..sub.1(2.0%)/ EGFR, .alpha.4B1(2.0)/
HuTfR(0.12%)/ HuTfR(0.12%)/ HuTfR(0.12%)/ MsTfR(0.12) MsTfR(0.12)
MsTfR(0.12) IV twice a week IV twice a week IV twice a week Amount
12.5 mg/kg (drug) 25 mg/kg (drug) 37.5 mg/kg (drug) of drug 2.5
mg/kg of each 2.5 mg/kg of each 2.5 mg/kg of each AON AON AON
[0171] It was observed that single-drug compound carrying AONs
specific for EGFR and the two-drug compound carrying both AONs
specific for EGFR and AONs specific a4.beta.-subunits of
laminin-411 inhibited tumor growth after six treatments that were
administered during days 19-52 after implantation of tumor cells
(FIG. 7).
[0172] FIG. 7 shows extent of tumor growth inhibition by
compositions herein in subjects bearing triple-negative breast
tumors. Referring to this figure, animals treated with each of
P/mPEG/LOEt/AON-EGFR/TfR(H/M; squares), or with
P/mPEG/LOEt/AON-EGFR/.alpha.4.beta.1/TfR(H/M; triangles) showed
significant inhibition compared with PBS negative control
(diamonds). P=0.002 vs. .alpha.4.beta.1; P=0.0001 vs. PBS.
P/mPEG/LOEt/AON-.alpha.4.beta.1/TfR(H/M) treatment inhibited tumor
growth compared to control PBS treatment, and was observed to be
less effective compared to data obtained with other
nanobiopolymers, shown in the figure. (P=0.01 vs. PBS). Error bars
denote SEM.
[0173] The two-drug compound was observed to have produced a
stronger therapeutic effect than the single-drug compound carrying
AONs specific for EGFR alone, and the data were statistically
significant for each single drug compound and the two-drug compound
(P=0.1 vs. PBS). Further, administration on a schedule of the eight
treatments was observed to be more effective than six treatments
for greater regression of tumors.
Example 19. Nanobiopolymer Conjugates Inhibited Expression of
Cancer Stem Cell Markers
[0174] Cancer stem cells represent a population of malignant cells
that give rise to the tumor. There are a number of reports
describing overexpression of stem cell markers, such as CD133,
CD44, Notch1 or C-myc, in human tumors, which coincides with tumor
drug resistance (Fan et al. 2004 Cancer Res. 2004; 64:7787-7793;
Estrach et al. 2011 Circ Res 109:172-182; Zhang et al. 2008 J Exp
Clin Cancer Res 27:85; Wang et al. 2008 PLoS One. 3:e3769;
Herschkowitz et al. 2012 Proc Natl Acad Sci USA 109:2778-2783). At
the same tim