U.S. patent application number 12/579281 was filed with the patent office on 2010-07-22 for targeted cargo protein combination therapy.
This patent application is currently assigned to THE UNITED STATES OF AMERICA, as represented by the Secretary. Invention is credited to Raj K. Puri.
Application Number | 20100183545 12/579281 |
Document ID | / |
Family ID | 42337122 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100183545 |
Kind Code |
A1 |
Puri; Raj K. |
July 22, 2010 |
TARGETED CARGO PROTEIN COMBINATION THERAPY
Abstract
The present invention combines a targeted cargo protein with an
active agent for the treatment of a disease or condition.
Inventors: |
Puri; Raj K.; (Potomac,
MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 SW SALMON STREET, SUITE 1600
PORTLAND
OR
97204
US
|
Assignee: |
THE UNITED STATES OF AMERICA, as
represented by the Secretary,
|
Family ID: |
42337122 |
Appl. No.: |
12/579281 |
Filed: |
October 14, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61105408 |
Oct 14, 2008 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
514/1.1 |
Current CPC
Class: |
A61K 49/0008 20130101;
A61K 31/704 20130101; A61K 31/519 20130101; A61K 31/555 20130101;
A61P 35/00 20180101; A61K 31/704 20130101; A61K 31/7068 20130101;
A61K 45/06 20130101; A61K 38/2026 20130101; A61K 47/642 20170801;
A61K 31/7068 20130101; A61K 33/24 20130101; A61K 31/513 20130101;
A61K 31/513 20130101; A61K 31/555 20130101; A61K 31/519 20130101;
A61K 33/24 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.2 ;
514/12 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 38/02 20060101 A61K038/02; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] This invention was created in the performance of a
Cooperative Research and Development Agreement with the Food and
Drug Administration, an Agency of the Department of Health and
Human Services. The Government of the United States has certain
rights in this invention.
Claims
1. A method of treating a disease or condition in a subject
comprising administering to the subject a targeted cargo protein in
combination with an active agent, wherein said targeted cargo
protein comprises a first portion that is specific for a target
cell surface molecule associated with the disease or condition and
a second portion that inhibits said cell, and wherein said active
agent comprises any therapeutically appropriate substance for
treating the disease or condition.
2. The method of claim 1, wherein the target cell surface molecule
is an IL-4 receptor.
3. The method of claim 1, wherein the targeted cargo protein is
selected from at least one of the group consisting of PRX321,
IL4-BAD, DT-IL-4, and IL-4 doxorubicin.
4. The method of claim 1, wherein the active agent is a
chemotherapeutic agent.
5. The method of claim 4, wherein said chemotherapeutic agent is an
active agent selected from the group consisting of gemcitabine,
cisplatin, FOLFOX (folinic acid, fluorouracil and oxaliplatin) and
doxorubicin.
6. The method of claim 1, wherein said disease is cancer.
7. The method of claim 1, wherein said disease is a cancer that
over-expresses IL-4 receptors.
8. The method of claim 1, wherein said disease is selected from the
group consisting of brain cancer, malignant astrocytoma,
gliobastoma multiforme, Kaposi sarcoma, bladder cancer, renal cell
cancer, breast cancer, pancreatic cancer, non-small cell lung
cancer, thyroid cancer, squamous cell carcinoma of the head and
neck, colon cancer, gastrointestinal system, mesothelioma and
prostate cancer.
9. The method of claim 8, wherein said disease is pancreatic
cancer.
10. The method of claim 9, wherein said pancreatic cancer is
pancreatic ductal adenocarcinoma (PDA).
11. The method of claim 1, wherein the targeted cargo protein is
administered concurrently with the active agent.
12. A method of treating pancreatic cancer in a subject comprising
administering to the subject a composition comprising PRX321 and a
composition comprising gemcitabine.
13. A method of inhibiting a target cell that is characterized by
over-expression of an IL-4 receptor, comprising a. contacting the
target cell with a targeted cargo protein, said targeted cargo
protein comprising a targeting moiety that specifically binds to an
IL-4 receptor and a cargo moiety that inhibits the target cell, and
b. contacting the target cells with an active agent.
14. The method of claim 13, wherein the target cell is contacted
with the targeted cargo protein concurrently with, or within 24
hours, within 48 hours, within 72 hours or within 96 hours of
contacting the target cell with the active agent.
15. The method of claim 13, wherein the targeted cargo protein is
selected from the group consisting of PRX321, IL4-BAD, DT-IL-4, and
IL-4 doxorubicin
16. The method of claim 13, wherein the target cell is a cancer
cell.
17. The method of claim 13, wherein the target cell is selected
from the group consisting of brain cancer cell, malignant
astrocytoma cell, gliobastoma multiforme cell, Kaposi sarcoma cell,
bladder cancer cell, renal cancer cell, breast cancer cell,
pancreatic cancer cell, lung cancer cell, thyroid cancer cell,
squamous cell carcinoma cell of the head and neck, colon cancer
cell gastrointestinal cancer cell, mesothelioma cell and prostate
cancer cell.
18. The method of claim 16, wherein the cancer cell is pancreatic
cancer cells.
19. The method of claim 13, wherein the targeted cargo protein is
PRX321.
20. The method of claim 13, wherein the active agent is selected
from the group consisting of gemcitabine, doxorubicin, FOLFOX
(folinic acid, fluorouracil and oxaliplatin) and cisplatin.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/105,408 filed Oct. 14, 2008. The contents of
that application is hereby incorporated by reference.
FIELD
[0003] This invention relates to compositions and methods for
treating diseases and conditions using targeted cargo proteins in
combination with another active agent.
BACKGROUND
[0004] Pancreatic ductal adenocarcinoma (PDA) is one of the most
lethal human malignancies (32,000 deaths per year). Because of its
aggressive growth and rapid metastasis to lymph nodes and liver,
only 10% to 15% of patients are found to be resectable at diagnosis
(1). Currently, the most common strategy for the treatment of
advanced pancreatic cancer is treatment with gemcitabine, although
the median survival time continues to be <6 months for these
patients (2, 3). Recently, several types of inhibitors targeting
the epidermal growth factor (EGF) receptor, platelet-derived growth
factor (PDGF) receptor, and nuclear factor-nB (NF-nB) have shown
their effectiveness in pancreatic cancer in murine models (4-7). In
clinical trials, EGF receptor tyrosine kinase inhibitor (Erlotinib,
Tarceva) plus gemcitabine enhances 1-year survival for patients
with advanced pancreatic cancer (8). However, the difference in
median survival between Erlotinib plus gemcitabine group and
gemcitabine alone group is <1 month. An effective new approach
is needed for management of patients with this disease.
[0005] Gemcitabine (Gemzar) is a widely accepted first-line therapy
for advanced pancreatic cancer, although the median survival time
continues to be <6 months for these patients (2, 3). As most
studies using single agent show low response rate and little effect
on patient survival in advanced adenocarcinoma, several clinical
trials using a combined approach of radiotherapy and/or molecular
target therapy with gemcitabine have been initiated (33). In vitro
studies have reported synergistic effect of gemcitabine with
cisplatin, fluvastatin hydroxymethylglutaryl-CoA reductase
inhibitor, CpG-oligodeoxynucleotides, EGFR, PDGF, and vascular
endothelial growth factor inhibitor targeting drugs (6, 34-37). In
addition, immunotoxins were shown to exert synergistic effect with
chemotherapeutic drugs, for example, doxorubicin plus
anti-B4-blocked ricin, Ara-C plus granulocyte macrophage
colony-stimulating factor fused to truncated diphtheria toxin
(DT388-GM-CSF), and fludarabine with rituximab saporin-S6
conjugated protein (38-40).
SUMMARY
[0006] The disclosure describes proteins and other moieties that
interact or bind to target cells such as cancer cells using a
targeting moiety that is linked to a protein or other toxic agent
that kills or inhibits growth or function of the target cells. The
protein or other toxic agent that kills or inhibits cancer cell
growth is referred to as a cargo moiety and the cargo moiety linked
to the targeting moiety is collectively referred to as a targeted
cargo protein. As described herein, these targeted cargo proteins
are used in combination with active agents know to be effective in
treating cancer to synergistically enhance the treatment of cancer
in a mammalian subject, such as a human. The active agents used in
combination with the targeted cargo proteins may be
chemotherapeutic agents, antibodies or other agents typically used
to treat cancer or other diseases or conditions. Targeting cell
surface receptors with targeted cargo proteins provides a unique
opportunity for tumor therapy.
[0007] The invention is based in part on the unexpected discovery
that a targeted cargo protein targeted against the IL-4 receptor,
interleukin-4 (IL-4) cytotoxin (an embodiment of which is also
known as PRX321), when combined with gemcitabine, a
chemotherapeutic agent currently used to treat advanced pancreatic
cancer, is shown to have a synergistic anti-tumor effect both in
vitro and in a clinically relevant mouse model of advanced
pancreatic cancer. Specifically, those mice treated with a
combination of PRX321 and gemcitabine showed a significant decrease
in tumor burden and improved survival compared to treatment with
either PRX321 or gemcitabine alone. This study demonstrates for the
first time the potential of combining an IL-4 cytotoxin such as
PRX321 with a chemotherapeutic agent for treating patients with
pancreatic cancer. The devices and methods of the present invention
are directed to alleviating the above-described problems with
previous treatments and, in addition, provide improved therapeutic
results in comparison to IL-4-cytotoxin (IL4-PE) alone. It is
believed that unique target expression on PDA and synergistic
effect of two drugs having independent mechanisms of action
contribute to the overall improved therapeutic results.
[0008] Here, we show the efficacy of the combination therapy of
gemcitabine with PRX321 in animal models of pancreatic ductal
adenocarcinoma (PDA). Targeting cell surface receptor with targeted
cargo proteins (e.g. cytotoxins or immunotoxins) provides a unique
opportunity for tumor therapy. Targeted cargo proteins offer the
advantage of enhanced specificity and direct toxicity for tumor
cells that over-express the receptor, thus limiting the potential
toxicity to normal tissues (9). Several clinical trials using
PRX321, IL-13 cytotoxin, and recombinant immunotoxin BL22 have
shown survival benefits in patients with glioblastoma multiforme,
chronic lymphocytic leukemia, and hairy cell leukemia (10-13).
[0009] Interleukin-4 (IL-4) is an important Th2-derived cytokine,
which is involved in mediating antitumor immune-modulating
activities (14). IL-4 has been shown to have a modest but direct
inhibitory effect on the growth of several tumor cells in vitro and
in vivo (15, 16). Based on these properties, IL-4 was tested in the
clinic as a treatment for hematopoietic and solid malignancies, but
it showed limited antitumor activity (17). To improve this limited
activity, we targeted IL-4 receptor (IL-4R) because a variety of
human tumor cells, including pancreatic cancer, express
high-affinity receptors for IL-4 (18-22).
[0010] We show herein that 60% of PDA samples express moderate- to
high density surface IL-4Rs, whereas normal pancreas express no or
very low levels of IL-4R. PRX321 is highly cytotoxic to pancreatic
cancer cell lines; however, it was not cytotoxic to HPDE cells,
fibroblasts, and HUVEC, which express no or low levels of IL-4R. We
also show that PRX321 synergizes with gemcitabine in mediating
cytotoxic activity in pancreatic cancer cell lines in vitro, and in
animal models of human pancreatic cancer in vivo. A significant
prolonged survival effect of PRX321 and its combination with
gemcitabine was shown in mice with early disease. Forty percent of
mice that received combination therapy showed complete eradication
of pancreatic tumors. In addition, this significant survival
benefit was also confirmed in animals implanted with the clinical
pancreatic cancer sample.
[0011] The foregoing and other features of the disclosure will
become more apparent from the following detailed description, which
proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a schematic representation of the structure and
amino acid sequence of an exemplary targeted cargo protein, a
circularly permuted IL-4-Pseudomonas toxin, PRX321 (SEQ ID NO: 1).
Disulfide bonds are indicated on the drawing.
[0013] FIG. 2A shows the visible fluorescent area quantification of
tumor size as a function of time (obtained from sequential
whole-body imaging) in an early pancreatic tumor model in which
tumor bearing mice received no treatment, treatment with
gemcitabine alone, treatment with IL-4 cytotoxin (PRX321) alone, or
a combination of gemcitabine and PRX321.
[0014] FIG. 2B shows Kaplan-Meier survival curves in an early
pancreatic tumor model.
[0015] FIG. 3A shows the visible fluorescent area quantification of
tumor size as a function of time (obtained from sequential
whole-body imaging) in an advanced pancreatic tumor model in which
tumor bearing mice received no treatment, treatment with
gemcitabine alone, treatment with IL-4 cytotoxin (PRX321) alone, or
a combination of gemcitabine and PRX321.
[0016] FIG. 3B shows Kaplan-Meier survival curves in an advanced
pancreatic tumor model.
[0017] FIG. 4 shows Kaplan-Meyer survival curves in an advanced
pancreatic cancer model generated from a clinical sample by
orthotopic transplantation in SCID mice.
DETAILED DESCRIPTION
[0018] While the invention has been described in some detail by way
of illustration and example, it should be understood that the
invention is susceptible to various modifications and alternative
forms, and is not restricted to the specific embodiments set forth
in the Examples. It should be understood that these specific
embodiments are not intended to limit the invention but, on the
contrary, the intention is to cover all modifications, equivalents,
and alternatives falling within the spirit and scope of the
invention.
[0019] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
[0020] The present invention involves treating a disease or
condition in a subject by administering to the subject at least one
targeted cargo protein of the present invention in combination with
an active agent known to be effective in treating the disease or
condition.
[0021] In some embodiments of the invention, the targeted cargo
protein comprises a toxin, and is thus a cytotoxin or immunotoxin.
Preferably, the targeted cargo protein is a cytotoxin or
immunotoxin that binds specifically to the IL-4 receptor, for
example, an IL-4-cytotoxin such as PRX321.
[0022] In accordance with the present invention, the active agent
may be any substance or the like, or treatment protocol or the
like, that provides a therapeutic benefit to the patient when
combined with the administration of a targeted cargo protein.
Active agents include, but are not limited to chemotherapeutic
agents. It is intended that an active agent may be a substance that
uses a different mechanism of action than the targeted cargo
protein, or it may be a substance that uses the same mechanism of
action. In some embodiments of the invention, the active agent is a
chemotherapeutic agent. In most preferred embodiments of the
invention, the active agent is gemcitabine or doxarubicin.
[0023] In some embodiments of the invention, the disease or
condition is any disease or condition characterized by cells having
a unique or identifying expression pattern of a surface molecule or
target. In some embodiments of the invention, the cell surface
molecule is a receptor. In some preferred embodiments of the
invention, the receptor is an IL-4 receptor (IL-4 R).
[0024] Although the IL-4 R is expressed at low levels by certain
normal cells, such as resting T lymphocytes, B lymphocytes and
resting or activated CD 34 bone marrow cells, it is over-expressed
in a wide range of solid tumors, including, for example, brain
cancer, including malignant astrocytoma and gliobastoma multiforme,
Kaposi sarcoma, bladder cancer, renal cell cancer, breast cancer,
pancreatic cancer, non-small cell lung cancer, thyroid cancer,
squamous cell carcinoma of the head and neck, colon cancer and
other cancers of the gastrointestinal system, mesothelioma and
prostate cancer. As used herein, when a cell surface molecule is
over-expressed or uniquely expressed in cells characterizing a
disease or condition such as cancer, a targeted cargo protein that
binds specifically to the cell surface molecule is said to be
specific for the cell associated with the disease or condition.
[0025] In some embodiments of the invention, the disease or
condition is a cancer or tumor producing disease. In some
embodiments of the invention, the cancer is a pancreatic cancer. In
preferred embodiments of the invention, the pancreatic cancer is
pancreatic ductal adenocarcinoma (PDA). In other preferred
embodiments of the invention, the disease or condition is any
disease or condition that over-expresses IL-4 receptors.
Conditions, as used herein, include, but are not limited to
inflammation.
[0026] In some embodiments, the invention provides methods and
compositions for inhibiting the growth of a target cell, said
target cell comprising a cell characterized by having
over-expression of an IL-4 receptor. The invention includes
contacting the cell with a targeted cargo protein, said targeted
cargo protein comprising a targeting moiety and a protein
synthesis-inhibiting moiety, and then, within a pre-determined and
medically appropriate time period (e.g., one week), at least one
active agent. In some embodiments of the invention, the
pre-determined time period may be selected from the time periods
consisting of within 96 hours, within 72 hours, within 48 hours,
and within 24 hours. In some embodiments, the cell is concurrently
contacted with both the targeted cargo protein and the first active
agent.
[0027] The present invention also includes a method of treating a
disease or condition by administering a targeted cargo protein in
combination with an active agent.
[0028] As used herein, "in combination" or variations on that
phrase, refer to administering the targeted cargo protein and the
active agent within a close enough time period that the patient
derives a beneficial result that would not have occurred if the
targeted cargo protein and the active agent were not administered
in combination. In some embodiments of the invention, in
combination refers to administering the targeted cargo protein and
the active agent in the same composition; in other embodiments the
targeted cargo protein and the active agent are administered
sequentially or serially. In some embodiments of the invention, the
targeted cargo protein and the active agent are administered are
part of an integrated therapeutic plan. In some of these exemplary
embodiments, the targeted cargo protein and the active agent may be
administered hours or days, weeks, or months apart, but the
combination of the two agents provides a beneficial result for the
patient.
[0029] The composition of the present invention may also be
administered repetitively. For example, a cargo protein may
administered alone or in combination with an active agent, then the
cargo protein may be administered again, alone or in combination
with an active agent, at any therapeutically appropriate interval
(e.g., the next day or after a week).
[0030] One skilled in the art will recognize that the invention as
described here may be reconfigured into different combinations,
elements, and processes which are included within the scope of the
invention.
[0031] It is possible to deliver IL4-PE through drug leaching
stents for localized cancer to avoid systemic exposure and stent
serve as a "reservoir"
[0032] The following explanations of terms and methods are provided
to better describe the present disclosure and to guide those of
ordinary skill in the art in the practice of the present
disclosure. The singular forms "a," "an," and "the" refer to one or
more than one, unless the context clearly dictates otherwise. For
example, the term "comprising a targeted cargo protein" includes
single or plural targeted cargo proteins and is considered
equivalent to the phrase "comprising at least about one targeted
cargo protein." The term "or" refers to a single element of stated
alternative elements or a combination of two or more elements,
unless the context clearly indicates otherwise. As used herein,
"comprises" means "includes." Thus, "comprising A or B," means
"including A, B, or A and B," without excluding additional
elements.
[0033] (A) Active agent, as used herein, refers to any substance
that provides a therapeutic benefit to the patient. Active agents
include but is not limited to chemotherapeutic agents; anti-disease
drugs; anti-disease chemicals, immune system mediators, including
enhancers and inhibitors; immune suppressive compounds such as
cyclosporine and retinoic acid which has shown to enhance the
cytotoxic activity of ricin A. Other therapeutic agents include
toxins which are not required to be internalized into the target
cell such as PRX302 which acts on the cell surface by creating
pores.
[0034] Exemplary active agents include but are not limited to those
disclosed in PCT/US2008/002747 (Pastan, et al.), incorporated
herein by reference. The preferred active agents are gemcitabine
and doxorubicin. Some additional exemplary active agents are
protein synthesis inhibitors such as L-asparaginase,
cyctotoxic/antitumor antibiotics, such as daunorubicin, dosorubicin
epirubicin, idarubicin, mitoxantrone, valrubicin, bleomycin,
hyroxyurea, and mitomycin. Other active agents include plant
alkaloids, such as docetaxel, paclitaxel, vinblastine, vincristine,
vindesine, and vinorelbine. Other active agents include alkylating
agents, such as nitrogen mustards (such as chlorambucil,
chlormethine, cyclophosphamide, ifosfamide or melphalan), a
nitrosoureas (such as carmustine, lomustine, spreptozocin),
platinum drugs (such as carboplatin, cisplatin, oxaliplatin,
BBR3464), busulfan, dacarbazine, mechlorethamne, procarbazine,
temozolomide, thioTEPA, uramustine), antimetabolites (such as
aminopterin, methotrexate, pemetrexed, ralititrexed, cladribine,
clofarabine, fludarabine, mercaptopurine, thioguanine, pentostatin,
capecitabine, cytarabine, fluorouracil and gemcitabine). Other
active also include topoisomerase inhibitors such as topotecan,
irinotecan, podophyllum, etoposide and teniposide. Active agents
also include therapeutic antibodies (such as alemtuzumab,
bevacizumab, cetuximab, gentuzumab, pantitumumab, rituximab,
tositmomab) and kinase inhibitors (such as imatinib, nilotinib,
dasatinib, erlotinib, gefitinib, lapatinib, sorafenib, sunitinib
and vandetanib).
[0035] Combinations of active agents may be used in the methods of
the invention. For example the FOLFOX protocol, which involves
administration of three agents, folinic acid, fluorouracil and
oxaliplatin, may be used in combination with one or more targeted
cargo proteins.
[0036] Adjuvants or immune-stimulants such as BCG (Bacillus
Calmette Geurin) may also be used in combination with targeted
cargo proteins.
[0037] (B) "Over-expresses" or similar terms, refers to the
presence of a receptor, or the presence of a receptor in an amount
significantly higher than normal. Typically, such over-expression
is an indicator of a disease or condition. Il-4 receptor is one
known example. EGFR and VEGFR receptors may also be targeted, in
accordance with the present invention.
[0038] (C) Targeted cargo protein consists of a targeting moiety
linked to a cargo moiety. In accordance with the present invention,
a targeting moiety is any compound that binds to a molecule (herein
referred to as a target) displayed on the target cell surface. A
targeting moiety can be an antibody that binds to a target (e.g.
receptor), a ligand (e.g., cytokine or growth factor) that bind to
a receptor, a permuted ligand that binds to a receptor or a peptide
sequence sensitive to cleavage by a tumor-associated protease.
Exemplary targeting moieties include but are not limited to those
disclosed in U.S. Pat. No. 6,011,002, incorporated herein by
reference. Some exemplary targeting moieties and exemplary GenBank
accession numbers are shown in Table 1 below. Typically, targeting
moieties selectively bind to one type of cell displaying a target
more effectively than they bind to other types of cell that do not
display the target, or that display the target at low levels.
TABLE-US-00001 TABLE 1 Exemplary targeting moiety sequences
Receptor or Antigen to be Targeted Accession Number* Epidermal
growth factor (EGF) NP_001954; EAX06257.1; AAR84237.1 Vascular
endothelial growth AAA35789; CAC19515 factor (VEGF) Interleukin 2
(IL-2) CAA07317; AAB46883.1; NP_000577.2 Interleukin 3 (IL-3)
AAC08706.1; AAA99502.1; CAE45598.1 Interleukin 4 (IL-4) AAH70123;
CAA57444.1; AAH67515.1 (also see SEQ ID NO: 2 and various
circularly permuted ligands in U.S. Pat. No. 6,011,002) IL-5
NP_000870.1; CAA01794.1; P32927.2 IL-13 AAH96141.2; AAH96138.1;
AAH96139.1 Granuclocyte-macrophage colony P04141.1; AAI13925.1;
AAI08725.1 stimulating factor (GMCSF) Granulocyte colony
stimulating Q99062; P09919 factor (GCSF) Tenascin AAA36728.1;
CAA39628.1; NP_002151.2 Mesothelin CAC37289.1; ABW03459.1;
AAH09272.1; AAH03512.1; as well as the mesothelins disclosed in
U.S. Pat. Nos. 7,081,518 and 6,051,405 (mesothelin sequences
therein herein incorporated by reference) CD22 BAA36575.1;
BAA36576.1; BAA36567.1 PSMA (also known as folate ABO93402.2;
AAC83972.1; NP_001014986.1; hydrolase) NP_004467.1 *GenBank Numbers
are herein incorporated by reference, as well as their
corresponding nucleic acid sequences.
[0039] The cargo moiety may be derived from plant, animal, or
bacteria. In accordance with the present invention, cargo moieties
function to significantly kill, reduce or inhibit the growth of
target cells. A cargo moiety may be a peptide (e.g. protein
fragment or full length protein) or other molecule that can
function to significantly reduce or inhibit a target cell. In some
examples, the cargo moiety is not a peptide, but another molecule
that can function to significantly reduce or inhibit the growth of
target cells, such as thapsigargin. Exemplary cargo moieties
include cytotoxins, such as Pseudomonas exotoxin (PE), diphtheria
toxin (DT), including but are not limited to those disclosed in
PCT/US2008/002747 (Pastan, et al.), incorporated herein by
reference. In other examples, cargo moieties are proteins that
normally contribute to the control of cell life cycles, for example
a cargo protein can trigger cell death, such as via apoptotic
pathways (e.g. Bad, Bax and other pro-apoptotic members of the
Bc1-2 family of proteins). Some exemplary cargo moieties and
exemplary GenBank accession numbers are provided in Table 2
below.
TABLE-US-00002 TABLE 2 Exemplary cargo moiety sequences Cargo
Moiety Accession Numbers Diphtheria ABU25232; CAA24778 toxin (DT)
Aerolysin ABR14715.1; ABR14714.1 Proaerolysin AAA21938.1; P09167.2;
U.S. Pat. No. 7,282,476 (proaerolysin sequences therein herein
incorporated by reference) Bouganin AAL35962 and SEQ ID NO: 9 in
U.S. Pat. No. 6.737,511, as well as variant sequences provided in
U.S. Pat. No. 7,339,031 and WO 2005/090579 (bouganin sequences
therein herein incorporated by reference) Pseudomonas 1IKP A;
AAB59097.1; AAF90003.1 (also see SEQ ID NO: 1 of U.S. Pat. No.
exotoxin (PE) 6,011,002) Bcl-2 pro- BAD: CAG46757; AAH01901.1;
CAG46733.1; and sequences provided in U.S. apoptotic Pat. No.
6,737,511 proteins such BAX: CAE52909.1; AAO22992.1; EAW52418.1 as
BAD and BAX Cholera toxin BAA06291.1; ACF35010.1; BAA06288.1; as
well as variant sequences provided in US patent application No.
61/058,872 (variant cholera toxin sequences therein herein
incorporated by reference) Ribonuclease A BAA05124.1; NP_937877.1;
NP_115961.2; Q5GAN4.1; and sequences provided in PCT Publication
No. WO 2007/041361 (rapLR1 sequences therein herein incorporated by
reference) *GenBank Numbers are herein incorporated by reference,
as well as their corresponding nucleic acid sequences.
[0040] In addition to native targeting moieties and cargo moieties,
variant sequences can also be used, such as mutant sequences with
greater biological activity than that of the native sequence.
[0041] The preferred targeted cargo proteins are IL-4-PE, IL-4-BAD,
IL-4-DT and IL-4-doxarubicin.
[0042] (D) Inhibiting or inhibition or similar terms refers to cell
killing, cell inhibition, loss of cell function, or any other
action, direct or indirect, that results in or mediates cell
viability and/or function. In preferred embodiments, the
compositions of the invention include at least two different
mechanisms of action, e.g., two mechanisms of cell killing (such as
apoptosis and necrosis).
[0043] (E) Contacting refers to placement in direct physical
association. With respect to therapeutic targeted cargo proteins
and active agents, such therapeutics are considered to contact a
target cancer cell, such as a pancreatic cancer cell, in a subject
if the therapeutics are administered to the subject by a route that
is generally accepted in the art for administering that type of
therapeutic.
[0044] (F) Specific or specific binding refers to a preferential
binding between an agent and a specific target. For example,
specific binding refers to the situation when a targeted cargo
protein that includes a targeting moiety specific for a molecule or
receptor displayed on a cancer cell binds to the cancer cell but
does not significantly bind to other cells that do not display the
target but are in close proximity to the cancer cell. Specific
binding interactions are mediated by one or, typically, more
noncovalent bonds between the binding molecules. In contrast to
non-specific binding sites, specific binding sites are saturable.
One exemplary way to characterize specific binding is by a specific
binding curve. A specific binding curve shows, for example, the
amount of one binding partner (the first binding partner) bound to
a fixed amount of the other binding partner as a function of the
first binding partner concentration. As the first binding partner
concentration increases under these conditions, the amount of the
first binding partner bound will saturate. In another contrast to
non-specific binding sites specific binding partners involved in a
direct association with each other (e.g., a protein-protein
interaction) can be competitively removed (or displaced) from such
association (e.g., protein complex) by excess amounts of either
specific binding partner. Such competition assays are very well
known in the art. If a targeted cargo protein exhibits specific
binding to a cell surface molecule on a cancer cell, it is said to
be specific for its target on the cancer cell.
[0045] (G) Antibody or antibodies refer to Immunoglobulin molecules
and immunologically active portions of immunoglobulin molecules,
that is, molecules that contain an antigen binding site that
specifically binds (immunoreacts with) an epitope, such as an
epitope displayed by cancer cells or another target cell.
Antibodies include monoclonal antibodies, polyclonal antibodies, as
well as humanized antibodies.
[0046] Antibodies also include affibodies. Affibodies mimic
monoclonal antibodies in function but are based on Protein A.
Affibodies can be engineered as high-affinity ligands for binding
to a targeting moiety.
[0047] (H) Subject refers to a living multi-cellular vertebrate
organisms, including human a non-human mammal.
[0048] Description of exemplary embodiments of targeting moieties
and cargo moieties and their combinations that are useful in the
methods of the invention are the following:
[0049] Targeting Moieties
[0050] In addition to the targeting moieties described above, it
will be appreciated that targeting moieties (as well as
protein-based cargo moieties) may be truncated or modified and
still have the same or even more biological activity. Therefore,
the invention includes variants of targeting moieties and cargo
moieties and portions, fragments or subunits thereof that have at
least 60% sequence identity, at least 75%, at least 80%, at least
85%, at least 90%, at least 98%, or even at least 99% sequence
identity to the native protein sequences or fragments from which
they are derived, as long as the variants retain, or have enhanced,
desired biological activity. In some examples, variant sequences
retain substantially the same amount of even more of the native
biological function of the parent protein, such as the ability to
activate an intracellular signal cascade. However, useful variant
targeting moiety molecules may in some examples retain little or no
biological activity, but retain the ability to bind the appropriate
target with high specificity, and such molecules are included
within the scope of the invention.
[0051] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. 215:403-10, 1990) is available from several
sources, including the National Center for Biological Information
(NCBI, National Library of Medicine, Building 38A, Room 8N805,
Bethesda, Md. 20894) and on the Internet, for use in connection
with the sequence analysis programs blastp, blastn, blastx, tblastn
and tblastx. Additional information can be found at the NCBI web
site.
[0052] BLASTN can be used to compare nucleic acid sequences, while
BLASTP can be used to compare amino acid sequences. To compare two
nucleic acid sequences, the options can be set as follows: -i is
set to a file containing the first nucleic acid sequence to be
compared (such as C:\seq1.txt); -j is set to a file containing the
second nucleic acid sequence to be compared (such as C:\seq2.txt);
-p is set to blastn; -o is set to any desired file name (such as
C:\output.txt); -q is set to -1; -r is set to 2; and all other
options are left at their default setting. For example, the
following command can be used to generate an output file containing
a comparison between two sequences: C:\Bl2seq-i c:\seq1.txt-j
c:\seq2.txt-p blastn-o c:\output.txt-q-1-r 2.
[0053] To compare two amino acid sequences, the options of Bl2seq
can be set as follows: -i is set to a file containing the first
amino acid sequence to be compared (such as C:\seq1.txt); -j is set
to a file containing the second amino acid sequence to be compared
(such as C:\seq2.txt); -p is set to blastp; -o is set to any
desired file name (such as C:\output.txt); and all other options
are left at their default setting. For example, the following
command can be used to generate an output file containing a
comparison between two amino acid sequences: C:\Bl2seq-i
c:\seq1.txt-j c:\seq2.txt-p blastp-o c:\output.txt. If the two
compared sequences share homology, then the designated output file
will present those regions of homology as aligned sequences. If the
two compared sequences do not share homology, then the designated
output file will not present aligned sequences.
[0054] Once aligned, the number of matches is determined by
counting the number of positions where an identical nucleotide or
amino acid residue is presented in both sequences. The percent
sequence identity is determined by dividing the number of matches
either by the length of the sequence set forth in the identified
sequence, or by an articulated length (such as 100 consecutive
nucleotides or amino acid residues from a sequence set forth in an
identified sequence), followed by multiplying the resulting value
by 100. For example, a nucleic acid sequence that has 1166 matches
when aligned with a test sequence having 1554 nucleotides is 75.0
percent identical to the test sequence (1166/1554*100=75.0). The
percent sequence identity value is rounded to the nearest tenth.
For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to
75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to
75.2. The length value will always be an integer.
[0055] For comparisons of amino acid sequences of greater than
about 30 amino acids, the Blast 2 sequences function is employed
using the default BLOSUM62 matrix set to default parameters, (gap
existence cost of 11, and a per residue gap cost of 1). Homologs
are typically characterized by possession of at least 70% sequence
identity counted over the full-length alignment with an amino acid
sequence using the NCBI Basic Blast 2.0, gapped blastp with
databases such as the nr or swissprot database. Queries searched
with the blastn program are filtered with DUST (Hancock and
Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs
use SEG. In addition, a manual alignment can be performed. Proteins
with even greater similarity will show increasing percentage
identities when assessed by this method, such as at least about
75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a cargo
protein or targeting moiety provided herein.
[0056] When aligning short peptides (fewer than around 30 amino
acids), the alignment is performed using the Blast 2 sequences
function, employing the PAM30 matrix set to default parameters
(open gap 9, extension gap 1 penalties). Proteins with even greater
similarity to the reference sequence will show increasing
percentage identities when assessed by this method, such as at
least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence
identity to a cargo moiety or targeting moiety provided herein.
When less than the entire sequence is being compared for sequence
identity, homologs will typically possess at least 75% sequence
identity over short windows of 10-20 amino acids, and can possess
sequence identities of at least 85%, 90%, 95% or 98% depending on
their identity to the reference sequence. Methods for determining
sequence identity over such short windows are described at the NCBI
web site.
[0057] Antibodies or fragments thereof may be used as targeting
moieties. A naturally occurring antibody (e.g., IgG, IgM, IgD)
includes four polypeptide chains, two heavy (H) chains and two
light (L) chains interconnected by disulfide bonds. However, it has
been shown that the antigen-binding function of an antibody can be
performed by fragments of a naturally occurring antibody. Thus,
these antigen-binding fragments are also intended to be designated
by the term "antibody." Specific, non-limiting examples of binding
fragments encompassed within the term antibody include (i) a Fab
fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd
fragment consisting of the VH and CH1 domains; (iii) an Fv fragment
consisting of the VL and VH domains of a single arm of an antibody
(scFv) and scFv molecules linked to each other to form a bivalent
dimer (diabody) or trivalent trimer (triabody); (iv) a dAb fragment
(Ward et al., Nature 341:544-546, 1989) which consists of a VH
domain; (v) an isolated complimentarity determining region (CDR);
and (vi) a F(ab')2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region.
[0058] Methods of producing polyclonal and monoclonal antibodies
are known to those of ordinary skill in the art, and many
antibodies are available. See, e.g., Coligan, Current Protocols in
Immunology Wiley/Greene, N.Y., 1991; and Harlow and Lane,
Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, 1989;
Stites et al., (eds.) Basic and Clinical Immunology (4th ed.) Lange
Medical Publications, Los Altos, Calif., and references cited
therein; Goding, Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York, N.Y., 1986; and Kohler and Milstein,
Nature 256: 495-497, 1975. Other suitable techniques for antibody
preparation include selection of libraries of recombinant
antibodies in phage or similar vectors. See, Huse et al., Science
246: 1275-1281, 1989; and Ward et al., Nature 341: 544-546,
1989.
[0059] Immunoglobulins and certain variants thereof are known and
many have been prepared in recombinant cell culture (e.g., see U.S.
Pat. No. 4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP
256,654; EP 120,694; EP 125,023; Faoulkner et al., Nature 298:286,
1982; Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann
Rev. Immunol 2:239, 1984). Detailed methods for preparation of
chimeric (humanized) antibodies can be found in U.S. Pat. No.
5,482,856. Additional details on humanization and other antibody
production and engineering techniques can be found in Borrebaeck
(ed), Antibody Engineering, 2nd Edition Freeman and Company, NY,
1995; McCafferty et al., Antibody Engineering, A Practical
Approach, IRL at Oxford Press, Oxford, England, 1996, and Paul
Antibody Engineering Protocols Humana Press, Towata, N.J.,
1995.
[0060] In some examples, an antibody specifically binds to a target
protein (e.g., a cell surface receptor such as an IL4 receptor)
with a binding constant that is at least 10.sup.3 M.sup.-1 greater,
10.sup.4 M.sup.-1 greater or 10.sup.5 M.sup.-1 greater than a
binding constant for other molecules in a sample. In some examples,
a specific binding reagent (such as an antibody (e.g., monoclonal
antibody) or fragments thereof) has an equilibrium constant
(K.sub.d) of 1 nM or less. For example, a specific binding agent
may bind to a target protein with a binding affinity of at least
about 0.1.times.10.sup.-8 M, at least about 0.3.times.10.sup.-8M,
at least about 0.5.times.10.sup.-8M, at least about
0.75.times.10.sup.-8 M, at least about 1.0.times.10.sup.-8 M, at
least about 1.3.times.10.sup.-8 M at least about
1.5.times.10.sup.-8M, or at least about 2.0.times.10.sup.-8 M. Kd
values can, for example, be determined by competitive ELISA
(enzyme-linked immunosorbent assay) or using a surface-plasmon
resonance device such as the Biacore T100, which is available from
Biacore, Inc., Piscataway, N.J.
[0061] IL-4 is a pleiotropic cytokine produced by activated T
cells, and is the ligand for the IL-4 receptor. The IL-4 receptor
also binds to IL-13. Thus, IL-13 can also be used as a targeting
moiety to target the IL-4 receptor. IL-4, IL-3, IL-5, IL-13, and
CSF2 form a cytokine gene cluster on human chromosome 5q, with this
gene particularly close to IL-13. Exemplary IL-4 and IL-13 proteins
that can be used in the targeted cargo proteins of the present
disclosure include those provided in Table 1, as well as sequences
having at least 60% sequence identity, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 98% or even at
least 99% sequence identity to such sequences, as long as the
variant retains the ability to bind the IL-4 receptor.
[0062] IL-4 (including IL-4 circularly permuted ligands and other
IL-4 receptor binding proteins such as IL-13) are targeting
moieties that can be linked to BCL-2 family proteins, such as BAX,
BAD, BAT, BAK, BIK, BOK, BID BIM, BMF and BOK, or a toxin such as
aerolysin, proaerolysin, Pseudomonas exotoxin, or combinations
thereof. Any form or derivative of IL-4 can be used as the
targeting moiety. For example, IL-4 or fragments of IL-4 that bind
to the IL-4 receptor can be used. Additionally, multiple cargo
moieties can be linked to IL-4 or multiple IL-4 proteins can be
linked to cargo moieties.
[0063] Antibodies (including fragments, humanized antibodies and
the like as described above) that specifically bind to IL-4
receptors can be linked to BCL-2 family proteins, such as BAX, BAD,
BAT, BAK, BIK, BOK, BID BIM, BMF and BOK, or a toxin such as
aerolysin, proaerolysin, Pseudomonas exotoxin, or combinations
thereof. Antibodies are commercially available from various
companies such as Millipore, Bedford, Mass. or custom made
antibodies can be ordered from companies such as Cambridge Research
Biochemicals.
[0064] Cargo Moieties
[0065] Cargo moieties that are useful in the methods of the
invention reduce or inhibit target cells. As described above, some
examples of cargo moieties are not proteins, but other molecules,
such as chemotherapeutic agents. For example, toxins and proteins
that function to control cell life cycles can be used as cargo
moieties. Toxins that can be used as cargo moieties include toxins
made by microorganisms, plants or animals, as well as toxins made
by human cells. Similarly, any natural cell growth controlling
protein can be used as a cargo moiety. For example, proteins that
trigger cell death during the normal life cycle of an organism can
be used as cargo moieties. In some examples, an oncolytic virus
(e.g., see Allen et al., Mol. Ther. 16:1556-64, 2008) or liposomes
carrying cytotoxic agents (e.g., see Madhankumar et al., Mol.
Cancer. Ther. 5:3162-9, 2006) is used as the cargo protein.
[0066] In one example, the cargo moiety is a toxin. Toxins that are
cytotoxic may be herein referred to as "cytotoxins." Exemplary
toxins that can be used include pore-forming toxins, and toxins
that upon internalization inhibit cell growth. In other examples,
cargo moieties are proteins that are apoptotic triggering proteins,
and cell growth inhibiting proteins. In some examples, the toxin is
a modified bacterial toxin such that the resulting toxin is less
immunogenic than the native toxin. Such modified toxins, such as a
modified Pseudomonas exotoxin A, can reduce the patient's
immunogenic response, thereby allowing repeated administration.
[0067] Pore forming toxins are toxins that form pores in the cell
membrane thereby killing the cell via cell lyses. Exemplary pore
forming toxins include but are not limited to human toxins such as
perforin or bacterial toxins such as aerolysin as well as modified
pore-forming protein toxins that are derived from naturally
occurring pore-forming protein toxins (nPPTs) such as aerolysin or
aerolysin-related polypeptides. Suitable aerolysin-related nPPTs
have the following features: a pore-forming activity that is
activated by removal of an inhibitory domain via protease cleavage,
and the ability to bind to receptors that are present on cell
membranes through one or more binding domains. In some examples the
linker can be engineered to be sensitive to a protease or be
chemically liable. Additional examples of pore forming toxins that
can be used as cargo moieties include, but are not limited to,
proaerolysin from Aeromonas hydrophila, Aeromonas trota and
Aeromonas salmonicida, alpha toxin from Clostridium septicum,
anthrax protective antigen, Vibrio cholerae VCC toxin, epsilon
toxin from Clostridium perfringens, and Bacillus thuringiensis
delta toxins. A detailed description of the engineering of
proaerolysin can be found in U.S. Pat. No. 7,282,476, which is
herein incorporated by reference.
[0068] Additional toxins that can be used as cargo moieties include
toxins that act within a cell. For example, anthrax, diphtheria,
cholera, and botulinum toxins include a portion that acts in the
cytoplasm, as well as a portion that acts to bind to the cell
surface. These toxins, or portions thereof, can be linked to a
targeting moiety and used to inhibit cancer cell growth. Select
members of the ribonuclease A (RNase A) superfamily are potent
cytotoxins. These cytotoxic ribonucleases enter the cytosol, where
they degrade cellular RNA and cause cell death.
[0069] In some examples ribosome inactivating proteins can be used
as toxins. In these examples the cargo moiety is a polypeptide
having ribosome-inactivating activity including, without
limitation, gelonin, bouganin, saporin, ricin, ricin A chain,
bryodin, restrictocin, and variants thereof. Diphtheria toxin and
Pseudomonas exotoxin A inhibit protein synthesis via
ADP-ribosylation of elongation factor 2. When the cargo moiety is a
ribosome-inactivating protein or inhibits protein synthesis via
ADP-ribosylation of elongation factor 2, the targeted cargo protein
can be internalized upon binding to the cancer cell.
[0070] Cargo moieties that induce apoptosis can also be used to
target cancer cells. Examples of cargo moieties that induce
apoptosis include caspases, granzymes and BCL-2 pro-apoptotic
related proteins such as BAX (e.g., Accession no: CAE52910), BAD
(e.g., Accession no: CAG46757), BAT (e.g., Accession no: AAI07425),
BAK (e.g., Accession no: AAA74466), BIK (e.g., Accession no:
CAG30276), BOK (e.g., Accession no: AAH06203), BID (e.g., Accession
no: CAG28531), BIM (e.g., Accession no: NP.sub.--619527) and BMF
(e.g., Accession no: AAH69328). These cargo moieties can be used
alone or in combination to reduce or inhibit cancer cell
growth.
[0071] Aerolysin is a channel-forming toxin produced as an inactive
protoxin called proaerolysin (PA). Exemplary aerolysin and PA
sequences that can be used in a targeted cargo protein are provided
in Table 2. The PA protein contains many discrete functionalities
that include a binding domain, a toxin domain, and a C-terminal
inhibitory peptide domain that contains a protease activation site.
The binding domain recognizes and binds to
glycophosphatidylinositol (GPI) membrane anchors, such as are found
in Thy-1 on T lymphocytes, the PIGA gene product found in
erythrocyte membranes and Prostate Stem Cell Antigen (PSCA). The
activation or proteolysis site within proaerolysin is a six amino
acid sequence that is recognized as a proteolytic substrate by the
furin family of proteases. PA is activated upon hydrolysis of a
C-terminal inhibitory segment by furin. Activated aerolysin binds
to GPI-anchored proteins in the cell membrane and forms a heptamer
that inserts into the membrane producing well-defined channels of
.about.17 .ANG.. Channel formation leads to rapid cell death.
Wild-type aerolysin is toxic to mammalian cells, including
erythrocytes, for example at 1 nanomolar or less.
[0072] In some examples, a target cargo protein is a PA molecule
with the native furin site replaced with a different cleavage site,
such as prostate-specific protease cleavage site (e.g., a
PSA-specific cleavage site, which permits activation of the variant
PA in the presence of a prostate-specific protease such as PSA,
PMSA, or HK2). In one example, a prostate-specific protease
cleavage site is inserted into the native furin cleavage site of
PA, such that PA is activated in the presence of a
prostate-specific protease, but not furin. In another example, a
variant PA molecule further includes a functionally deleted binding
domain (e.g., about amino acids 1-83 of a native PA protein
sequence). Functional deletions can be made using any method known
in the art, such as deletions, insertions, mutations, or
substitutions. In some examples, targeted cargo proteins include
variant PA molecules in which the native binding domain is
functionally deleted and replaced with a prostate-tissue or other
tissue-specific binding domain. In other examples, variant PA
molecules include a furin cleavage site and a functionally deleted
binding domain which is replaced with a prostate-tissue specific
binding domain. Such variant PA molecules are targeted to prostate
cells via the prostate-tissue specific binding domain, and
activated in the presence of furin.
[0073] Bouganin is a ribosome-binding protein originally isolated
from Bougainvillea speotabilis (see U.S. Pat. No. 6,680,296).
Exemplary modified bouganins are described in WO 2005/090579 and
U.S. Pat. No. 7,339,031. Bouganin damages ribosomes and leads to a
cessation of protein synthesis and cell death. Exemplary bouganin
proteins that can be used in the targeted cargo proteins of the
present disclosure include those in GenBank Accession No. AAL35962,
as well as those native and modified bouganin sequences provided in
U.S. Pat. Nos. 6,680,296; 7,339,031 and PCT publication WO
2005/090579 (bouganin sequences herein incorporated by reference),
as well as sequences having at least 60% sequence identity, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98% or even at least 99% sequence identity to such
sequences.
[0074] BAD, BCL2-associated agonist of cell death, is a regulator
of programmed cell death (apoptosis). BAD positively regulates cell
apoptosis by forming heterodimers with BCL-xL and BCL-2, and
reversing their death repressor activity. Proapoptotic activity of
BAD is regulated through its phosphorylation. Exemplary BAD
proteins that can be used in the targeted cargo proteins of the
present disclosure include those in GenBank Accession Nos.
CAG46757; AAH01901.1; and CAG46733.1, as well as those sequences
provided in U.S. Pat. No. 6,737,511 (sequences herein incorporated
by reference), as well as sequences having at least 60% sequence
identity, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98% or even at least 99% sequence identity
to such sequences, as long as the variant retains or has enhanced
biological activity of the native BAD protein.
[0075] BAX, BCL2-associated X protein, is a regulator of programmed
cell death (apoptosis). This protein forms a heterodimer with BCL2,
and functions as an apoptotic activator. BAX interacts with, and
increases the opening of, the mitochondrial voltage-dependent anion
channel (VDAC), which leads to the loss in membrane potential and
the release of cytochrome c. Exemplary BAX proteins that can be
used in the targeted cargo proteins of the present disclosure
include those provided by GenBank Accession Nos. CAE52909.1;
AA022992.1; EAW52418.1, U.S. Pat. No. 6,645,490 (Bax in the IL2-Bax
construct is a Bax-alpha variant that can be used in the present
disclosure), as well as sequences having at least 60% sequence
identity, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98% or even at least 99% sequence identity
to such sequences, as long as the variant retains or has enhanced
biological activity of the native BAX protein.
[0076] In some examples, the BAX protein of a targeted cargo
protein may be modified such that the C-terminal anchor domain has
been deleted and replaced with a CaaX sequence. CaaX is a peptide
with the sequence Cysteine-a-a-X where "X" is any amino acid and
"a" is an aliphatic amino acid. Because membrane association of BAX
is needed for optimal apoptosis activity, addition of membrane
binding domains such as CaaX can enhance their pro-apoptotic
activities. Proteins with CaaX sequence are farnesylated.
Farnesylated proteins are targeted to membranes (e.g., see Wright
and Philip, J. Lipid Res., 2006, 47(5): 883-91). Potential BAX
variants containing a CaaX sequence may or may not contain the
C-terminal anchor domain.
[0077] Pseudomonas exotoxin (PE) is a toxin secreted by
Pseudomonas. Native PE is cytotoxic for mammalian cells due to its
ability to enter cells by receptor-mediated endocytosis and then,
after a series of intracellular processing steps, translocate to
the cell cytosol and ADP-ribosylate elongation factor 2. This
results in the inhibition of protein synthesis and cell death. PE
has three functional domains: an amino-terminal receptor-binding
domain, a middle translocation domain, and a carboxyl-terminal
ADP-ribosylation domain. Modified PE molecules can include
elimination of domain Ia, as well as deletions in domains II and
III. Exemplary PE proteins that can be used in the targeted cargo
proteins of the present disclosure include those provided in Table
1, as well as sequences having at least 60% sequence identity, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98% or even at least 99% sequence identity to such
sequences as long as the variant retains or has enhanced biological
activity of the native PE protein.
[0078] Thapsigargin is an inhibitor of sarco/endoplasmic reticulum
Ca2+ ATPases. Thapsigargin is classified as a sesquiterpene
lactone, and raises cytosolic calcium concentration by blocking the
ability of the cell to pump calcium into the sarcoplasmic and
endoplasmic reticulum which causes these stores to become depleted.
Store-depletion can secondarily activate plasma membrane calcium
channels, allowing an influx of calcium into the cytosol.
[0079] Ribonuclease A (RNAseA) is an endonuclease that cleaves
single-stranded RNA. RNAse A toxins can be obtained from mammals
and reptiles. Exemplary RNAse A proteins that can be used in the
targeted cargo proteins of the present disclosure include those
provided in Table 2, as well as sequences having at least 60%
sequence identity, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98% or even at least 99% sequence
identity to such sequences, as long as the variant retains or has
enhanced biological activity of the native RNAseA toxin.
[0080] The cargo moiety used can include native sequences (such as
the GenBank Accession Nos. and sequences present in the patents
referenced in Table 2 and listed above), as well as variants
thereof, such as a variant having at least 98%, at least 95%, at
least 90%, at least 80%, at least 70%, or at least 60% sequence
identity with the native cargo moiety, as long as the variant
retains or has enhanced biological activity of the native cargo
moiety (e.g., at least about this amount of sequence identity to
the GenBank Accession Nos. listed in Table 2 and listed above). In
some examples, variant sequences retain substantially the same
amount (or even more) of the native biological function of the
cargo moiety, such as the ability to kill or inhibit the growth of
a target cell. A cargo moiety can also be a fragment of the native
sequence that retains a substantial amount of the native biological
function of the protein.
[0081] The cargo moieties are engineered to target cells by linking
them to targeting moieties. Targeting moieties include agents that
can bind to cell surface molecules or targets.
[0082] Making Targeted Cargo Proteins
[0083] Targeted cargo proteins can be prepared by many routine
methods as known in the art. Targeted cargo proteins, as well as
modifications thereto, can be made, for example, by engineering the
nucleic acid encoding the targeted cargo protein using recombinant
DNA technology or by peptide synthesis. Modifications to the
targeted cargo protein may be made, for example, by modifying the
targeted cargo protein polypeptide itself, using chemical
modifications and/or limited proteolysis. Combinations of these
methods may also be used to prepare the targeted cargo
proteins.
[0084] Methods of cloning and expressing proteins are well-known in
the art, detailed descriptions of techniques and systems for the
expression of recombinant proteins can be found, for example, in
Current Protocols in Protein Science (Coligan, J. E., et al., Wiley
& Sons, New York). Those skilled in the art will understand
that a wide variety of expression systems can be used to provide
the recombinant protein. Accordingly, the targeted cargo proteins
can be produced in a prokaryotic host (e.g., E. coli, A.
salmonicida or B. subtilis) or in a eukaryotic host (e.g.,
Saccharomyces or Pichia; mammalian cells, e.g., COS, NIH 3T3, CHO,
BHK, 293, or HeLa cells; or insect cells). The targeted cargo
proteins can be purified from the host cells by standard techniques
known in the art.
[0085] Sequences for various exemplary targeting moieties and cargo
moieties are provided in the Tables 1 and 2. Variants and homologs
of these sequences can be cloned, if an alternative sequence is
desired, using standard techniques [see, for example, Ausubel et
al., Current Protocols in Molecular Biology, Wiley & Sons, NY
(1997 and updates); Sambrook et al., supra]. For example, the
nucleic acid sequence can be obtained directly from a suitable
organism, such as Aeromonas hydrophila, by extracting mRNA and then
synthesizing cDNA from the mRNA template (for example by RT-PCR) or
by PCR-amplifying the gene from genomic DNA. Alternatively, the
nucleic acid sequence encoding either the targeting moiety or the
cargo moiety can be obtained from an appropriate cDNA library by
standard procedures. The isolated cDNA is then inserted into a
suitable vector, such as a cloning vector or an expression
vector.
[0086] Mutations (if desired) can be introduced at specific,
pre-selected locations by in vitro site-directed mutagenesis
techniques well-known in the art. Mutations can be introduced by
deletion, insertion, substitution, inversion, or a combination
thereof, of one or more of the appropriate nucleotides making up
the coding sequence.
[0087] The expression vector can further include regulatory
elements, such as transcriptional elements, required for efficient
transcription of the targeted cargo protein-encoding sequences.
Examples of regulatory elements that can be incorporated into the
vector include, but are not limited to, promoters, enhancers,
terminators, and polyadenylation signals. Vectors that include a
regulatory element operatively linked to a nucleic acid sequence
encoding a genetically engineered targeted cargo protein can be
used to produce the targeted cargo protein.
[0088] The expression vector may additionally contain heterologous
nucleic acid sequences that facilitate the purification of the
expressed targeted cargo protein, such as affinity tags such (e.g.,
metal-affinity tags, histidine tags, avidin/streptavidin encoding
sequences, glutathione-S-transferase (GST) encoding sequences, and
biotin encoding sequences). In one example, such tags are attached
to the N- or C-terminus of a targeted cargo protein, or can be
located within the targeted cargo protein. The tags can be removed
from the expressed targeted cargo protein prior to use according to
methods known in the art. Alternatively, the tags can be retained
on the targeted cargo protein, providing that they do not interfere
with the ability of the targeted cargo protein to target and kill
(or decrease growth of) cancer cells.
[0089] As an alternative to a directed approach to introducing
mutations into naturally occurring pore-forming proteins, a cloned
gene expressing a pore-forming protein can be subjected to random
mutagenesis by techniques known in the art. Subsequent expression
and screening of the mutant forms of the protein thus generated
would allow the identification and isolation of targeted cargo
moieties.
[0090] The targeted cargo proteins can also be prepared as
fragments or fusion proteins. A fusion protein is one which
includes a targeted cargo protein linked to other amino acid
sequences that do not inhibit the ability of the targeted cargo
protein to selectively target and inhibit cancer cell growth or
kill cancer cells. In an alternative example, the other amino acid
sequences are short sequences of, for example, up to about 5, about
6, about 7, about 8, about 9, about 10, about 20, about 30, about
50 or about 100 amino acid residues in length. These short
sequences can be linker sequences as described above.
[0091] Methods for making fusion proteins are well known to those
skilled in the art. For example U.S. Pat. No. 6,057,133 discloses
methods for making fusion molecules composed of human interleukin-3
(hIL-3) variant or mutant proteins functionally joined to a second
colony stimulating factor, cytokine, lymphokine, interleukin,
hematopoietic growth factor or IL-3 variant. U.S. Pat. No.
6,072,041 to Davis et al. discloses the generation of fusion
proteins comprising a single chain Fv molecule directed against a
transcytotic receptor covalently linked to a therapeutic
protein.
[0092] The targeted cargo protein can include one or more linkers,
as well as other moieties, as desired. These can include a binding
region, such as avidin or an epitope, or a tag such as a
polyhistidine tag, which can be useful for purification and
processing of the fusion protein. In addition, detectable markers
can be attached to the fusion protein, so that the traffic of the
fusion protein through a body or cell can be monitored
conveniently. Such markers include radionuclides, enzymes,
fluorophores, chromophores, and the like.
[0093] One of ordinary skill in the art will appreciate that the
DNA can be altered in numerous ways without affecting the
biological activity of the encoded protein. For example, PCR can be
used to produce variations in the DNA sequence which encodes a
targeted cargo protein. Such variations in the DNA sequence
encoding a targeted cargo protein can be used to optimize for codon
preference in a host cell used to express the protein, or may
contain other sequence changes that facilitate expression.
[0094] A covalent linkage of a targeting moiety directly to a cargo
moiety or via a linker may take various forms as is known in the
art. For example, the covalent linkage may be in the form of a
disulfide bond. The DNA encoding one of the components can be
engineered to contain a unique cysteine codon. The second component
can be derivatized with a sulfhydryl group reactive with the
cysteine of the first component. Alternatively, a sulfhydryl group,
either by itself or as part of a cysteine residue, can be
introduced using solid phase polypeptide techniques. For example,
the introduction of sulfhydryl groups into peptides is described by
Hiskey (Peptides 3:137, 1981).
[0095] Proteins also can be chemically modified by standard
techniques to add a sulfhydryl group. For example, Traut's reagent
(2-iminothiolane-HCl) (Pierce Chemicals, Rockford, Ill.) can be
used to introduce a sulfhydryl group on primary amines, such as
lysine residues or N-terminal amines. A protein or peptide modified
with Traut's reagent can then react with a protein or peptide which
has been modified with reagents such as N-succinimidyl
3-(2-pyridyldithio) propionate (SPDP) or succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Pierce
Chemicals, Rockford, Ill.).
[0096] The components can also be joined using the polymer,
monomethoxy-polyethylene glycol (mPEG), as described in Maiti et
al., Int. J. Cancer Suppl., 3:17-22, 1988.
[0097] The targeting moiety and the cargo moiety can also be
conjugated through the use of standard conjugation chemistries as
is known in the art, such as carbodiimide-mediated coupling (for
example, DCC, EDC or activated EDC), and the use of 2-iminothiolane
to convert epsilon amino groups to thiols for crosslinking and
m-maleimidobenzoyl-n-hydroxysuccinimidyl ester (MBS) as a
crosslinking agent.
[0098] Linking of a cargo moiety to a targeting moiety may be
direct meaning that one portion of the cargo moiety is directly
attached to a portion of the targeting moiety. For example, one end
of the amino acid sequence of a cargo protein can be directly
attached to an end of the amino acid sequence of the targeting
moiety. For example, the C-terminus of the cargo protein can be
linked to the N-terminus of the targeting moiety, or the C-terminus
of the targeting moiety can be linked to the N-terminus of the
cargo protein. Methods of generating such fusion proteins are
routine in the art, for example using recombinant molecular biology
methods.
[0099] In another example, the cargo moiety is linked to the
targeting moiety indirectly through a linker. The linker can serve,
for example, simply as a convenient way to link the two entities,
as a means to spatially separate the two entities, to provide an
additional functionality to the targeted cargo protein, or a
combination thereof.
[0100] In general, the linker joining the targeting moiety and the
cargo moiety can be designed to (1) allow the two molecules to fold
and act independently of each other, (2) not have a propensity for
developing an ordered secondary structure which could interfere
with the functional domains of the two moieties, (3) have minimal
hydrophobic or charged characteristic which could interact with the
functional protein domains and/or (4) provide steric separation of
the two regions. For example in some instances it may be desirable
to spatially separate the targeting moiety and the cargo moiety to
prevent the targeting moiety from interfering with the inhibitory
activity of the targeted cargo moiety and/or the cargo moiety
interfering with the targeting activity of the targeting moiety.
The linker can also be used to provide, for example, lability to
the connection between the targeting moiety and the cargo moiety,
an enzyme cleavage site (for example a cleavage site for a
protease), a stability sequence, a molecular tag, a detectable
label, or various combinations thereof.
[0101] The linker can be bifunctional or polyfunctional, e.g.
contains at least about a first reactive functionality at, or
proximal to, a first end of the linker that is capable of bonding
to, or being modified to bond to, the targeting moiety and a second
reactive functionality at, or proximal to, the opposite end of the
linker that is capable of bonding to, or being modified to bond to,
the cargo moiety being modified. The two or more reactive
functionalities can be the same (i.e. the linker is
homobifunctional) or they can be different (i.e. the linker is
heterobifunctional). A variety of bifunctional or polyfunctional
cross-linking agents are known in the art that are suitable for use
as linkers (for example, those commercially available from Pierce
Chemical Co., Rockford, Ill.), such as avidin and biotin.
Alternatively, these reagents can be used to add the linker to the
targeting moiety and/or cargo moiety.
[0102] The length and composition of the linker can be varied
considerably provided that it can fulfill its purpose as a
molecular bridge. The length and composition of the linker are
generally selected taking into consideration the intended function
of the linker, and optionally other factors such as ease of
synthesis, stability, resistance to certain chemical and/or
temperature parameters, and biocompatibility. For example, the
linker should not significantly interfere with the ability of the
targeting moiety to target the targeted cargo protein to a cancer
cell, or with the activity of the targeted cargo protein relating
to activation, pore-forming ability, or toxin activity.
[0103] Linkers suitable for use may be branched, unbranched,
saturated, or unsaturated hydrocarbon chains, as well as peptides
as noted above. Furthermore, if the linker is a peptide, the linker
can be attached to the targeting moiety and/or the cargo moiety
using recombinant DNA technology. Such methods are well-known in
the art and details of this technology can be found, for example,
in Sambrook et al., supra.
[0104] In one example, the linker is a branched or unbranched,
saturated or unsaturated, hydrocarbon chain having from 1 to 100
carbon atoms, wherein one or more of the carbon atoms is optionally
replaced by --O-- or --NR-- (wherein R is H, or C1 to C6 alkyl),
and wherein the chain is optionally substituted on carbon with one
or more substituents selected from the group of (C1-C6) alkoxy,
(C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6)
alkoxycarbonyl, (C1-C6) alkylthio, amide, azido, cyano, nitro,
halo, hydroxy, oxo (.dbd.O), carboxy, aryl, aryloxy, heteroaryl,
and heteroaryloxy.
[0105] Examples of suitable linkers include, but are not limited
to, peptides having a chain length of 1 to 500 amino acid residues
(such as 1 to 100, 1 to 50, 6 to 30, such as less than 30 amino
acids). Typically surface amino acids in flexible protein regions
include Gly, Asn and Ser. Other neutral amino acids, such as Thr
and Ala, can also be used in the linker sequence. Additional amino
acids can be included in the linker to provide unique restriction
sites in the linker sequence to facilitate construction of the
fusions. Other exemplary linkers include those derived from groups
such as ethanolamine, ethylene glycol, polyethylene with a chain
length of 6 to 100 carbon atoms, polyethylene glycol with 3 to 30
repeating units, phenoxyethanol, propanolamide, butylene glycol,
butyleneglycolamide, propyl phenyl, and ethyl, propyl, hexyl,
steryl, cetyl, and palmitoyl alkyl chains.
[0106] In one example, the linker is a branched or unbranched,
saturated or unsaturated, hydrocarbon chain, having from 1 to 50
carbon atoms, wherein one or more of the carbon atoms is optionally
replaced by --O-- or --NR-- (wherein R is as defined above), and
wherein the chain is optionally substituted on carbon with one or
more substituents selected from the group of (C1-C6) alkoxy,
(C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl,
(C1-C6) alkylthio, amide, hydroxy, oxo (.dbd.O), carboxy, aryl and
aryloxy.
[0107] In a specific example, the linker is a peptide having a
chain length of 1 to 50 amino acid residues, such as 1 to 40, 1 to
20, or 5 to 10 amino acid residues.
[0108] Peptide linkers that are susceptible to cleavage by enzymes
of the complement system, urokinase, tissue plasminogen activator,
trypsin, plasmin, or another enzyme having proteolytic activity may
be used in one example. According to another example, the targeted
cargo protein includes a targeting moiety attached via a linker
susceptible to cleavage by enzymes having a proteolytic activity
such as a urokinase, a tissue plasminogen activator, plasmin,
thrombin or trypsin. In addition, targeting moieties may be
attached to the cargo moiety via disulfide bonds (for example, the
disulfide bonds on a cysteine molecule). Since many tumors
naturally release high levels of glutathione (a reducing agent)
this can reduce the disulfide bonds with subsequent release of the
cargo moiety at the site of delivery.
[0109] In one example, the targeted cargo protein includes a
targeting moiety linked by a cleavable linker region. In another
example, the cleavable linker region is a protease-cleavable
linker, although other linkers, cleavable for example by small
molecules, may be used. Examples of protease cleavage sites are
those cleaved by factor Xa, thrombin and collagenase. In one
example, the protease cleavage site is one that is cleaved by a
protease that is associated with a disease. In another example, the
protease cleavage site is one that is cleaved by a protease that is
up-regulated or associated with cancers in general. Examples of
such proteases are uPA, the matrix metalloproteinase (MMP) family,
the caspases, elastase, prostate specific antigen (PSA, a serine
protease), and the plasminogen activator family, as well as
fibroblast activation protein. In still another example, the
cleavage site is cleaved by a protease secreted by
cancer-associated cells. Examples of these proteases include
matrixmetalloproteases, elastase, plasmin, thrombin, and uPA. In
another example, the protease cleavage site is one that is
up-regulated or associated with a specific cancer. The precise
sequences are available in the art and the skilled person will have
no difficulty in selecting a suitable cleavage site. By way of
example, the protease cleavage region targeted by Factor Xa is I E
G R. The protease cleavage region targeted by enterokinase is D D D
D K. The protease cleavage region targeted by thrombin is L V P R
G. In one example, the cleavable linker region is one which is
targeted by endocellular proteases.
[0110] As known in the art, the attachment of a linker to cargo
moiety (or of a linker element to a cleavable element, or a
cleavable element to another cargo moiety) need not be a particular
mode of attachment or reaction.
[0111] Testing Targeted Cargo Proteins
[0112] Targeted cargo proteins can be tested using standard
techniques known in the art. Exemplary methods of testing candidate
targeted cargo proteins are provided below and in the examples
included herein. One of ordinary skill in the art will understand
that other methods of testing the targeted cargo proteins are known
in the art and are also suitable for testing candidate targeted
cargo proteins. For example, methods known in the art for testing
for anti-tumor activity can be used. The targeted cargo proteins
can initially be screened against a panel of tumor cell lines. A
cell proliferation assay, such as the WST-1 kit sold by Roche, can
be used. Potency can be evaluated using different drug
concentrations in the presence or absence of active agents that
inhibit cancer cells. Selected drug candidates from the initial
tumor cell screen can be further characterized through additional
in vitro assays and in relevant xenograft models to examine
anti-tumor activity, such as those described in the Examples
herein.
[0113] Use of Targeted Cargo Proteins in Combination with Active
Agents
[0114] Targeted cargo proteins of the invention may be administered
by parenteral means, including subcutaneous, intravenous or
intramuscular injection, or by injection into a body cavity.
Parenteral administration by intravenous injection or infusion is
preferred. Alternatively, targeted cargo proteins may be
administered by direct injection or infusion into a tumor, for
example a brain tumor or a prostate tumor. Alternative methods of
administration of the targeted cargo proteins will be evident to
one of ordinary skill in the art. Such methods may include for
example, the use of catheters or implantable pumps to provide
continuous infusion over a period of several hours to several days
into the subject in need of treatment. It is anticipated that
active agents will be administered by the routes that are currently
in use for their administration in clinical settings.
[0115] The dosages of targeted cargo proteins to be administered to
a subject are not subject to absolute limits, but will depend on
the nature of the targeted cargo protein and its unwanted side
effects, the subject being treated and the type of condition being
treated and the manner of administration. Generally the dose will
be a therapeutically effective amount. (A therapeutically effective
amount of a targeted cargo protein can be determined in various
ways, such as assaying for improvement of the condition of a
subject having cancer by monitoring the size of a tumor in a
subject, the partial or complete alleviation of symptoms, halting
the growth of a tumor, or decreasing the size of a tumor. Effective
amounts may also be determined through various in vitro or in vivo
assays similar to the ones described in the Examples provided
herein.)
[0116] The therapeutically effective dose will also depend on
whether administration is parenteral or local. For parenteral
administration of targeted cargo proteins, exemplary dosages for
administration to a subject for a single treatment may range from
10 ng to 10 mg per square meter (m.sup.2) of body surface area,
from 1 .mu.g to 1 mg per m.sup.2 of body surface area, and from 10
.mu.g to 100 .mu.g per square meter (m.sup.2) of body surface area.
For localized treatment (such as injection or infusion into a brain
tumor) a single treatment may comprise a dosage of targeted cargo
protein ranging from 10 ng to 10 mg, from 10 .mu.g to about 1 mg,
or from 25 .mu.g to 0.5 mg.
[0117] Treatments with targeted cargo proteins may be completed in
a single day, or may be done repeatedly on multiple days with the
same or a different dosage. Repeated treatments may be done on the
same day, on successive days, or every 1-3 days, every 3-7 days,
every 1-2 weeks, every 2-4 weeks, every 1-2 months, or at even
longer intervals.
[0118] Dosages of the active agents are determined in accordance
with current clinical protocols for the active agent being
used.
[0119] It is anticipated that the therapeutic dosages of either the
targeted cargo proteins or the active agents when used in
combination may be reduced from what would otherwise be determined
to be the optimal level for each agent administered alone due to
the synergy between the targeted cargo protein and the active
agent.
[0120] The active agents may be administered concurrently with the
targeted cargo proteins, or within hours or days. In some
embodiments, the active agent is administered within 24, 48, 72 or
96 hours of administration of the targeted cargo protein. The
active agents may be administered more or less frequently than the
targeted cargo proteins. For example, when repeat treatments with a
targeted cargo protein are given, some treatments, but not others,
may be done in conjunction with an active agent. Treatment with the
active agent may be done more of less frequently than treatment
with the targeted cargo protein.
[0121] Pharmaceutical Compositions
[0122] Pharmaceutical compositions can include one or more targeted
cargo proteins and/or one or more active agents, and one or more
non-toxic pharmaceutically acceptable carriers, diluents,
excipients and/or adjuvants. If desired, other active ingredients
may be included in the compositions. As indicated above, such
compositions are suitable for use in the treatment of cancer. The
term "pharmaceutically acceptable carrier" refers to a carrier
medium which does not interfere with the effectiveness of the
biological activity of the active ingredients and which is not
toxic to the host or patient. Representative examples are provided
below.
[0123] The pharmaceutical compositions may comprise, for example,
from about 1% to about 95% of a targeted cargo protein.
Compositions formulated for administration in a single dose form
may comprise, for example, about 20% to about 90% of the targeted
cargo proteins, whereas compositions that are not in a single dose
form may comprise, for example, from about 5% to about 20% of the
targeted cargo proteins. Concentration of the targeted cargo
protein in the final formulation can be at least 1 ng/mL, such as
at least 1 .mu.g/mL or at least 1 mg/mL. For example, the
concentration in the final formulation can be between about 0.01
.mu.g/mL and about 1,000 .mu.g/mL. In one example, the
concentration in the final formulation is between about 0.01 mg/mL
and about 100 mg/mL.
[0124] The targeted cargo proteins can be delivered along with a
pharmaceutically acceptable vehicle. In one example, the vehicle
may enhance the stability and/or delivery properties. Thus, the
disclosure also provides for formulation of the targeted cargo
protein with a suitable vehicle, such as an artificial membrane
vesicle (including a liposome, noisome, nanosome and the like),
microparticle or microcapsule, or as a colloidal formulation that
comprises a pharmaceutically acceptable polymer. The use of such
vehicles/polymers may be beneficial in achieving sustained release
of the targeted cargo proteins. Alternatively, or in addition, the
targeted cargo protein formulations can include additives to
stabilize the protein in vivo, such as human serum albumin, or
other stabilizers for protein therapeutics known in the art.
Targeted cargo protein formulations can also include one or more
viscosity enhancing agents which act to prevent backflow of the
formulation when it is administered, for example by injection or
via catheter. Such viscosity enhancing agents include, but are not
limited to, biocompatible glycols and sucrose.
[0125] Pharmaceutical compositions formulated as aqueous
suspensions contain the active compound(s) in admixture with one or
more suitable excipients, for example, with suspending agents, such
as sodium carboxymethylcellulose, methyl cellulose,
hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone,
hydroxypropyl-.mu.-cyclodextrin, gum tragacanth and gum acacia;
dispersing or wetting agents such as a naturally-occurring
phosphatide, for example, lecithin, or condensation products of an
alkylene oxide with fatty acids, for example, polyoxyethyene
stearate, or condensation products of ethylene oxide with long
chain aliphatic alcohols, for example,
hepta-decaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
for example, polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example, polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxy-benzoate,
or one or more coloring agents.
[0126] Compositions can be preserved by the addition of an
anti-oxidant such as ascorbic acid.
[0127] The pharmaceutical compositions can be formulated as a
dispersible powder or granules, which can subsequently be used to
prepare an aqueous suspension by the addition of water. Such
dispersible powders or granules provide the active ingredient in
admixture with one or more dispersing or wetting agents, suspending
agents and/or preservatives. Suitable dispersing or wetting agents
and suspending agents are exemplified by those already mentioned
above.
[0128] Pharmaceutical compositions can also be formulated as
oil-in-water emulsions. The oil phase can be a vegetable oil, for
example, olive oil or arachis oil, or a mineral oil, for example,
liquid paraffin, or it may be a mixture of these oils. Suitable
emulsifying agents for inclusion in these compositions include
naturally-occurring gums, for example, gum acacia or gum
tragacanth; naturally-occurring phosphatides, for example, soy
bean, lecithin; or esters or partial esters derived from fatty
acids and hexitol, anhydrides, for example, sorbitan monoleate, and
condensation products of the said partial esters with ethylene
oxide, for example, polyoxyethylene sorbitan monoleate.
[0129] The pharmaceutical compositions containing one or more
targeted cargo proteins and/or one or more active agents can be
formulated as a sterile injectable aqueous or oleaginous suspension
according to methods known in the art and using suitable one or
more dispersing or wetting agents and/or suspending agents, such as
those mentioned above. The sterile injectable preparation can be a
sterile injectable solution or suspension in a non-toxic parentally
acceptable diluent or solvent, for example, as a solution in
1,3-butanediol. Acceptable vehicles and solvents that can be
employed include, but are not limited to, water, Ringer's solution,
lactated Ringer's solution and isotonic sodium chloride solution.
Other examples include, sterile, fixed oils, which are
conventionally employed as a solvent or suspending medium, and a
variety of bland fixed oils including, for example, synthetic mono-
or diglycerides. Fatty acids such as oleic acid can also be used in
the preparation of injectables.
[0130] In one example, the targeted cargo protein is conjugated to
a water-soluble polymer, e.g., to increase stability or circulating
half life or reduce immunogenicity. Clinically acceptable,
water-soluble polymers include, but are not limited to,
polyethylene glycol (PEG), polyethylene glycol propionaldehyde,
carboxymethylcellulose, dextran, polyvinyl alcohol (PVA),
polyvinylpyrrolidone (PVP), polypropylene glycol homopolymers
(PPG), polyoxyethylated polyols (POG) (e.g., glycerol) and other
polyoxyethylated polyols, polyoxyethylated sorbitol, or
polyoxyethylated glucose, and other carbohydrate polymers. Methods
for conjugating polypeptides to water-soluble polymers such as PEG
are described, e.g., in U.S. patent Pub. No. 20050106148 and
references cited therein. In one example the polymer is a
pH-sensitive polymers designed to enhance the release of drugs from
the acidic endosomal compartment to the cytoplasm (see for example,
Henry et al., Biomacromolecules 7(8):2407-14, 2006).
[0131] Active agents may be included in a pharmaceutical
formulation together with target cargo proteins for
co-administration, or may be formulated separately. They may be
formulated in conventional pharmaceutically acceptable carriers.
(vehicles) such as those found in Remington's Pharmaceutical
Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th
Edition (1975), describes compositions and formulations suitable
for pharmaceutical delivery of active agents or the targeted cargo
protein molecules provided herein.
[0132] Diseases or Conditions that May be Treated by the Methods of
the Disclosure
[0133] Diseases or conditions that may be treated using the methods
of the disclosure are characterized by cells that uniquely express,
or over-express, at least one target molecule that specifically
binds to a targeted cargo protein. Such diseases or conditions may
include various inflammatory conditions as well as benign tumors or
malignant tumors (cancer). Tumors can be solid or hematological.
Examples of hematological tumors include, but are not limited to:
leukemias, including acute leukemias (such as acute lymphocytic
leukemia, acute myelocytic leukemia, acute myelogenous leukemia and
myeloblastic, promyelocytic, myelomonocytic, monocytic and
erythroleukemia), chronic leukemias (such as chronic myelogenous
leukemia, and chronic lymphocytic leukemia), myelodysplastic
syndrome, and myelodysplasia, polycythemia vera, lymphoma, (such as
Hodgkin's disease, all forms of non-Hodgkin's lymphoma), multiple
myeloma, Waldenstrom's macroglobulinemia, and heavy chain
disease.
[0134] Examples of solid tumors, such as sarcomas and carcinomas,
include, but are not limited to: fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, and other
sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, lung cancer, ovarian cancer, prostate cancer, benign
prostatic hyperplasia, hepatocellular carcinoma, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, medullary carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, Wilms' tumor, epithelial tumors (e.g., cervical
cancer, gastric cancer, skin cancer, head and neck tumors),
testicular tumor, bladder carcinoma, melanoma, brain tumors, and
CNS tumors (such as a glioma, astrocytoma, medulloblastoma,
craniopharyogioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, menangioma, meningioma,
neuroblastoma and retinoblastoma).
[0135] Preferred diseases and conditions that may be treated by the
methods of the invention include brain cancer, including malignant
astrocytoma and gliobastoma multiforme, Kaposi sarcoma, bladder
cancer, renal cell cancer, breast cancer, pancreatic cancer,
non-small cell lung cancer, thyroid cancer, squamous cell carcinoma
of the head and neck, colon cancer and other cancers of the
gastrointestinal system, mesothelioma and prostate cancer.
EXAMPLES
Example 1
[0136] We have observed that 42 of 70 (60%) tumor samples from
patients with PDA express moderate- to high-density surface IL-4
receptor (IL-4R), whereas normal pancreatic samples express no or
low-density IL-4R. PRX321 was specifically and highly cytotoxic
[50% protein synthesis inhibition (IC50) ranging from >0.1 to 13
ng/mL] to six of eight pancreatic cancer cell lines, whereas no
cytotoxicity (IC50 >1,000 ng/mL) was observed in normal human
pancreatic duct epithelium cells, fibroblasts, and human umbilical
vein endothelial cells (HUVEC). We also showed that PRX321 in
combination with gemcitabine exhibited synergistic antitumor
activity in vitro. To confirm synergistic antitumor activity in
vivo and monitor precise real-time disease progression, we used a
novel metastatic and orthotopic mouse model using green fluorescent
protein-transfected cancer cells and whole-body imaging system. The
combination of both agents caused complete eradication of tumors in
40% of nude mice with small established PDA tumors. In addition,
combined treatment significantly prolonged the survival of nude
mice bearing day 14 advanced distant metastatic PDA tumors. Similar
results were observed in mice xenografted with PDA obtained from a
patient undergoing surgical resection. These results indicate that
PRX321 combined with gemcitabine may provide effective therapy for
the treatment of patients with PDA.
Example 2
[0137] In this study, we examined expression of IL-4R in samples
derived from PDA and the efficacy of PRX321, gemcitabine, and
combination of both in primary and metastatic tumor models. To
imitate aggressive clinical situation and to monitor precise
real-time disease progression, we used a novel metastatic and
orthotopic advanced pancreatic cancer model using retroviral green
fluorescent protein (GFP)--transfected pancreatic cancer cell line
and whole-body imaging system (27). Together, our study shows that
PRX321 synergizes with gemcitabine, significantly inhibiting the
growth of primary and metastatic tumor lesions, prolonging the
survival time, and completely eradicating tumors in 40% of mice in
an early pancreatic cancer model.
Example 3
Materials and Methods
[0138] Cell culture, reagents, and tissue specimens. Cell lines
were obtained from the American Type Culture Collection and
Sciencell. Human pancreatic duct epithelium (HPDE) cells were
cultured routinely in keratinocyte serum-free medium supplemented
with bovine pituitary extract and epidermal growth factor (Life
Technologies; ref. 28). PRX321 [IL4(38-37)-PE38KDEL] was produced
as described previously (23). Fifteen paraffin-embedded tissue
sections and tissue arrays containing 70 tumor specimens were
obtained from Cooperative Human Tissue Network and U.S. Biomax,
respectively. Gemcitabine was procured through the pharmacy of the
clinical center (NIH).
[0139] Immunohistochemistry and flow cytometry.
Immunohistochemistry was done as described previously (24).
Deparafinized tissue sections were incubated with anti-human IL-4Ra
polyclonal antibody (Santa Cruz Biotechnology) or isotype control
(IgG). The results were scored on the basis of the density of
staining 0%, 0% to 10%, 11% to 50%, 51% to 100% as negative, weak,
moderate, and strong, respectively. Tissue sections for IL-4R were
evaluated by Dr. Satoru Takahashi who is a pathologist at Nagoya
City University in Japan.
[0140] Expression of IL-4Ra on pancreatic cancer cell lines and
HPDE cells was assessed by flow cytometry using
phycoerythrin-conjugated anti-IL-4Ra monoclonal antibody as
previously described (29). Staining with isotype Matched IgG served
as control. Protein synthesis inhibition assay and assessment of
synergism or antagonism. The in vitro cytotoxic activity of PRX321,
gemcitabine, and their combination was measured by the inhibition
of protein synthesis (18). Drug interaction between PRX321 and
gemcitabine was assessed at a concentration ratio of 1:1, using the
combination index (CI), where CI<1, CI=1, and CI>1 indicate
synergistic, additive, and antagonistic effects, respectively (30).
On the basis of the isobologram analysis for mutually exclusive
effects, the CI value was calculated as follows:
CI = ( D ) 1 ( D x ) 1 + ( D ) 2 ( D x ) 2 ##EQU00001##
[0141] where (Dx)1 and (Dx)2 are the concentrations of PRX321 and
gemcitabine, respectively, required to inhibit cell growth by 50%,
and (D)1 and (D)2 are the drug concentrations in combination
treatments that also inhibit cell growth by 50% (isoeffective
compared with the single drugs). Semiquantitative and real-time
TaqMan reverse transcription-PCR. Semiquantitative reverse
transcription-PCR (RT-PCR) was done as described previously (31).
Quantification of IL-4Ra mRNA expression levels in pancreatic
cancer cell lines was determined by real-time RT-PCR using a set of
IL-4Ra-specific TaqMan probe (5-FAM, 3-MGB) and primers (Applied
Biosystems; ref. 24). Gene expression was normalized to
glyceraldehyde-3-phosphate dehydrogenase or h-actin before the fold
change in gene expression was calculated.
[0142] Retroviral transduction and selection of high-GFP-expressing
MIAPaCa-2 pancreatic cancer cells. MIA-PaCa-2 cells expressing GFP
were established using a 1:1 precipitated mixture of retroviral
supernatants of the HEK293 cells and RPMI 1640 (Life Technologies,
Inc.), as described previously (32).
[0143] Animals. Severe combined immunodeficient (SCID) mice and
nude nu/nu mice between age 5 and 6 weeks were maintained in a
barrier facility on HEPA-filtered racks. All animal studies were
conducted under an approved protocol in accordance with the
principles and procedures outlined in the NIH Guideline for the
Care and Use of Laboratory Animals.
[0144] Whole-body imaging. The tumor-bearing mice were periodically
examined in a fluorescence light box illuminated by fiberoptic
light at 440/20 nm wavelength (Lightools Research, Inc.). Emitted
fluorescence was collected through a long-pass filter GG475 (Chroma
Technology) on a Hamamatsu C5810 3-chip cooled color charge coupled
device camera (Hamamatsu Photonics Systems). Real-time
determination of tumor burden was done by quantifying fluorescent
surface area as described previously (32).
[0145] Surgical orthotopic implantation of MIA-PaCa-2-GFP tumors.
MIA-PaCa-2-GFP cells were injected s.c. into the right dorsal flank
of nude mice. Pancreatic tumors, grown s.c. in nude mice, were cut
with scissors and minced into f3% 3% 3-mm pieces. For orthotopic
surgery, the pancreas was carefully exposed, and tumor chunks were
transplanted on the middle of the pancreas with a 6-0 Dexon
surgical suture (Davis-Geck, Inc.). The pancreas was then returned
to the peritoneal cavity, the abdominal wall, and the skin was
closed with 6-0 Dexon sutures.
[0146] Experimental design and treatment. For early pancreatic
cancer model, primary tumor lesions were detected by external
whole-body imaging on day 4 after transplantation. Once the tumors
were visualized, mice were randomized into four groups of 10 each.
Treatment was initiated on day 5. For advanced pancreatic cancer
model, primary and metastasis tumor lesions were detected by
external whole-body imaging on day 14 post-transplantation of tumor
chunk and randomized into four groups of 10 mice each. Treatment
was initiated on day 15.
[0147] Primary and orthotopic pancreatic cancer model using a
clinical sample. Primary pancreatic cancer specimens were obtained
from a patient undergoing radical pancreatectomy at National Cancer
Institute under institutional review board-approved protocol.
Viable tumor tissue from specimen was cut into small pieces (3% 3%
3 mm) and implanted in the pancreas of 5- to 6-week-old male SCID
mice. Primary xenografts were propagated continuously in SCID mice
for in vivo testing. Clinical sample-bearing mice were also treated
after day 31 by the same protocol as described above.
[0148] Statistical analysis. The mean tumor volume in therapeutic
and control groups was analyzed by ANOVA. Survival curves were
generated by Kaplan-Meier method and compared by using the log-rank
test.
Results
Expression of IL-4R in PDA Tissues.
[0149] Tissue sections from 15 normal pancreas and 70 PDA specimens
were analyzed by immunohistochemical analysis for the expression of
IL-4Ra (data not shown). Tumor specimens showed weak to strong
staining for IL-4Ra in PDAs. Only weak staining was observed in
tumor-infiltrating stromal fibroblasts and endothelial cells. When
the proportion of IL-4Ra-positive cancer cells was counted, 23 of
70 primary tumors classified into strong expression group, 19 into
moderate expression group, 11 into weak expression group, and 17
into the negative expression group. Thus, 42 of 70 (60%) PDA
samples expressed moderate to high density IL-4Ra. In contrast,
only 2 of 15 normal pancreas samples showed weak staining for
IL-4Ra in normal acinar and ductal cells.
Pancreatic Cancer Cell Lines Expressing IL-4R are Sensitive to
PRX321.
[0150] We examined the expression of IL-4Ra mRNA by RT-PCR and
real-time RT-PCR in eight pancreatic cancer and one normal HPDE
cell lines. Six of eight cancer cell lines showed varied density of
IL-4Ra mRNA expression, whereas HPAF-II, PK-1, and HPDE cell lines
showed no expression (data not shown). Real-time RT-PCR analysis
confirmed conventional RT-PCR results and showed that MIA-PaCa-2
and SW1990 cell lines expresses highest level of IL-4Ra mRNA,
followed by Capan-1, ASPC-1, Panc-1, and HS766T cell lines. Flow
cytometric analysis confirmed mRNA expression data and showed that
IL-4Ra is expressed on the cell surface of three pancreatic cancer
cell lines but not in normal HPDE cells.
[0151] Next, we determined the sensitivity of pancreatic cancer
cell lines to PRX321 by protein synthesis inhibition assay, which
has been shown to be directly proportional to cell death (19).
PRX321 inhibited protein synthesis of pancreatic cancer cell lines
in a concentration-dependent manner. MIA-PaCa-2 and SW1990 cell
lines were extremely sensitive to the cytotoxin (IC50 0.08 and 0.36
ng/mL, respectively), followed by Capan-1 (IC50 7 ng/mL) and HS766T
(IC50 13 ng/mL). IC50 in Panc-1 and ASPC-1 cell lines was <10
ng/mL. Consistent with the lack of IL-4Ra mRNA expression, HPAF-II
and PK-1 cell lines were not sensitive to PRX321 (IC50 z 1,000
ng/mL; data not shown).
[0152] The cytotoxic activity of PRX321 was neutralized by
incubation with an excess of IL-4, suggesting specific cytotoxicity
through binding of PRX321 to IL-4R (data not shown). We also
examined the cytotoxicity of PRX321 in fibroblast, HUVEC, and HPDE
cell lines, because some of the specimens revealed weak expression
of IL-4Ra in nontumor cells. However, PRX321 was not found to be
cytotoxic to these cells (IC50 z 1,000 ng/mL). The PRX321 cytotoxic
activity correlated with extent of IL-4Ra expression. For example,
MIAPaCa-2 cells showed lowest IC50 and highest density IL-4R
expression as determined by flow cytometric and real-time PCR
analyses whereas PK-1 cell line showed highest IC50 as this cell
line showed undetectable level of mRNA expression. We also used
another cytotoxin IL-13 Pseudomonas exotoxin, an IL-13 receptor
specific fusion protein (12), to assess the cytotoxicity to
pancreatic cancer cell line. However, IL-13 cytotoxin was not
cytotoxic to HPAF-II cells (IC50 z1,000 ng/mL).
Synergistic Cytotoxicity of PRX321 and Gemcitabine in Pancreatic
Cancer Cell Lines.
[0153] Gemcitabine alone mediated a dose-dependent inhibition of
protein synthesis with IC50 of 22 nmol/L in MIA-PaCa-2 cells, 3.2
nmol/L in Capan-1 cells, 1,000 nmol/L in SW1990 cells, and 14
nmol/L in HS766T cells (Table 3). When it was combined with PRX321,
the protein synthesis inhibition in MIA-PaCa-2 cells was greatly
enhanced: IC50 of PRX321 became 0.012, 0.001, and 0.00004 ng/mL by
adding 0.03, 0.3, and 3 nmol/L gemcitabine, respectively. These
same phenomena were also observed in SW1990 and Capan-1 cells, but
not in HS766T cells. The combination index at IC50 and IC75
(concentration of drug causing 75% inhibition of protein synthesis)
in MIA-PaCa-2, SW1990, and Capan-1 cells was <1 at all
concentrations of gemcitabine (Table 3).
TABLE-US-00003 TABLE 3 Cytotoxicity of IL-4 cytotoxin (PRX321),
gemcitabine, and their combination in pancreatic cancer cell lines
Cancer cell line Drug IC50* IC75** MIA-PaCa-2 IL-4 cytotoxin 0.065
ng/mL 0.32 ng/mL Gemcitabine 22 nmol/L 280 nmol/L Capan-1 IL-4
cytotoxin 3.5 ng/mL 22 ng/mL Gemcitabine 3.2 nmol/L 9 nmol/L SW1990
IL-4 cytotoxin 0.36 ng/mL 1 ng/mL Gemcitabine 1,000 nmol/L 3,000
nmol/L CI## IC50 IC75 MIA-PaCa-2 IL-4 cytotoxin + gemcitabine 0.03
nmol/L 0.153 0.563 IL-4 cytotoxin + gemcitabine 0.3 nmol/L 0.0336
0.094 IL-4 cytotoxin + gemcitabine 3 nmol/L 0.137 0.0138 Capan-1
IL-4 cytotoxin + gemcitabine 0.287 0.34 0.003 nmol/L IL-4 cytotoxin
+ gemcitabine 0.03 nmol/L 0.026 0.0713 IL-4 cytotoxin + gemcitabine
0.3 nmol/L 0.096 0.0603 IL-4 cytotoxin + gemcitabine 3 nmol/L 0.938
0.401 SW1990 IL-4 cytotoxin + gemcitabine 0.5 0.65 0.003 nmol/L
IL-4 cytotoxin + gemcitabine 0.03 nmol/L 0.27 0.5 IL-4 cytotoxin +
gemcitabine 0.3 nmol/L 0.021 0.3 IL-4 cytotoxin + gemcitabine 3
nmol/L 0.014 0.19 IL-4 cytotoxin + gemcitabine 30 nmol/L 0.03 0.11
IL-4 cytotoxin + gemcitabine 300 nmol/L 0.3 0.31 NOTE: CI < 1,
CI = 1, and CI > 1 indicate synergistic, additive, and
antagonistic effects, respectively. *Fifty percent protein
synthesis inhibition. **Seventy-five percent protein synthesis
inhibition. ##CI values were calculated using the formula described
in Materials and Methods.
Example 4
In Vivo Whole-Body Optical Imaging of PDA
[0154] We developed pancreatic cancer models to investigate
antitumor effects of PRX321 and showed its correlation with imaging
studies in vivo. Pancreatic cancer cells were transfected with GFP.
Our transfection technique using retroviral vector revealed
consistent bright GFP fluorescence of MIA-PaCa-2 cells. There was
no significant difference in morphology, growth rate, and
sensitivity to PRX321 between parent and GFP-transfected cells
(data not shown). GFP-transfected MIA-PaCa-2 tumor chunks were
orthotopically transplanted to pancreas of nude mice. These tumor
pieces were derived from MIA-PaCa-2-GFP cells transplanted s.c. GFP
fluorescence enabled real-time and sequential whole-body imaging of
tumors. Noninvasive quantitative measurements of external visible
fluorescent area enabled the construction of in vivo tumor growth
curves, which seem to correlate with visible tumor growth (see
FIGS. 2A and 3A).
Example 5
Complete Eradication of Tumors by Combination of PRX321 and
Gemcitabine in an Early Tumor Model
[0155] Small primary tumor lesions on day 4 after transplantation
were observed in all mice by the real-time whole-body imaging
(average fluorescent area 26.17 F 4.19 mm2). Treatment was
initiated on day 5 after transplantation. Group 1 animals (negative
control) did not receive any treatment. Group 2 animals received
gemcitabine (150 mg/kg) by i.p. administration twice a week as long
as the experiment lasted. Group 3 animals received PRX321 (100
ug/kg by i.p. route twice a day for 5 days. Group 4 animals were
treated with the combination of gemcitabine and PRX321. The results
are shown in FIG. 2A. Imaging studies on days 14 and 24 confirmed
the significant primary tumor growth and metastasis in the
non-treatment group. In contrast, gemcitabine or PRX321 treatment
group showed a reduction in the rate of tumor growth, compared with
non-treatment group. The PRX321 treatment group showed no tumor
lesions in 6 of 10 mice on day 14, although tumor recurred by day
34. Remarkably, the combination treatment group revealed
significant suppression of tumor growth of primary tumor lesions.
Tumor lesions were undetectable in all 10 mice on day 14. By day
44, 6 of 10 mice showed local recurrence and distant metastasis.
The rest of the four mice showed complete eradication of tumor and
mice remained tumor-free through day 94 when the experiment was
terminated.
Example 6
Synergistic Increase in Survival of Mice Treated with a Combination
of PRX321 and Gemcitabine in an Early Tumor Model
[0156] Median survival time of the animals treated in Example 5 was
27 days in non-treatment group, whereas it was significantly
increased to 54, 64, and 92 days in gemcitabine group
(P<0.0001), PRX321 group (P<0.0001), and their combination
group (P<0.0001) compared with non-treatment group,
respectively. Compared with gemcitabine group, significant
prolonged survival time was observed in PRX321 group (P=0.017) and
the combination group (P<0.0001). Increase in significant
survival advantage correlated with tumor area as detected by GFP
fluorescence. Prolonged survival time in the combination group was
341% compared with the non-treatment group. Kaplan-Meier survival
curves are shown in FIG. 2B. In addition, we did not observe any
organ toxicity in heart, liver, lung, kidney, and spleen of
PRX321-injected mice evaluated by histologic examination.
Example 7
Real-Time Imaging of Tumor Growth of the Primary and Metastasis
Lesion in an Advanced In Vivo Model
[0157] As approximately 85% patients with PDA are diagnosed at an
advanced stage at initial diagnosis, an advanced PDA in vivo model
needs to be established to imitate the clinical situation and to
monitor the disease and treatment effect (33). Fluorescence imaging
on day 14 post-transplantation confirmed the tumor growth of
primary lesions in all mice and also detected the metastasis
lesions to liver, lymph nodes, and peritoneal locations in 40 of 62
mice. Six mice showed metastatic lesions to liver or lymph nodes
around hepatoduodenum ligament, 8 showed metastasis lesions
corresponding to peritoneal locations, and 26 with both metastasis
lesions. We did not include mice with the GFP spot at spleen as a
metastasis group. Forty mice with confirmed primary and metastasis
tumor lesions on day 14 post-transplantation were divided into four
groups and treated as described in Materials and Methods (average
fluorescent area 94.67 F 8.31 mm2). Group 1 animals (negative
control) did not receive any treatment. Group 2 animals received
gemcitabine (150 mg/kg) by i.p. administration twice a week as long
as the experiment lasted. Group 3 animals received PRX321 (100
ug/kg by i.p. route twice a day for 5 days. Group 4 animals were
treated with the combination of gemcitabine and PRX321. Treatment
was done after the confirmation of metastasis lesions on day 14.
The results are shown in FIG. 3A. The real-time whole-body imaging
of tumor growth confirmed the significant primary tumor growth and
metastatic spread on days 14, 21, and 28 after transplantation of
tumor in non-treatment control group. Gemcitabine and PRX321
treatment group showed a reduction in the rate of tumor growth
compared with the non-treatment group. Especially, the combination
treatment group revealed significant suppression of tumor growth at
primary and metastasis tumor lesions. The reduction in tumor size
on day 28 was 39.8% in the gemcitabine group (P<0.001), 71.2% in
the PRX321 group (P<0.001), and 79.6% in the combination group
(P<0.001) compared with the no treatment group.
Example 8
Combination of PRX321 and Gemcitabine Prolongs the Survival of Mice
with Advanced Orthotopic Pancreatic Tumor
[0158] We examined the efficacy of PRX321 on the survival of
animals in the advanced PDA model in Example 7. Median survival
time of animals was 28 days in non-treatment group, whereas it was
significantly increased to 34, 43, and 52 days in gemcitabine group
(P=0.0089), PRX321 group (P<0.0001), and their combination group
(P<0.0001), respectively. Compared with gemcitabine group,
significant prolonged survival time was also observed in the PRX321
group (P=0.0047) and the combination group (P=0.0002). Prolonged
survival time in the combination group was 186% compared with the
non-treatment group. Increase in significant prolongation of
survival correlated with tumor area as detected by whole-body
imaging. Kaplan Meier survival curves are shown in FIG. 3B.
Example 9
Expression of IL-4R in a Clinical Sample and Development of
Orthotopic Xenograft Tumor Model
[0159] We obtained a tumor tissue sample that was surgically
resected at Surgery Branch at NIH and pathologically diagnosed as
moderately differentiated adenocarcinom. This tumor section showed
strong staining for IL-4Ra in the ductal adenocarcinoma cells and
faint staining of fibroblasts. We also established tumor and
fibroblast cells cultured from this sample to examine the antitumor
activity of PRX321. The cancer cells expressing IL-4R were highly
sensitive to PRX321 (IC50 0.32 ng/mL), whereas fibroblast cells
were not sensitive (IC50 z1,000 ng/mL).
Example 10
PRX321, Gemcitabine, and Their Combination Significantly Prolonged
Survival of Mice Transplanted with a Clinical Pancreatic Cancer
Sample
[0160] The clinical sample described in Example 9 was
orthotopically transplanted on the pancreas of SCID mice and when
tumors grew, they were harvested and then orthotopically propagated
in the next set of SCID mice. All mice showed growth of primary
tumor and metastasis to lymph nodes in peritoneum, hepatoduodenum
ligament, and para-aortic areas. Seventy-five percent of these mice
showed the metastasis lesion to liver when mice were sacrificed 30
days after tumor implantation. To assess the effect of PRX321 in an
advanced metastasis model, a third set of SCID mice were
orthotopically implanted with tumor pieces obtained from the second
set of mice. These mice, when advanced disease developed, were
divided into four groups on day 31 and treated as described in
Materials and Methods. Group 1 animals (negative control) did not
receive any treatment. Group 2 animals received gemcitabine (150
mg/kg) by i.p. administration twice a week as long as the
experiment lasted. Group 3 animals received PRX321 (100 ug/kg by
i.p. route twice a day for 5 days (days 31-35). Group 4 animals
were treated with the combination of gemcitabine and PRX321. Median
survival time of animals was 62 days in the non-treatment group,
whereas it was significantly increased to 86, 102, and 134 days in
the gemcitabine group (P=0.0081), PRX321 group (P=0.0006), and
combination group (P<0.0001), respectively. Compared with
gemcitabine, significant prolonged survival time was observed of
PRX321-treated mice (P=0.0037) and the combination group
(P<0.0001). Prolonged survival time in the combination group was
216% compared with the non-treatment group. Kaplan-Meier survival
curves are shown in FIG. 4.
Example 11
Conclusions
[0161] These studies support our observations of gemcitabine
synergizing with PRX321. Despite synergistic effect with
gemcitabine, few combinations have shown clinical advantage (4-7).
For example, although EGFR inhibitor showed synergistic antitumor
effect in preclinical models, the survival benefits for patients
with advanced pancreatic cancer seem very modest at best. It was
later found that mutations in the EGFR gene, which correlate with
clinical response, are found in <5% of pancreatic cancer
patients (8, 41). Therefore, new effective therapies that do not
depend on receptor mutation are needed. As our results show the
survival benefit by PRX321 when combined with gemcitabine in both
early and advanced pancreatic cancer models, it is possible that
this novel approach will afford better tumor responses than
previously observed. The precise mechanism of synergistic effect of
gemcitabine with PRX321 is not known. Gemcitabine is a synthetic
pyrimidine antimetabolite structurally related to cytarabine (42).
Gemcitabine inhibits DNA synthesis through inhibition of
ribonucleotide reductase and depletion of deoxynucleotide pools. On
the other hand, PRX321 inhibits protein synthesis after
internalization into an endosome. In addition, we have previously
shown that PRX321 can cause apoptotic cell death of cancer cells
regardless of the cell cycle status (43). It is possible that
gemcitabine enhances apoptotic cell death induced by PRX321.
Because apoptosis is a prominent mechanism of cancer cell death,
the combination therapy of these drugs, which act through different
mechanism, may be a beneficial treatment option for patients with
PDA.
[0162] We studied two types of advanced pancreatic cancer models to
show the anti-tumor activity of PRX321 and gemcitabine. In
orthotopic model, the freshly resected clinical tumor was implanted
to pancreas of SCID mice. It has been reported that this model
recapitulates the natural history of the clinical disease,
including the invasive and metastatic pattern (44). Accordingly,
the peritoneal organs, lymph nodes, liver, and spleen of mice in
our model showed tumor metastasis and invasion 1 month after
transplantation. PRX321 and gemcitabine showed remarkable antitumor
effects in this model. In future studies, it will be of interest to
determine whether metastatic lesions to various organs express
IL-4R, and after treatment with PRX321 these receptor levels
decrease along with disappearing tumor. In other orthotopic tumor
model, tumor pieces developed from MIPaCa-2-GFP cells by s.c. is
implanted to pancreas of nude mice. In this model, PRX321 as well
as gemcitabine caused profound antitumor effects. These data are
compatible with our previous report that showed the survival
benefit by PRX321 alone in orthotopic early and advanced animal
models using Panc-1 and BxPC-3 pancreatic cancer cell lines (19).
Although we did not test IL-4R-negative tumor in vivo models, our
previous studies have shown that non-small cell lung cancer cell
line expressing no or low IL-4R are not sensitive to PRX321 in vivo
(20). Similar conclusions were drawn in squamous cell carcinoma of
head and neck tumor models (45). Thus, PRX321 and gemcitabine show
better survival benefit compared with either agent alone in two
pancreatic tumor models, one derived from clinical sample and the
other derived from MIPaCa-2 cell line.
[0163] The whole-body imaging of host visualizes the real-time
tumor growth at the primary site and tumor development at
metastasis sites without the invasive procedures, surgery,
anesthesia, or use of contrast medium. Due to the fact that
whole-body imaging has the potential of high correlation with MRI
in quantifying tumor volume, the precise evaluation of tumor growth
rate, metastatic situation, and effectiveness of drugs could all be
monitored without sacrificing animals (32, 46). In addition,
imaging may identify biomarker of tumor response in preclinical
models that can be validated in the clinical trial (47). A recent
article reported that red fluorescent protein showed brighter and
less background image compared with GFP, when animals were imaged
(48). In our study, we used GFP-transfected cells. Therefore, it is
possible that we were not able to detect micrometastasis lesions.
Nevertheless, we could show that mice developed spontaneous tumor
metastasis within the short time after orthotopic transplantation,
which correlated with short survival time. In addition, our model
showed that PRX321 reduced the rate of tumor growth, including
primary and metastasis lesions for 15 and 9 days after treatment in
early and advanced model, respectively.
[0164] Although PRX321 mediated remarkable antitumor effects in
vivo, no visible signs of toxicity and features such as weight loss
and inactivity were observed in mice receiving optimal doses of
PRX321 and/or gemcitabine (data not shown). These results are
compatible with previous studies related to both agents (data not
shown; refs. 19, 24, 49). Previous studies have shown that low
density IL-4R are expressed on normal immunologic and
nonhematopoietic cells (22). Consequently, PRX321 is not cytotoxic
to these cells. Preclinical toxicity studies in mice have shown
that PRX321 is well tolerated up to 475 Ag/kg dose given i.v. (50).
As human IL-4 does not bind murine IL-4R, PRX321 has also been
administered to cynomolgus monkeys, whose IL-4R binds human IL-4.
In these animals, PRX321 was reasonably tolerated up to a dose of
200 Ag/kg given i.v. every alternate day for three injections (21).
In a phase 1 clinical trial, reversible elevation of liver enzymes
and injection site inflammatory reactions were reported after i.v.
administration of PRX321 at 0.027 mg/m2 (25). As our study shows
synergistic effects when PRX321 is combined with gemcitabine
against pancreatic cancer in vitro and in vivo, lower doses of
PRX321 may be effective for the treatment of patients with PDA when
combined with gemcitabine.
[0165] In conclusion, these studies provide a novel approach for
monitoring tumor response by whole-body imaging of the host.
Further studies should be done to evaluate the safety,
tolerability, and efficacy of PRX321 when combined with gemcitabine
in various pancreatic cancer models. In addition, because of their
synergistic effect, PRX321 in combination with gemcitabine should
be tested in patients with PDA.
Example 12
Repetitive Therapy of Orthotopic Human Pancreatic Cancer by of
IL4-PE
[0166] Immunodeficient nude mice were transplanted with Green
Fluorescence Protein (GFP) transfected human pancreatic tumor cells
(Hs766T) orthotropically on pancreas. Five days after tumor
implantation, mice were treated with IL4-PE 100 ug/kg/day, i.p. for
one week or every alternate week for 3 weeks. Tumor size was
measured by imaging of visible fluorescence area in live
animals.
[0167] One week administration of IL-4-PE significantly decreased
the tumor volume compared to control. However, repetitive therapy
with IL4-PE caused dramatic regression of established pancreatic
tumor growth. We did not observe any visible side effects with
repetitive therapy of IL4-PE.
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Sequence CWU 1
1
11485PRTArtificial Sequencea circularly permuted IL-4-Pseudomonas
toxin, PRX321 1Met Asp Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala Ala
Thr Val Leu1 5 10 15Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg
Cys Leu Gly Ala 20 25 30Thr Ala Gln Gln Phe His Arg His Lys Gln Leu
Ile Arg Phe Leu Lys 35 40 45Leu Arg Asp Arg Asn Leu Trp Gly Leu Ala
Gly Leu Asn Ser Cys Pro 50 55 60Val Lys Glu Ala Asn Gln Ser Thr Leu
Glu Asn Phe Leu Glu Arg Leu65 70 75 80Lys Thr Ile Met Arg Glu Lys
Tyr Ser Lys Cys Ser Ser Gly Gly Asn 85 90 95Gly Gly His Lys Cys Asp
Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu 100 105 110Asn Ser Leu Thr
Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr 115 120 125Asp Ile
Phe Ala Ala Ser Lys Ala Ser Gly Gly Pro Glu Gly Gly Ser 130 135
140Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu
Thr145 150 155 160Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln
Leu Glu Gln Cys 165 170 175Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
Tyr Leu Ala Ala Arg Leu 180 185 190Ser Trp Asn Gln Val Asp Gln Val
Ile Arg Asn Ala Leu Ala Ser Pro 195 200 205Gly Ser Gly Gly Asp Leu
Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln 210 215 220Ala Arg Leu Ala
Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val225 230 235 240Arg
Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala 245 250
255Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu
260 265 270Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly
Thr Gln 275 280 285Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg
Gln Leu Glu Glu 290 295 300Arg Gly Tyr Val Phe Val Gly Tyr His Gly
Thr Phe Leu Glu Ala Ala305 310 315 320Gln Ser Ile Val Phe Gly Gly
Val Arg Ala Arg Ser Gln Asp Leu Asp 325 330 335Ala Ile Trp Arg Gly
Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr 340 345 350Gly Tyr Ala
Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn 355 360 365Gly
Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe 370 375
380Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu
Val385 390 395 400Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu
Asp Ala Ile Thr 405 410 415Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu
Thr Ile Leu Gly Trp Pro 420 425 430Leu Ala Glu Arg Thr Val Val Ile
Pro Ser Ala Ile Pro Thr Asp Pro 435 440 445Arg Asn Val Gly Gly Asp
Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu 450 455 460Gln Ala Ile Ser
Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro465 470 475 480Pro
Lys Asp Glu Leu 485
* * * * *