U.S. patent application number 12/122807 was filed with the patent office on 2008-10-16 for treatment of cancer using tlr3 agonists.
This patent application is currently assigned to INSTITUT GUSTAVE ROUSSY. Invention is credited to FABRICE ANDRE, Jean-Christophe Sabourin, Laurence Zitvogel.
Application Number | 20080253998 12/122807 |
Document ID | / |
Family ID | 35717497 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080253998 |
Kind Code |
A1 |
ANDRE; FABRICE ; et
al. |
October 16, 2008 |
TREATMENT OF CANCER USING TLR3 AGONISTS
Abstract
The present invention relates generally to the fields of
genetics and medicine. More specifically, the present invention
relates to improved methods of treating cancers using a TLR3
agonist, by assessing the expression of a TLR3 receptor by cancer
cells.
Inventors: |
ANDRE; FABRICE; (Paris,
FR) ; Zitvogel; Laurence; (Antony, FR) ;
Sabourin; Jean-Christophe; (Paris, FR) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Assignee: |
INSTITUT GUSTAVE ROUSSY
Villejuif Cedex
FR
|
Family ID: |
35717497 |
Appl. No.: |
12/122807 |
Filed: |
May 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11144322 |
Jun 3, 2005 |
7378249 |
|
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12122807 |
|
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|
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PCT/IB04/04093 |
Nov 19, 2004 |
|
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11144322 |
|
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Current U.S.
Class: |
424/85.7 ;
424/138.1; 424/178.1; 424/422; 435/7.23; 514/44A |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 2800/52 20130101; C12N 15/117 20130101; C12N 15/1135 20130101;
C12Q 2600/158 20130101; C12Q 2600/106 20130101; G01N 33/57492
20130101; C12N 2310/13 20130101; C12N 15/111 20130101; A61P 35/04
20180101; C12Q 2600/118 20130101; A61P 31/00 20180101; C12Q 1/6886
20130101; C12N 2310/17 20130101; C12N 2310/53 20130101; C12N
2320/50 20130101; C12Q 2600/112 20130101; C12N 2310/14
20130101 |
Class at
Publication: |
424/85.7 ;
514/44; 424/138.1; 424/422; 435/7.23; 424/178.1 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 31/713 20060101 A61K031/713; A61K 31/7105 20060101
A61K031/7105; G01N 33/574 20060101 G01N033/574; A61P 35/04 20060101
A61P035/04; A61K 9/00 20060101 A61K009/00; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for treating a subject having a cancer comprising: a.
determining whether cancer cells in said subject express a TLR3
receptor, the expression of a TLR3 receptor being indicative of a
subject responding to a TLR3 agonist, and b. administering to said
subject whose cancer cells are determined to express a TLR3
receptor a pharmaceutical composition comprising an effective
amount of a TLR3 agonist and a pharmaceutically acceptable
carrier.
2. The method according to claim 1, wherein said cancer in said
subject comprises at least 10% of cancer cells expressing a TLR3
receptor.
3. The method according to claim 1, wherein said TLR3 agonist is a
dsRNA TLR agonist.
4. The method according to claim 3, wherein said TLR3 agonist is an
siRNA or an shRNA that specifically binds to mRNA encoding a tumor
antigen or another protein involved in tumor proliferation.
5. The method according to claim 1, wherein said TLR3 agonist is an
antibody or an antigen binding fragment thereof.
6. The method according to claim 1, wherein said determining
whether cells of said cancer in said subject express a TLR3
receptor is performed ex vivo on a biopsied sample of said cancer
using a TLR3-specific ligand.
7. The method according to claim 5, wherein said TLR3-specific
ligand is capable of detecting TLR3 expressed on the surface and in
the cytoplasm of cancer cells.
8. The method according to claim 5, wherein said TLR3-specific
ligand is an antibody or an antigen binding fragment thereof.
9. A method for treating a subject having a cancer, the method
comprising the steps of: a. administering to said subject an
effective amount of an agent that causes upregulation of TLR3
expression, and b. administering to said subject an effective
amount of a TLR3 agonist.
10. The method according to claim 9, comprising the additional step
of determining whether cells of said cancer in said subject express
a TLR3 receptor, said additional step being performed prior to step
b.
11. The method according to claim 1, comprising the additional step
of administering to said subject a second therapeutic agent
selected from a TLR3 agonist of different molecular composition
than the TLR3 agonist administered in step b; a cytotoxin; a
cytotoxin-TLR3 ligand complex; an agent that inhibits expression or
an activity of a tumor antigen; an agent that inhibits expression
or an activity of a tumor proliferative protein; a chemotherapy
agent, an agent or combination of agents typically used for the
treatment of the specific cancer to be treated; an
immunostimulatory agent; an immunosuppression agent; a cytokine or
cytokine analog; a chemokine; an agent that affects upregulation of
a cell surface receptor; an agent that affects GAP junctions; a
cytostatic agent; an agent that inhibits differentiation; or an
agent that inhibits cell adhesion, wherein said second therapeutic
agent is administered separately as part of a multiple dosage
regimen, or as part of said pharmaceutical composition.
12. The method according to claim 14, wherein said
immunostimulatory agent is IFN.alpha..
13. A pharmaceutical composition comprising: a. a TLR3 agonist; b.
a second therapeutic agent selected from a second TLR3 agonist TLR3
agonist different from the TLR3 agonist of a; a cytotoxin; a
cytotoxin-TLR3 ligand complex; an agent that inhibits expression or
an activity of a tumor antigen; an agent that inhibits expression
or an activity of a tumor proliferative protein; a chemotherapy
agent; an agent or combination of agents typically used for the
treatment of the specific cancer to be treated; an
immunostimulatory agent; an immunosuppression agent; a cytokine or
cytokine analog; a chemokines, an agent that affects upregulation
of a cell surface receptor; an agent that affects GAP junctions; a
cytostatic agent; an agent that inhibits differentiation; an agent
that inhibits cell adhesion; or an endocytosis inhibitor; and c. a
pharmaceutically acceptable carrier.
14. The composition according to claim 13 wherein the active
ingredients of said composition are in separate dosage forms.
15. An implantable drug release device impregnated with or
containing a TLR3 agonist or a composition comprising a TLR3
agonist, such that said TLR3 agonist is released from said device
and is therapeutically active.
16. A kit comprising: a. a cancer cell or cancer cell line that
expresses TLR3; b. a TLR3-specific ligand; and c. a reagent for
detecting the binding of said TLR3-specific ligand to a TLR3
expressed by said cancer cell wherein said kit is for determining
if an agent is capable of increasing the susceptibility of a cancer
to treatment with a TLR3 agonist; or a. a TLR3-specific ligand; and
b. additional reagents for detecting the expression of a TLR3
receptor in a cancer cell in a sample, wherein said kit is for
assessing the response of a subject having cancer to a treatment
using a TLR3 agonist or for selecting subjects having a cancer that
responds to a treatment using a TLR3 agonist; or a. reagents for
determining the expression of a TLR3 receptor in a cancer cell in a
sample; b. a cancer cell characterized by the presence of TLR3 on
its cell surface and a reagent for detecting if said test compound
causes TLR3-mediated apoptosis or cell death of said cancer cell;
and c. optionally comprising a reagent that detects agonism of TLR3
by said test compound, wherein said kit is for determining if a
test compound is useful for the treatment of cancer.
17. The kit according to claim 16, wherein the TLR3-specific ligand
selected from an antibody or a fragment or a derivative thereof; a
TLR3-specific nucleic acid primer or probe; or a double-stranded
RNA molecule.
18. A complex comprising: a. a TLR3 ligand; and b. a cytotoxic
agent, wherein said agonist and said cytotoxic agent are bound to
one another directly through a covalent bond, bond to one another
through a non-covalent bond, conjugated to one another directly or
through a linking moiety or are present in a single pharmaceutical
carrier or vehicle.
19. The complex according to claim 18, wherein said TLR3 ligand is
selected from an antibody, or a fragment or derivative thereof,
that specifically binds to TLR3; or a double-stranded RNA
molecule.
20. A method for assessing the response of a subject having cancer
to a treatment using a TLR3 agonist or selecting a subject having a
cancer that responds to a treatment using a TLR3 agonist, the
method comprising the step of determining whether cancer cells in
said subject express a TLR3 receptor, the expression of a TLR3
receptor being indicative of a responder subject.
21. A method of determining if a test compound is useful for the
treatment of cancer, said method comprising the step of determining
if said test compound is a TLR3 agonist.
22. The method according to claim 21, wherein said determining if a
test compound is useful for the treatment of cancer comprises the
steps of: a. contacting said test compound with a cancer cell
characterized by the presence of TLR3 on its cell surface; and b.
determining if said test compound induces a TLR3-mediated
biological effect.
23. A method for assessing the response of a subject having cancer
to a treatment using a TLR3 agonist, the method comprising
determining whether cancer cells in said subject express a TLR3
receptor, the expression of a TLR3 receptor being indicative of a
responder subject.
24. A method for selecting subjects having a cancer that respond to
a treatment using a TLR3 agonist, the method comprising determining
whether cancer cells in said subject express a TLR3 receptor, the
expression of a TLR3 receptor being indicative of a responder
subject.
25. A method for treating a subject having a cancer, the method
comprising determining whether cancer cells in said subject express
a TLR3 receptor, the expression of a TLR3 receptor being indicative
of a subject responding to a TLR3 agonist, and treating said
subject whose cancer cells express a TLR3 receptor with a TLR3
agonist.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/144,322, filed on Jun. 3, 2005, now U.S. Pat. No. 7,378,249,
which is a continuation-in-part of PCT/IB2004/004093, filed on Nov.
19, 2004, which are incorporated herein by reference in their
entireties, including all references, Figures, Tables and nucleic
acid and polynucleotide sequences.
[0002] The present invention relates generally to the fields of
genetics and medicine. More specifically, the present invention
relates to improved methods of treating cancers using TLR3
agonists.
INTRODUCTION
[0003] Cancer is one of the leading causes of death in Europe and
in the United States. There are more than 1.2 million cancer cases
each year in the U.S. and cancer is expected to be the leading
cause of the death by the year 2010. Lung and prostate cancer are
the top cancer killers for men in the United States. Lung and
breast cancer are the top cancer killers for women in the United
States. A cure for cancer has yet to be found. Current treatment
options, such as surgery, chemotherapy and radiation treatment, are
oftentimes either ineffective or present serious side effects.
[0004] Currently, cancer therapy may involve surgery, chemotherapy,
hormonal therapy and/or radiation treatment to eradicate neoplastic
cells in a patient. Recently, cancer therapy could also involve
biological therapy or immunotherapy. All of these approaches pose
significant drawbacks for the patient. Surgery, for example, may be
contraindicated due to the health of the patient or may be
unacceptable to the patient. Additionally, surgery may not
completely remove the neoplastic tissue.
[0005] Despite the availability of a variety of chemotherapeutic
agents, chemotherapy has many drawbacks. Almost all
chemotherapeutic agents are toxic, and chemotherapy causes
significant, and often dangerous, side effects, including severe
nausea, bone marrow toxicity, immunosuppression, etc. Additionally,
even with administration of combinations of chemotherapeutic
agents, many tumor cells are resistant or develop resistance to the
chemotherapeutic agents. In fact, those cells resistant to the
particular chemotherapeutic agents used in the treatment protocol
often prove to be resistant to other drugs, even those agents that
act by mechanisms different from the mechanisms of action of the
drugs used in the specific treatment; this phenomenon is termed
pleiotropic drug or multidrug resistance. Thus, because of drug
resistance, many cancers prove refractory to standard
chemotherapeutic treatment protocols.
[0006] Double-stranded RNA molecules, such as poly A-polyU and poly
I-poly U, are immunostimulating agents. Preclinical studies
performed in 1970-1980's showed that the incubation of blood
mononuclear cells with poly A-poly U induces interferon alpha
secretion, and that the injection of poly A-poly U activates
natural killer cells in vitro (EP281 380; EP 113 162).
[0007] It has recently been demonstrated that the double-stranded
RNA receptor is Toll Like receptor 3 (TLR3) (Alexopoulou et al.,
2001). This receptor has been described to be expressed in
membranes of dendritic cells and of cells from colic mucosa. The
binding of double-stranded RNA to this receptor activates dendritic
cells and activates T lymphocytes. Consequently, the use of
double-stranded RNA for treating cancer has been attempted.
However, the response rate was not high and no therapeutic
applications were developed.
[0008] Therefore, a method allowing to select responding patient
would greatly enhance the therapeutic efficacy of double-stranded
therapies.
SUMMARY OF THE INVENTION
[0009] The present invention demonstrates the existence of a
correlation between the expression of a TLR3 in cells from a tumor
sample in a subject and the ability of said subject to respond to
treatment with a composition comprising a TLR3 agonist, preferably
an antibody or other TLR3 binding protein, a phosphate containing
small molecule, an organophospho-ester, a nucleotide,
nucleotide-like, nucleotide analog, or nucleic acid molecule, or
most preferably a double-stranded RNA. More specifically, the
present invention shows, for the first time, that TLR3 is expressed
in tumoral cell membranes and that the binding of a TLR3 agonist,
in particular double-stranded RNA, on said tumoral cells through
the TLR leads to tumoral cells lysis and tumor regression. In
contrast, tumoral cells that do not express TLR are not sensitive
to a TLR3 agonist treatment, in particular the double-stranded RNA
treatment. The finding is particularly surprising and important for
the management of cancer patients because an increase in survival
is rarely if ever achieved in any known active regimen in
metastatic breast cancer. The usual finding is an improvement in
early endpoints (response rate and/or time to progression,
otherwise known as progression-free survival) but the improvement
almost never translates into a significant increase in overall
survival, as exhibited in patients treated in accordance with the
present invention.
[0010] Furthermore, the finding that TLR3 agonist therapy can
increase survival will permit therapeutic strategies that are
expected to be efficacious and a suitable treatment for tumors,
including particularly breast tumors, whether they involve
auxiliary lymph nodes or not, that is beyond only breast cancers
presenting auxiliary lymph node involvement. Likewise, the TLR3
agonists may find use in breast (and other) cancers that are
metastatic, recurring and/or refractory.
[0011] Accordingly, in another aspect, the present invention
provides a method of treating a human subject having a tumor
comprising TLR3-expressing cells, which method comprises
administering to said patient a therapeutically effective amount of
at least one TLR3 agonist. In a preferred embodiment, said human
patient has a solid tumor. In a preferred embodiment, said human
patient has a breast cancer.
[0012] Therefore, the present invention concerns the use of a TLR3
agonist for the manufacture of a medicament for treating cancer in
a subject, wherein said cancer in said subject comprises cancer
cells expressing a TLR3 receptor. Preferably, the TLR agonist is a
TLR3 agonist. More preferably, the TLR3 agonist is a
double-stranded RNA molecule. Optionally, the cancer is a
metastatic cancer. In a particular embodiment, the present
invention concerns the use of a double-stranded polyA/polyU RNA
molecule for the manufacture of a medicament for treating a cancer
in a subject, wherein said cancer in said subject comprises cancer
cells expressing a TLR3 receptor. Preferably the cancer is a solid
tumor or a carcinoma, for example a breast cancer.
[0013] The present invention also concerns a method for assessing
the response of a subject having cancer to a treatment using a TLR3
agonist, the method comprising determining whether cancer cells in
said subject express a TLR receptor, the expression of a TLR3
receptor being indicative of a responder subject. More preferably,
the TLR3 agonist is a double-stranded RNA molecule.
[0014] The present invention further concerns a method for
selecting subjects having a cancer that respond to a treatment
using a TLR3 agonist, the method comprising determining whether
cancer cells in said subject express a TLR3 receptor, the
expression of a TLR3 receptor being indicative of a responder
subject. More preferably, the TLR3 agonist is a double-stranded RNA
molecule.
[0015] In addition, the present invention concerns a method for
treating a subject having a cancer, the method comprising
determining whether cancer cells in said subject express a TLR3
receptor, the expression of a TLR3 receptor being indicative of a
subject responding to a TLR3 agonist, and treating said subject
whose cancer cells express a TLR3 receptor with a TLR3 agonist.
More preferably, the TLR3 agonist is a double-stranded RNA
molecule.
[0016] Moreover, the inventors have surprisingly found in the
follow up study to the 300 patient clinical trials that patients
having a solid tumor and treated with a TLR3 agonist in accordance
with a repeat-dose and with an intravenous therapeutic regimen
exhibited greater survival than other patients not treated with the
TLR3 agonist.
[0017] Accordingly, in one aspect, the present invention provides a
method of achieving enhanced or prolonged survival in human
patients with cancer, which comprises administering to said patient
a therapeutically effective amount of at least one TLR3 agonist. In
a preferred embodiment, said at least one TLR3 agonist is
intravenously administered. In a preferred embodiment, the cancer
is a breast cancer. In a more particular embodiment, the cancer is
a metastatic or recurrent breast cancer. In another preferred
embodiment, the cancer is a node positive breast cancer.
[0018] Accordingly, in one aspect, the present invention provides a
method of achieving enhanced survival in human patients with cancer
or a method for treating a subject having a cancer, which comprises
administering to said patient (a) at least a first dose
therapeutically effective amount of at least one TLR3 agonist; and
(b) at least a second dose of a therapeutically effective amount of
at least one TLR3 agonist. In a preferred embodiment, said first
and said second doses are administered at an interval of less than
one month, less than three weeks, less than two weeks, or less than
one week.
[0019] In a preferred embodiment of the methods and uses according
to the present invention, the subject is a human subject.
[0020] It will be appreciated that the methods of treatment
mentioned herein can be used as prophylactic treatment; in any of
the embodiments herein, a prophylactically effective amount of the
TLR3 agonist can be interchanged with a therapeutically effective
amount of a TLR3 agonist.
[0021] In a preferred embodiment of the methods and uses according
to the present invention, the cancer is a solid tumor or a
carcinoma. Preferably, the solid tumor is selected from breast
cancer, colon cancer, lung cancer, prostate cancer, renal cancer,
metastatic or invasive malignant melanoma, brain tumor, ladder
cancer and liver cancer. Carcinoma includes bladder, breast, colon,
kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid or
skin carcinoma, including squamous cell carcinoma. In a most
preferred embodiment, the solid tumor is a breast cancer. However,
the present invention also contemplates hematopoietic tumors such
as leukemia, acute lymphocytic leukemia, acute lymphoblastic
leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma,
non-Hodgkins lymphoma, hairy cell lymphoma, Burketts lymphoma,
acute and chronic myelogenous leukemias and promyelocytic leukemia.
The present invention is also relevant for the treatment of
metastasis.
[0022] In a preferred embodiment, the expression of a TLR3 receptor
in said cancer cell is determined using a TLR3-specific ligand.
Preferably, the ligand is an antibody, or a fragment or derivative
thereof.
[0023] In an alternative embodiment, the expression of a TLR3
receptor in said cancer cell is determined using a TLR3-specific
primer or probe.
[0024] Preferably, the expression of a TLR3 receptor in said cancer
cell is determined in vitro or ex vivo. However, the determination
in vivo is also encompassed by the present invention.
[0025] In a preferred embodiment of the methods and uses according
to the present invention, the double-stranded RNA molecule is a
polyA/polyU molecule. In another preferred embodiment of the
methods and uses according to the present invention, the
double-stranded RNA molecule is a polyI/polyC molecule.
[0026] The present invention further concerns a kit for selecting
subjects that respond to a treatment using a TLR3 agonist, more
preferably a double-stranded RNA molecule, the kit comprising
reagents for determining the expression of a TLR3 receptor in a
cancer cell in a sample.
DESCRIPTION OF THE FIGURES
[0027] The file of this patent contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Patent and
Trademark Office upon request and payment of the necessary fee.
[0028] FIG. 1 illustrates the TLR3 expression by primary tumor.
TLR3 is overexpressed by tumor cells in 10% of samples (n=18)
[0029] FIG. 2 illustrates survival of patients with TLR3-tumors
(FIG. 2a) or with TLR3+ tumors (FIG. 2b) according to treatment
with a placebo (observation) or with dsRNA.
DETAILED DESCRIPTION OF THE INVENTION
[0030] A marker-based approach to tumor identification and
characterization promises improved diagnostic and prognostic
reliability. Typically, the diagnosis of breast cancer and other
types of cancer requires histopathological proof of the presence of
the tumor. In addition to diagnosis, histopathological examinations
also provide information about prognosis and selection of treatment
regimens. Prognosis may also be established based upon clinical
parameters such as tumor size, tumor grade, the age of the patient,
and lymph node metastasis.
[0031] With the available and potent conventional drug regimens as
well as the advent of novel therapy approaches targeting specific
biological pathways, the determination of optimal treatment of a
primary cancer is becoming increasingly complex. Moreover, the
outcome of a treatment of a patient with cancer is often
unpredictable. Only a portion of the patients respond to a certain
type of treatment. The patients receiving a specific type of
treatment are subjected to an unnecessary suffering since adverse
reactions often are obtained from certain treatment used. Some
treatments elicit more severe reaction from the patient than other
treatments. Mostly, the effect of a treatment is not shown until
3-6 months after treatment. It would therefore be of great
importance if patients with a high probability to respond could be
identified before the onset of treatment. To date, no set of
satisfactory predictors for prognosis or therapeutic response based
on the clinical information alone has been identified for TLR
agonist compounds in cancer therapy.
[0032] As further described herein, the studies disclosed herein
present results from 300 patients with early breast cancer that had
been included from 1972 to 1979 in a randomized trial comparing
post-operative administration of TLR agonist polyAU with no
treatment. When the TLR was investigated as a potential biomarker,
it was observed that tumor biopsies positive for TLR demonstrated
high long term survival rates, and presumably that this survival is
in response to therapy with the TLR3 agonist. Patient biopsies were
stained with a TLR3-specific mAb and correlation of TLR3 expression
with polyAU efficacy was determined. 182 tumor samples (91%) were
available from the 200 patients included in the randomized trial.
TLR3 was strongly expressed by tumor cells in 18 patients (10%).
TLR3 expression correlated with a significantly increased 20-year
survival rate.
[0033] Accordingly, in one aspect, the present invention provides a
method of determining whether a patient will respond to therapy
with a TLR3 agonist. In one embodiment the present invention
concerns a method for selecting or identifying a subject having a
tumor that respond to a treatment using a TLR3 agonist, the method
comprising determining whether tumor cells in said subject express
a TLR3, the expression of a TLR3 receptor being indicative of a
responder subject. In a preferred embodiment, TLR3 is determined
using an immunohistochemical assay, as used in the Examples.
Preferably TLR3 expression is determined using an antibody that
specifically binds TLR3.
[0034] In another embodiment, the present invention provides a
method for characterizing a cell or a tumor in a patient, the
method comprising: obtaining or providing a biological sample from
the patient, wherein said sample comprises a tumor cell or
biological material derived therefrom, and determining whether said
cell expresses a TLR3. In a preferred embodiment, the present
invention provides a method for characterizing a cell or a tumor in
a patient, the method comprising: obtaining or providing a tumor
biopsy from the patient, and determining whether said biopsy
comprises a cell expressing a TLR3 polypeptide.
DEFINITIONS
[0035] As used in the specification, "a" or "an" may mean one or
more. As used in the claim(s), when used in conjunction with the
word "comprising", the words "a" or "an" may mean one or more than
one. As used herein "another" may mean at least a second or
more.
[0036] Where "comprising" is used, this can preferably be replaced
by "consisting essentially of", more preferably by "consisting
of".
[0037] Whenever within this whole specification "treatment of a
proliferative disease" or "treatment of a tumor", or "treatment of
cancer" or the like is mentioned with reference to a TLR3 agonist,
there is meant:
[0038] a method of treatment (=for treating) of a proliferative
disease, said method comprising the step of administering (for at
least one treatment) a TLR3 agonist, (preferably in a
pharmaceutically acceptable carrier material) to a warm-blooded
animal, especially a human, in need of such treatment, in a dose
that allows for the treatment of said disease (=a therapeutically
effective amount), preferably in a dose (amount) as specified to be
preferred hereinabove and herein below;
[0039] the use of a TLR3 agonist for the treatment of a
proliferative disease; or a TLR3 agonist, for use in said treatment
(especially in a human);
[0040] the use of a TLR3 agonist, for the manufacture of a
pharmaceutical preparation for the treatment of a proliferative
disease; and/or
[0041] a pharmaceutical preparation comprising a dose of a TLR3
agonist that is appropriate for the treatment of a proliferative
disease; or any combination of a), b), c) and d), in accordance
with the subject matter allowable for patenting in a country where
this application is filed;
[0042] a method of using a TLR3 agonist for the manufacture of a
pharmaceutical preparation for the treatment of a proliferative
disease, comprising admixing said TLR3 agonist with a
pharmaceutically acceptable carrier. In cases where a tumor disease
or a specific tumor (e.g. breast tumor, colon tumor, colon
carcinoma or colon cancer; or prostate tumor, prostate carcinoma or
prostate cancer) are mentioned instead of "proliferative disease",
categories a) to e) are also encompassed, meaning that the
respective tumor disease can be filled in under a) to e) above
instead of "proliferative disease", in accordance with the
patentable subject matter.
[0043] The terms "cancer" and "tumor" as used herein are defined as
a new growth of cells or tissue comprising uncontrolled and
progressive multiplication. In a specific embodiment, upon a
natural course the cancer is fatal. In specific embodiments, a
cancer is invasive, metastatic, and/or anaplastic (loss of
differentiation and of orientation to one another and to their
axial framework).
[0044] The term "breast cancer" as used herein is defined as cancer
which originates in the breast. In a specific embodiment, the
breast cancer spreads to other organs, such as lymph nodes. In a
specific embodiment, the breast cancer is invasive and may be
metastatic.
[0045] The term "invasive" as used herein refers to cells which
have the ability to infiltrate surrounding tissue. In a specific
embodiment, the infiltration results in destruction of the
surrounding tissue. In another specific embodiment, the cells are
cancer cells. In a preferred embodiment, the cells are breast
cancer cells, and the cancer spreads out of a duct into surrounding
breast epithelium. In a specific embodiment, "metastatic" breast
cancer is within the scope of "invasive."
[0046] The term "metastatic" as used herein is defined as the
transfer of cancer cells from one organ or part to another not
directly connected with it. In a specific embodiment, breast cancer
cells spread to another organ or body part, such as lymph
nodes.
[0047] "Weekly" stands for "about once a week" (meaning that more
than one treatment is made with an interval of about one week
between treatments), the about here preferably meaning +/-1 day
(that is, translating into "every 6 to 8 days"); most preferably,
"weekly" stands for "once every 7 days".
[0048] The term "biopsy" as used herein is defined as removal of a
tissue from an organ (e.g., breast) for the purpose of examination,
such as to establish diagnosis. Examples of types of biopsies
include by application of suction, such as through a needle
attached to a syringe; by instrumental removal of a fragment of
tissue; by removal with appropriate instruments through an
endoscope; by surgical excision, such as of the whole lesion; and
the like.
[0049] As used herein, the term "adjunctive" is used
interchangeably with "in combination" or "combinatorial". Such
terms are also used where two or more therapeutic or prophylactic
agents affect the treatment or prevention of the same disease. For
the avoidance of doubt, two agents used "in combination" may be,
but are not necessarily, co-administered.
[0050] As used herein, the term "in combination" refers to the use
of more than one therapies (e.g., more than one prophylactic agent
and/or therapeutic agent). The use of the term "in combination"
does not restrict the order in which therapies (e.g., prophylactic
or therapeutic agents) are administered to a subject with cancer. A
first therapy can be administered prior to (e.g., 5 minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,
12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks,
3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of a second therapy to a subject with cancer.
[0051] By "co-administration" or "co-administering" we mean that
the two agents are administered in temporal juxtaposition. The
co-administration may be effected by the two agents being mixed
into a single formulation, or by the two agents being administered
separately but simultaneously, or separately and within a short
time of each other. For example, in general the two agents are
co-administered within the time range of 24-72-12 hours. In this
case, the agents may be administered in either order, i.e. a TLR3
agonist may be administered first, or the inhibitor of a
compensatory cytoprotective pathway may be administered first. In a
preferred embodiment of the instant invention, the two agents are
co-administered in a single formulation, or are co-administered
simultaneously. Further, more than one cell cycle checkpoint
abrogation agent or more than one inhibitor of a compensatory
cytoprotective pathway may be administered together, and inhibitors
of different compensatory cytoprotective pathways may be
co-administered together.
[0052] As used herein, the terms "prevent", "preventing" and
prevention refer the inhibition of the development or onset of
cancer (particularly, a T-cell malignancy) or the prevention,
recurrence, onset, or development of one or more symptoms of
cancer, particularly a T-cell malignancy, in a subject resulting
from the administration of therapy (e.g., a prophylactic or
therapeutic agent) or a combination of therapies (e.g., a
combination of prophylactic and/or therapeutic agents).
[0053] As used herein, the term "prophylactically effective amount"
refers to that amount of the prophylactic agent sufficient to
result in the prevention of the recurrence or onset of cancer or
one or more symptoms thereof.
[0054] A used herein, a "protocol" includes dosing schedules and
dosing regimens. The protocols herein are methods of use and
include prophylactic and therapeutic protocols.
[0055] As used herein, a "prophylactic protocol" refers to a
regimen for dosing and timing the administration of one or more
prophylactic agents.
[0056] As used herein, the term "therapeutic protocol" refers to a
regimen for dosing and timing the administration of one or more
therapeutic agents.
[0057] As used herein, the phrase "side effects" encompasses
unwanted and adverse effects of a therapy (e.g., prophylactic
and/or therapeutic agent). Adverse effects are always unwanted, but
unwanted effects are not necessarily adverse. An adverse effect
from a prophylactic or therapeutic agent might be harmful or
uncomfortable or risky.
[0058] As used herein, the term "small molecules" and analogous
terms include, but are not limited to, organic or inorganic
compounds (i.e., including heteroorganic and organometallic
compounds) having a molecular weight less than 1,000 grams per
mole. In a preferred embodiment, "small molecules" encompass
organic or inorganic compounds having a molecular weight less than
750 grams per mole. In yet another specific embodiment, "small
molecules" encompass organic or inorganic compounds having a
molecular weight less than 500 grams per mole. Salts, esters, and
other pharmaceutically acceptable forms of such compounds are also
encompassed.
[0059] As used herein, the terms "subject" and "patient" are used
interchangeably.
[0060] As used herein, the terms "subject" and "subjects" refer to
an animal, preferably a mammal including, but not limited to, a
non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and
a non-primate (e.g., a monkey such as a cynomolgous monkey and a
human), and more preferably a human. In a specific embodiment, the
subject is a human with cancer. In a preferred embodiment, the
subject is a human with a solid tumor, for example a breast
cancer.
[0061] As used herein, the term "synergistic" refers to a
combination of therapies (e.g., prophylactic or therapeutic agents)
which is more effective than the additive effects of any two or
more single agents. For example, a synergistic effect of a
combination of therapies (e.g., prophylactic or therapeutic agents)
permits the use of lower dosages of one or more of the agents
and/or less frequent administration of said therapies to a subject
with cancer.
[0062] The ability to utilize lower dosages of therapies (e.g.,
prophylactic or therapeutic agents) and/or to administer said
therapies less frequently reduces the toxicity associated with the
administration of said therapies to a subject without reducing the
efficacy of said therapies in the prevention or treatment of
cancer. In addition, a synergistic effect can result in improved
efficacy of therapies in the prevention or treatment of cancer.
Finally, synergistic effect of a combination of therapies may avoid
or reduce adverse or unwanted side effects associated with the use
of any single therapy.
[0063] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any agent (s) which can be used in
the treatment, management, or amelioration of cancer or one or more
symptoms thereof. In certain embodiments, the term "therapeutic
agent" refers to a TLR3 agonist. In certain other embodiments, the
term "therapeutic agent" does not refer to a TLR3 agonist. In yet
other embodiments, the term "therapeutic agents" refers to a TLR3
agonist and a cancer therapy other than a TLR3 agonist. Preferably,
a therapeutic agent is an agent which is mown to be useful for, or
has been or is currently being used for the treatment, management,
or amelioration of cancer or one or more symptoms thereof.
Therapeutic agents may be characterized as different agents based
upon one or more effects the agents have in vivo and/or in
vitro.
[0064] As used herein, the term "effective amount" refers to the
amount of a therapy (e.g. a prophylactic or therapeutic agent)
which is sufficient to reduce or ameliorate the severity, duration
and/or progression of cancer or one or more symptoms thereof,
ameliorate one or more symptoms of cancer, prevent the advancement
of cancer, cause regression of cancer, prevent the recurrence,
development, or onset of cancer or one or more symptoms thereof, or
enhance or improve the prophylactic or therapeutic effect (s) of
another therapy (e.g., prophylactic or therapeutic agent).
[0065] As used herein, the term "therapeutically effective amount"
refers to that amount of a therapy (e.g., a therapeutic agent)
which is sufficient to destroy, modify, control or remove primary,
regional or metastatic cancer tissue, ameliorate cancer or one or
more symptoms thereof, or prevent the advancement of cancer, cause
regression of cancer, or enhance or improve the therapeutic effect
(s) of another therapy (e.g., a therapeutic agent).
[0066] A therapeutically effective amount may refer to the amount
of a therapy (e.g., a therapeutic agent) sufficient to delay or
minimize the spread of cancer. A therapeutically effective amount
may also refer to the amount of a therapy (e.g., a therapeutic
agent) that provides a therapeutic benefit in the treatment or
management of cancer. Further, a therapeutically effective amount
with respect to a therapeutic agent of the invention means that
amount of therapeutic agent alone, or in combination with other
therapies, that provides a therapeutic benefit in the treatment or
management of cancer. Used in connection with an amount of a TLR3
agonist, the term can encompass an amount that improves overall
therapy, reduces or avoids unwanted effects, or enhances the
therapeutic efficacy of or synergizes with another therapy (e.g., a
therapeutic agent).
[0067] As used herein, the terms "treat", "treatment" and
"treating" refer to the reduction or amelioration of the
progression, severity, and/or duration of cancer, particularly a
solid tumor, for example a breast cancer, or one or more symptoms
thereof that results from the administration of one or more
therapies (e.g., one or more prophylactic and/or therapeutic
agents).
[0068] As used herein, the terms "prevent", "preventing", and
"prevention" refer to the prevention of the recurrence, onset, or
development of cancer or one or more symptoms. thereof in a
subject, said prevention resulting from a therapy (e.g., the
administration of a prophylactic or therapeutic agent), or a
combination therapy (e.g., the administration of a combination of
prophylactic or therapeutic agents).
[0069] As used herein, the terms "therapies" and "therapy" can
refer to any protocol (s), method (s) and/or agent (s) that can be
used in the prevention, treatment, management or amelioration of
cancer or one or more symptoms thereof. In certain embodiments, the
terms "therapy" and "therapies" refer to cancer chemotherapy,
radiation therapy, hormonal therapy, biological therapy, and/or
other therapies useful for the prevention, management, or treatment
of cancer known to an oncologist skilled in the art.
[0070] As used herein, the terms "manage", "managing", and
"management" refer to the beneficial effects that a subject derives
from a therapy (e.g., a prophylactic or therapeutic agent), which
does not result in a cure of the disease. In certain embodiments, a
subject is administered one or more therapies (e.g., prophylactic
or therapeutic agents) to "manage" a disease so as to prevent the
progression or worsening of the disease.
[0071] The term "TLR3 agonist" refers to an affinity agent (i.e., a
molecule that binds a target molecule) capable of activating a TLR3
polypeptide to induce a full or partial receptor-mediated response.
For example, an agonist of TLR3 induces TLR3-mediated signalling,
either directly or indirectly. A TLR3 agonist, as used herein, may
but is not required to bind a TLR3 polypeptide, and may or may not
interact directly with the TLR3 polypeptide.
[0072] A "nucleotide agonist" or "nucleic acid agonist" refers to
the situation where the affinity agent comprises or consists of
nucleotides and/or nucleic acid(s). "Antibody agonist" refers to
the situation where the affinity agent is an antibody.
[0073] The terms "polynucleotide" and "nucleic acid", used
interchangeably herein, refer to polymeric forms of nucleotides of
any length, either ribonucleotides or deoxynucleotides. Thus, these
terms include, but are not limited to, single-, double-, or
multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a
polymer comprising purine and pyrimidine bases or other natural,
chemically or biochemically modified, non-natural, or derivatized
nucleotide bases. These terms further include, but are not limited
to, mRNA or cDNA that comprise intronic sequences (see, e.g., Niwa
et al. (1999) Cell 99(7):691-702). The backbone of the
polynucleotide can comprise sugars and phosphate groups (as may
typically be found in RNA or DNA), or modified or substituted sugar
or phosphate groups. Alternatively, the backbone of the
polynucleotide can comprise a polymer of synthetic subunits such as
phosphoramidites and thus can be an oligodeoxynucleoside
phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer.
Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi
et al. (1996) Nucl. Acids Res. 24:23181 0 2323. A polynucleotide
may comprise modified nucleotides, such as methylated nucleotides
and nucleotide analogs, uracyl, other sugars, and linking groups
such as fluororibose and thioate, and nucleotide branches. The
sequence of nucleotides may be interrupted by nonnucleotide
components. A polynucleotide may be further modified after
polymerization, such as by conjugation with a labeling component.
Other types of modifications included in this definition are caps,
substitution of one or more of the naturally occurring nucleotides
with an analog, and introduction of means for attaching the
polynucleotide to proteins, metal ions, labeling components, other
polynucleotides, or a solid support.
[0074] As used herein, the term "host cell" includes a particular
subject cell transfected with a nucleic acid molecule and the
progeny or potential progeny of such a cell.
[0075] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences (i.e.,
% identity number of identical overlapping positions/total number
of positions.times.100%). In one embodiment, the two sequences are
the same length. The determination of percent identity between two
sequences can also be accomplished using a mathematical algorithm.
A preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A.
87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl.
Acad. Sci. U.S.A. 90:5873. Such an algorithm is incorporated into
the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol.
Biol. 215:403. BLAST nucleotide searches can be performed with the
NBLAST nucleotide program parameters set, e.g., for score=100,
wordlength=12 to obtain nucleotide sequences used. The percent
identity between two sequences can be determined using techniques
similar to those described above, with or without allowing gaps. In
calculating percent identity, typically only exact matches are
counted.
[0076] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing
under which nucleotide sequences at least 30% (preferably, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%)
identical to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989). In one, non-limiting example stringent
hybridization conditions are hybridization at 6.times. sodium
chloride/sodium citrate (SSQ at about 45.degree. C., followed by
one or more washes in 0.1.times.SSC, 0.2% SDS at about 68.degree.
C. In a preferred, non-limiting example stringent hybridization
conditions are hybridization in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. (i.e., one or more washes at 50.degree. C.,
55.degree. C., 60.degree. C. or 65.degree. C.). It is understood
that the nucleic acids of the invention do not include nucleic acid
molecules that hybridize under these conditions solely to a
nucleotide sequence consisting of only A or T nucleotides. In a
particular embodiment, typical stringent hybridisation conditions
include temperatures above 30.degree. C., preferably above
35.degree. C., more preferably in excess of 42.degree. C., and/or
salinity of less than about 500 mM, preferably less than 200 mM.
Hybridization conditions may be adjusted by the skilled person by
modifying the temperature, salinity and/or the concentration of
other reagents such as SDS, SSC, etc.
TLR3
[0077] "TLR3", "TLR3 polypeptide" and "TLR3 receptor", used
interchangeably, are used herein to refer to Toll Like Receptor 3,
a member of the Toll-like receptor (TLRs) family. Its amino acid
sequence of is shown in SEQ ID NO: 2 (NCBI accession number
NP.sub.--003256, the disclosure of which is incorporated herein by
reference). As mentioned, it will be appreciated that any TLR3
polypeptide fragment or homologue can be used in accordance with
the present methods. In one aspect, the TLR3 polypeptide may
comprise an amino acid sequence of at least about 25, 30, 35, 40,
45, 50, 60, 70, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900 or
904 amino acid residues in length, of which at least about 50-80%,
preferably at least about 60-70%. more preferably at least about
65%, 75%, 80%, 85% or 90%. 95%, 98%, 99% or 99.5% of the amino acid
residues are identical or similar amino acids to the sequence of
the full-length native human TLR3 polypeptide (for example SEQ ID
NO: 2 for human TLR3). Identity or similarity may be determined
using any desired algorithm, including the algorithms and
parameters for determining homology which are described herein.
[0078] Toll Like Receptor 3 is a member of the Toll-like receptor
(TLR) family which plays a fundamental role in pathogen recognition
and activation of innate immunity. TLRs are highly conserved from
Drosophila to humans and share structural and functional
similarities. They recognize pathogen-associated molecular patterns
(PAMPs) that are expressed on infectious agents, and mediate the
production of cytokines necessary for the development of effective
immunity. The various TLRs exhibit different patterns of
expression. This receptor is most abundantly expressed in placenta
and pancreas, and is restricted to the dendritic subpopulation of
the leukocytes. It recognizes dsRNA associated with viral
infection, and induces the activation of NF-kappaB and the
production of type I interferons. It may thus play a role in host
defense against viruses. TLR3 mRNA sequence is described in NCBI
accession number NM.sub.--003265, the sequence of which is shown in
NO: 1. TLR3 is described in WO 98/50547 (the disclosure of which is
incorporated herein by reference).
[0079] As used in the present application, the term "TLR3 gene"
designates the Toll Like Receptor 3 gene, as well as variants,
analogs and fragments thereof, including alleles thereof (e.g.,
germline mutations). Such variants include, for instance,
naturally-occurring variants due to allelic variations between
individuals (e.g., polymorphisms), alternative splicing forms, etc.
Variants are preferably substantially homologous to NM.sub.--003265
sequence, i.e., exhibit a nucleotide sequence identity of at least
about 65%, typically at least about 75%, preferably at least about
85%, more preferably at least about 95% with NM.sub.--003265
sequence. A particular example of a TLR3 gene comprises
NM.sub.--003265 sequence. Variants and analogs of a TLR3 gene also
include nucleic acid sequences, which hybridize to a sequence as
defined above (or a complementary strand thereof) under stringent
hybridization conditions.
[0080] A fragment of a TLR3 gene designates any portion of at least
about 8 consecutive nucleotides of a sequence as disclosed above,
preferably at least about 15, more preferably at least about 20
nucleotides, further preferably of at least 30 nucleotides.
Fragments include all possible nucleotide lengths between 8 and 100
nucleotides, preferably between 15 and 100, more preferably between
20 and 100.
[0081] The term "gene" shall be construed to include any type of
coding nucleic acid, including genomic DNA (gDNA), complementary
DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of
corresponding RNA. The term gene particularly includes recombinant
nucleic acids encoding TLR3, i.e., any non naturally occurring
nucleic acid molecule created artificially, e.g., by assembling,
cutting, ligating or amplifying sequences. A TLR3 gene is typically
double-stranded, although other forms may be contemplated, such as
single-stranded. TLR3 genes may be obtained from various sources
and according to various techniques known in the art, such as by
screening DNA libraries or by amplification from various natural
sources. Recombinant nucleic acids may be prepared by conventional
techniques, including chemical synthesis, genetic engineering,
enzymatic techniques, or a combination thereof.
[0082] A TLR3 polypeptide designates any protein or polypeptide
encoded by a TLR3 gene as disclosed above. The term "polypeptide"
refers to any molecule comprising a stretch of amino acids. This
term includes molecules of various lengths, such as peptides and
proteins. The polypeptide may be modified, such as by
glycosylations and/or acetylations and/or chemical reaction or
coupling, and may contain one or several non-natural or synthetic
amino acids. A specific example of a TLR3 polypeptide comprises all
or part of NP.sub.--003256 sequence.
TLR3-Expressing Tumors Types
[0083] It has also been found that certain classes of patients with
cancer treated in accordance with the procedure described using a
TLR3 agonist exhibit greater survival than other patients. One
class of patients in which enhanced survival was found were
patients having tumors that express a TLR3 protein.
[0084] Determining whether tumor types express TLR3 can be carried
out as described in the examples, e.g. by detecting the presence of
one or more TLR3 polypeptides in a biological sample from a cancer
patient, generally from a tumor biopsy. The inventors provide
herein that several tumor types can express TLR3 proteins and that
these types of tumors can be treated with a TLR3 agonist according
to the invention.
[0085] In other specific embodiments, a diagnostic assay is
performed on a tumor sample from a patient to determine whether the
tumor sample comprises TLR3-expressing cells. Such assays are
described herein; for example antibody-based immunohistochemistry
assays can be used advantageously. Preferably a tumor biopsy is
performed, yielding a biological sample. A determination that said
biological sample comprises TLR3 expressing cells indicates that
the patient can benefit from the TLR3 agonist administration. The
patient is then treated with the TLR3 agonist.
[0086] Preferably, the step of determining whether cancer cells in
said subject express a TLR3 receptor is performed on a tumoral
sample derived from a patient. For example, the sample can be a
biopsy of the patient's tumor, a cell or tissue culture, etc. Such
sample can be obtained by conventional methods. In a particular
embodiment, the sample is obtained by non-invasive methods and/or
from tissue collections.
[0087] Therefore, in one embodiment of the methods and uses
according to the present invention, the step of determining whether
cancer cells in said subject express a TLR3 receptor comprises
providing a tumoral sample from the patient and detecting the
expression of a TLR3. The expression of a TLR3 may be detected at
the nucleic acid level or at the polypeptide level.
[0088] Various techniques known in the art may be used to detect or
quantify TLR3, including sequencing, hybridisation, amplification
and/or binding to specific ligands (such as antibodies). Suitable
methods include Southern blot (for DNAs), Northern blot (for RNAs),
fluorescent in situ hybridization (FISH), gel migration, ELISA,
radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA).
[0089] Some of these approaches are particularly suited for
assessing a polypeptide sequence or expression level, such as
Northern blot, ELISA and RIA. These latter require the use of a
ligand specific for the polypeptide, more preferably of a specific
antibody.
[0090] Different types of ligands may be used, such as specific
antibodies. In a specific embodiment, the sample is contacted with
an antibody specific for a TLR3 polypeptide and the formation of an
immune complex is determined. Various methods for detecting an
immune complex can be used, such as ELISA, radioimmunoassays (RIA)
and immuno-enzymatic assays (IEMA).
[0091] Within the context of this invention, an antibody designates
a polyclonal antibody, a monoclonal antibody, as well as fragments
or derivatives thereof having substantially the same antigen
specificity. Fragments include Fab, Fab'2, CDR regions, etc.
Derivatives include single-chain antibodies, humanized antibodies,
poly-functional antibodies, etc. TLR3-specific antibodies suitable
for use in the present invention are commercially available, such
as (TLR3 monoclonal antibodies, Ref 12-9039 and 12-9039,
eBioscience, USA; or polyclonal anti TLR3, Ref abl3555, abcam, UK;
etc).
[0092] In a specific embodiment, the method comprises contacting a
sample from the subject with (a support coated with) an antibody
specific for TLR3 polypeptide, and determining the presence of an
immune complex.
[0093] In an alternative embodiment, the expression of a TLR3
receptor in said cancer cell is determined using a TLR3-specific
primer or probe. Such primer or probes are designed to specifically
hybridise with a TLR3 gene, under suitable hybridisation
conditions, thereby allowing detection of a gene or RNA coding for
TLR3. A particular embodiment comprises contacting a tumor sample
from the patient with a TLR3-specific primer or probe, and
determining the existence of a hybrid or amplification product. The
presence (or amount) of TLR3 mRNA in a sample can provide an
indication as to the expression of said receptor. Such
determination may be accomplished by various techniques known in
the art, including through RT-PCR. To that purpose, total RNA is
isolated from cancer cells using commercially available kits, such
as the RNeasy Mini kit (Qiagen, Valencia, Calif.). DNase I-treated
total RNA (3 .mu.g) is reverse-transcribed by using random primers
with RNaseH-fiee reverse transcriptase (Invitrogen, San Diego,
Calif.). TLR3 can be amplified using specific primers described
below. TLR3 (5'-CTCAGAAGATTACCAGCCGCC-3' (SEQ ID NO:
3)/5'-CCATTATGAGACAGATCTAATG-3' (SEQ ID NO: 4)) (see
US2003/0165479, the disclosure of which is incorporated herein by
reference).
[0094] Prior to determining expression of TLR3, the sample may be
treated to improve availability of TLR3 nucleic acids or
polypeptides. Such treatment may include, for instance, a lysis of
the cells or tissue (e.g., mechanical, enzymatic or physical).
[0095] The invention also relates to a diagnostic kit comprising
products and reagents for detecting in a tumoral sample from a
subject the expression of a TLR3 gene or polypeptide. Said
diagnostic kit according to the present invention comprises any
primer, any pair of primers, any nucleic acid probe and/or any
ligand, preferably antibody, described in the present invention.
Said diagnostic kit according to the present invention can further
comprise reagents and/or protocols for performing a hybridization,
amplification or antigen-antibody immune reaction.
[0096] In addition to the discovery that breast cancer patients
treated with TLR3 agonist demonstrate increased survival, it was
found that a class of patients in which enhanced survival was found
when treated in accordance with the procedure described using a
TLR3 agonist were patients having metastatic or recurrent, or
aggressive breast cancers. Another class of patients in which
enhanced survival was found when treated in accordance with the
procedure described using a TLR3 agonist were patients having lymph
node positive breast cancers.
[0097] It is provided that cells from breast cancer samples express
TLR3. However, it is also envisaged that samples from subjects
having other tumor types will contain cells expressing TLR3, and
that these subjects can be treated with TLR3 agonists; it will be
appreciated that the skilled person may determine which tumors are
suitable for treatment using available methods, including the
methods described herein.
TLR3 Agonists
[0098] The TLR3 agonists according to the present invention can be
selected from any suitable agent. For example, TLR3 agonists can be
selected from a range of nucleic acid agonists; other agonists,
whether nucleic acid based, proteinaceous or small molecules, can
be tested using known assays.
[0099] Generally, any proteinaceous, nucleic acid or small molecule
candidate TLR3 agonist can be identified using known assays. For
example, assays for detecting TLR3 agonism of test compounds are
described, for example, in PCT publication nos. WO 03/31573, WO
04/053057, WO 04/053452, and WO 04/094671, the disclosures of each
of which are incorporated herein by reference.
[0100] Regardless of the particular assay employed, a compound can
be identified as an agonist of TLR3 if performing the assay with a
compound, which results in at least a threshold increase of some
biological activity mediated by TLR3. Conversely, a compound may be
identified as not acting as an agonist of TLR3 if, when used to
perform an assay designed to detect biological activity mediated by
TLR3, the compound fails to elicit a threshold increase in the
biological activity. Unless otherwise indicated, an increase in
biological activity refers to an increase in the same biological
activity over that observed in an appropriate control. An assay may
or may not be performed in conjunction with the appropriate
control. With experience, one skilled in the art may develop
sufficient familiarity with a particular assay (e.g., the range of
values observed in an appropriate control under specific assay
conditions) that performing a control may not always be necessary
to determine the TLR3 agonism of a compound in a particular assay.
The precise threshold increase of TLR3-mediated biological activity
for determining whether a particular compound is or is not an
agonist of TLR3 in a given assay may vary according to factors
known in the art including but not limited to the biological
activity observed as the endpoint of the assay, the method used to
measure or detect the endpoint of the assay, the signal-to-noise
ratio of the assay, the precision of the assay. For example,
regardless of the particular assay employed, a compound can
generally be identified as an agonist of TLR3 if performing the
assay with a compound results in at least a threshold increase of
some biological activity mediated by TLR3.
[0101] An assay may or may not be performed in conjunction with the
appropriate control. With experience, one skilled in the art may
develop sufficient familiarity with a particular assay (e.g., the
range of values observed in an appropriate control under specific
assay conditions) that performing a control may not always be
necessary to determine the TLR3 agonism of a compound in a
particular assay, and whether the same assay is being used to
determine the agonism of a compound for multiple TLRs.
[0102] The precise threshold increase of TLR3-mediated biological
activity for determining whether a particular compound is or is not
an agonist of TLR3 in a given assay may vary according to factors
known in the art including but not limited to the biological
activity observed as the endpoint of the assay, the method used to
measure or detect the endpoint of the assay, the signal-to-noise
ratio of the assay, the precision of the assay, and whether the
same assay is being used to determine the agonism of a compound for
multiple TLRs. Accordingly it is not practical to set forth
generally the threshold increase of TLR3-mediated biological
activity required to identify a compound as being an agonist or a
non-agonist of TLR3 for all possible assays. Those of ordinary
skill in the art, however, can readily determine the appropriate
threshold with due consideration of such factors.
[0103] Assays employing HEK293 cells transfected with an
expressible TLR3 structural gene may use a threshold of, for
example, at least a three-fold increase in a TLR3-mediated
biological activity (e.g., NF-KB activation) when the compound is
provided at a concentration of, for example, from about 1 microM to
about 10 microM for identifying a compound as an agonist of the
TLR3 transfected into the cell. However, different thresholds
and/or different concentration ranges may be suitable in certain
circumstances. Also, different thresholds may be appropriate for
different assays.
[0104] In certain embodiments, the TLR3 agonist can be a natural
agonist of a TLR3 or a synthetic TLR3 agonist compound.
[0105] In preferred embodiments of the invention, a TLR3 agonist is
used to treat a patient. TLR3 agonists are well known in the art
and suitable TLR3 agonists are available. Further TLR3 agonists, or
derivatives or analogs of known TLR3 agonists can be readily
identified, made and/or assessed.
[0106] The most commonly used TLR3 agonist are nucleic acid based
agonists. Thus in preferred aspects, a TLR3 agonist for use
according to the present invention are nucleotide or nucleic acid
based. Nucleotide or nucleic-acid based compounds can be assessed
for their ability to act as an TLR3 agonist using readily available
methods. The nucleic acid based TLR3 agonist can be single-stranded
or double-stranded or a mixture thereof. The nucleic acid based
TLR3 agonist can comprise deoxyribonucleotides, or ribonucleotides
or a mixture thereof. The nucleotides can be natural or synthetic,
and may be derivatives or analogs of natural nucleotides, such as
for example in Kandimalla et al. ((2003) Nucl. Acid. Res. 31(9):
2393-2400).
[0107] The particular TLR3 agonist used in the clinical study, the
analysis of which was at the origin of the observations on which
the present invention is based was a double stranded RNA compound
(dsRNA). The specific compound was polyadenylic-polyuridylic acid,
i.e., poly (A): poly (U), polyAU or poly A: U. Double-stranded RNA
which represents either genomic or life cycle intermediate material
of many viruses activates cells through binding to the
dsRNA-dependent protein kinase (PKR), a kinase that initiates a
complex molecular anti-viral program (Gil, J., Alcami, J., and
Esteban, M. 1999. Induction of apoptosis by
double-stranded-RNA-dependent protein kinase (PKR) involves the
alpha subunit of eukaryotic translation initiation factor 2 and
NF-kappaB. Mol Cell Biol 19:4653-4663). dsRNA was however only
recently suggested to act through TLR3 (Alexopoulou, L., Holt, A.
C., Medzhitov, R., and Flavell, R. A. 2001. Recognition of
double-stranded RNA and activation of NF-kappaB by Toll-like
receptor 3. Nature 413:732-738). It was reported that dsRNA
triggers the production of type 1 IFN, and dsRNA has been reported
to have promise for certain clinical applications such as
anti-viral therapies. A dsRNA compound referred to as Ampligen, for
example, has been studied for its ability induce type 1 IFN
production and as a consequence to treat viral infection.
[0108] Within the context of the present invention, the term
"double-stranded RNA" molecule designates any therapeutically or
prophylactically effective (synthetic) double-stranded RNA
compound. Such compounds are typically active per se, i.e., they do
not encode a polypeptide or do not require translation to be
active. dsRNA TLR3 agonists can have any suitable length.
Preferably, a dsRNA molecule TLR3 agonist has a length of at least
about 10 base pairs (bp), 20 bp, 30 bp, 50 bp, 80 bp, 100 bp, 200
bp, 400 bp, 600 bp, 800 bp or 1000 bp. In one aspect the dsRNA
molecule is a short dsRNA having a chain length of less than 30 bp,
50 bp, 80 bp, 100 bp or 200 bp. In another embodiment, the dsRNA
molecule is a longer dsRNA, but having a chain length of less than
400 bp, 600 bp, 800 bp or 1000 bp. In another embodiment, the dsRNA
molecule is a long dsRNA having a chain length of greater than 1000
bp. In one aspect, a dsRNA composition comprises a heterogenous
mixture of dsRNA molecules, wherein a plurality of molecules have
differing lengths. Preferably the dsRNA molecules have on average a
length of at least about 10 bp, 20 bp, 30 bp, 50 bp, 80 bp, 100 bp,
200 bp, 400 bp, 600 bp, 800 bp or 1000 bp. In another embodiment, a
dsRNA composition comprises a plurality dsRNA molecules where at
least 20%, 50%, 80%, 90% or 98% of dsRNA molecules have a length of
at least about 10 bp, 20 bp, 30 bp, 50 bp, 80 bp, 100 bp, 200 bp,
400 bp, 600 bp, 800 bp or 1000 bp. In a preferred embodiment dsRNA
composition has a substantially homogenous mixture of dsRNA
molecules, where substantially all the molecules do not differ in
chain length by more than 30 bp, 50 bp, 80 bp, 100 bp or 200 bp.
Average chain length of nucleic acid TLR3 agonists can be
determined easily, for example, by gel permeation
chromatography.
[0109] Previous studies of double-stranded RNA (dsRNA) assessing
their ability to be effective interferon inducers suggested that
dsRNA agents must possess the secondary structure of a double
stranded helix. Other dsRNA agents which have also been shown to be
suitable as TLR3 agonist include double-stranded polynucleotides
which are not complementary or not perfectly complementary; these
have been known as, so-called "mismatched" or "loop-out" structures
and exist in naturally occurring RNAs such as transfer tRNAs,
ribosomal RNAs and the viral RNA secondary structures. One commonly
cited dsRNA compound, Ampligen, comprises a structure where a few
parts of cytidine in the poly I: poly C structure are replaced with
uridine (i.e. mismatched RNA); this compound has been reported to
have physiological activity similar to that of the parent poly I:
poly C. However it will be appreciated that TLR3 agonists of any
type and configuration can be used in accordance with this
invention.
[0110] Generally, the polynucleotides need to be resistant to
nucleases in order to remain as macromolecules for a sufficient
length of time; polynucleotides are less sensitive to nuclease
attack when they are in a helical complex. However, certain analogs
such as Ampligen.TM. appear to retain their TLR3 agonist
activity.
[0111] In a particular embodiment, each strand of these dsRNAs can
have a length comprised between about 5 and 50 bases, more
preferably between 5 and 40, 35, 30, 25 or 20 bases. Each strand is
preferably perfectly complementary to the other. Preferred examples
of such dsRNAs are homopolyRNAs, i.e., dsRNAs in which each strand
comprises essentially a repeat of the same base; or comprise a
homopolyRNA region.
[0112] The base may be any naturally occurring base (e.g., polyA,
polyU, polyC, polyG) or non-naturally occurring (e.g., chemically
synthesized or modified) base (e.g., polyI). Polynucleotides
typified by polyinosinic--polycytidylic acid, i.e., poly (I):
poly(C) or poly I: C and polyadenylic-polyuridylic acid, i.e., poly
(A): poly (U) or poly A: U, are well-known compounds in the art and
have been known to induce interferon production by immune cells.
Thus in preferred embodiments, the TLR3 agonist for use according
to the invention is a double stranded nucleic acid selected from
the group consisting of: polyinosinic acid and polycytidylic acid,
polyadenylic acid and polyuridylic acid, polyinosinic acid analogue
and polycytidylic acid, polyinosinic acid and polycytidylic acid
analogue, polyinosinic acid analogue and polycytidylic acid
analogue, polyadenylic acid analogue and polyuridylic acid,
polyadenylic acid and polyuridylic acid analogue, and polyadenylic
acid analogue and polyuridylic acid analogue.
[0113] It will be appreciated that nucleic acid-based agonists of
TLR3 can be designed using any suitable method. Preferably, the
basic requirement of stability and resistance to nuclease attack
and the preferences for chain length are taken into account, and
that structural changes can be tested and assessed with reference
to the a rA.sub.n:rU.sub.n or rI.sub.n:rC.sub.n complex for
example. Measures can be taken to increase stability and resistance
to nucleases, or to increase or optionally decrease
interferon-inducing action.
[0114] Other examples of dsRNA include nucleic acids described in
U.S. Pat. Nos. 5,298,614 and 6,780,429. U.S. Pat. No. 5,298,614
reports that when chain length of the double stranded nucleic acid
derivatives is limited to certain ranges, the resulting substances
exhibit desired physiological activity with markedly less toxicity,
providing polynucleotides having a length of about 50 to 10,000 as
calculated by base pair numbers. Also described are derivative
wherein the purine or pyrimidine ring in the nucleic acid polymer
is substituted with at least one SH group, or said derivative
contains a disulphide bond, or both (preferred ratio of number of
sulphur atoms to cytidylic acid present in the poly C are 1:6 to
39). U.S. Pat. No. 6,780,429 describes a particular type of dsRNA
compounds that are "chain-shortened" having lengths of about 100 to
1,000 as calculated by base pair numbers, or preferably from 200 to
800, and more preferably from 300 to 600. The latter compounds are
reported to contain low numbers of 2'-5' phosphodiester bonds by a
method designed to avoid phosphate groups causing intramolecular
rearrangement from 3' position to 2' position through a mechanism
called pseudo rotation simultaneously that can occur during
hydrolysis of polynucleotides, resulting in a portion of 3'-5'
phosphodiester bonds in the chain-shortened polynucleotide molecule
being replaced by 2'-5' phosphodiester bonds. The disclosures of
each of these references is incorporated herein by reference.
[0115] Other nucleic acid agonists that can be suitable for use as
TLR3 agonists are provided in: Field et al.: Proc. Nat. Acad. Sci.
U.S. 58, 1004, (1967); Field et al.: Proc. Nat. Acad. Sci. U.S. 58,
2102, (1967); Field et al.: Proc. Nat. Acad. Sci. U.S. 61, 340,
(1968); Tytell et al.: Proc. Nat. Acad. Sci. U.S. 58, 1719, (1967);
Field et al.: J. Gen. Physiol. 56, 905 (1970); De Clercq et al.:
Methods in Enzymology, 78, 291 (1981). A number of synthetic
nucleic acid derivatives have been described, including
homopolymer-homopolymer complexes (Double Strand Nucleic Acid
Polymer such as those in which poly I:C or poly A:U are a parent
structure, where these homopolymer-homopolymer complexes contain:
(1) base modifications, exemplified by Polyinosinic
acid-poly(5-bromocytidylic acid), Polyinosinic
acid-poly(2-thiocytidylic acid), Poly(7-deazainosinic
acid)-polycytidylic acid, Poly(7-deazainosinic
acid)-poly(5-bromocytidylic acid), and Polyinosinic
acid-poly(5-thiouridylic acid); (2) Sugar Modifications,
exemplified by Poly(2'-azidoinosinic acid)-polycytidylic acid; and
(3) Phosphoric Acid Modifications, exemplified by Polyinosinic
acid-poly(cytidyl-5'-thiophosphoric acid). Other synthetic nucleic
acid derivatives that have been described include interchanged
copolymers, exemplified by Poly (adenylic acid-uridylic acid); and
homopolymer-copolymer complexes, exemplified by Polyinosinic
acid-poly (cytidylic acid-uridylic acid) and Polyinosinic acid-poly
(citydylic acid-4-thiouridylic acid). Other synthetic nucleic acid
derivatives that have been described include complexes of synthetic
nucleic acid with polycation, exemplified by Polyinosinic
acid-polycytidylic acid-poly-L-lysinecarboxy-methylcellulose
complex (called "Poly ICLC"). Yet another example of synthetic
nucleic acid derivative is Polyinosinic acid-poly
(1-vinylcytosine).
[0116] One example of a TLR3 agonist is Ampligen.TM. (Hemispherx,
Inc., of Rockville, Md., U.S.A.), a dsRNA formed by complexes of
polyriboinosinic and polyribocytidylic/uridylic acid, such as
rI.sub.n: r (C.sub.x, U or G).sub.n where x has a value from 4 to
29, e.g., rI.sub.n: r (C.sub.12 U).sub.n. Many mismatched dsRNA
polymers which behave similarly to Ampligen have been studied;
mismatched dsRNA based on poly I: C has included complexes of a
polyinosinate and a polycytidylate containing a proportion of
uracil bases or guanidine bases, e.g., from 1 in 5 to 1 in 30 such
bases. The key therapeutic advantage of mismatched dsRNAs over
other forms of natural and/or synthetic dsRNAs a reported reduction
in toxicity over compounds such as those described in Lampson et al
in U.S. Pat. No. 3,666,646.
[0117] Specific examples of double-stranded RNA according to the
present invention further include Polyadenur (Ipsen) and Ampligen
(Hemispherx). Polyadenur is a polyA/U RNA molecule, i.e., contains
a polyA strand and a polyU strand. Polyadenur has been developed
for the potential treatment of hepatitis B virus (HBV) infection.
Ampligen is of a polyI/polyC compound (or a variant thereof
comprising a polyI/polyC12U RNA molecule). Ampligen is disclosed
for instance in EP 281 380 or EP 113 162. Ampligen has been
proposed for the treatment of cancer, viral infections and immune
disorders. It was developed primarily for the potential treatment
of myalgic encephalomyelitis (ME, or chronic fatigue
syndrome/chronic fatigue immune dysfunction syndrome,
CFS/CFIDS).
[0118] A particular example of a dsRNA for use in the present
invention is a dsRNA comprising a polyA/polyU region, wherein each
strand of said dsRNA contains less than 25 bases.
[0119] Another particular example of a dsRNA for use in the present
invention is a dsRNA comprising a polyI/polyC (U) region, wherein
each strand of said dsRNA contains less than 25 bases.
[0120] Further dsRNAs have been disclosed in the literature or may
be developed, which can be used within the present invention. More
generally, any synthetic double-stranded homopolyRNA may be used in
the context of this invention.
[0121] TLR3 agonist can also be any organic or inorganic substance,
such as a lipid, peptide, polypeptide, small molecule, etc., in
isolated or in mixture with other substances. The TLR3 agonist
candidate may be selected from a combinatorial library of products,
for instance. In a preferred embodiment, the TLR3 agonist is an
antibody directed against TLR3 receptor and which is capable of
activating a TLR3 receptor to induce a full or partial
receptor-mediated response. The TLR3 agonist can also be an
antibody fragment or derivative of an antibody directed against
TLR3 receptor and which is capable of activating a TLR3 receptor to
induce a full or partial receptor-mediated response.
Anti-TLR3 Antibodies
[0122] The present invention involves the production and use of
antibodies, antibody fragments, or antibody derivatives that are
suitable for use in humans and that target the TLR3 protein. The
antibodies of this invention may be produced by any of a variety of
techniques known in the art. Typically, they are produced by
immunization of a non-human animal, preferably a mouse, with an
immunogen comprising a TLR3 protein present on the surface of tumor
cells. The receptor may comprise entire TLR3 expressing tumor
cells, cell membranes, the full length sequence of the TLR3
protein, or a fragment or derivative thereof, typically an
immunogenic fragment, i.e., a portion of the polypeptide comprising
an epitope exposed on the surface of cells expressing the TLR3.
Such fragments typically contain at least 7 consecutive amino acids
of the mature polypeptide sequence, even more preferably at least
10 consecutive amino acids thereof. They are essentially derived
from the extracellular domain of the TLR3 protein. In preferred
embodiments, the TLR3 protein or peptide used to generate
antibodies is a human TLR3 protein or peptide.
[0123] In a most preferred embodiment, the immunogen comprises a
wild-type human TLR3 polypeptide in a lipid membrane, typically at
the surface of a cell. In a specific embodiment, the immunogen
comprises intact TLR3 expressing tumor cells, optionally treated or
lysed.
[0124] In one embodiment, the antibodies are derived from one or
more already-existing monoclonal antibodies that recognize the
TLR3. Such antibodies can be directly or indirectly labeled (i.e.,
used with a labeled secondary antibody) for use as diagnostic
antibodies for the herein-described typing step to determine the
TLR3 status of patients. In addition, the antibodies can be made
suitable for human administration (preferably chimeric,
CDR-grafted, human or humanized antibodies) and, optionally, made
toxic as described herein for use as cytotoxic antibodies in the
present therapeutic methods.
[0125] The present diagnostic or therapeutic (e.g., cytotoxic)
antibodies can be full length antibodies or antibody fragments or
derivatives. Examples of antibody fragments include Fab, Fab',
Fab'-SH, F(ab').sub.2, and Fv fragments; diabodies; single-chain Fv
(scFv) molecules; single chain polypeptides containing only one
light chain variable domain, or a fragment thereof that contains
the three CDRs of the light chain variable domain, without an
associated heavy chain moiety; single chain polypeptides containing
only one heavy chain variable region, or a fragment thereof
containing the three CDRs of the heavy chain variable region,
without an associated light chain moiety; and multispecific
antibodies formed from antibody fragments. Such fragments and
derivatives and methods of preparing them are well known in the
art. For example, pepsin can be used to digest an antibody below
the disulfide linkages in the hinge region to produce F
(ab)'.sub.2, a dimer of Fab which itself is a light chain joined to
V.sub.H--C.sub.H1 by a disulfide bond. The F (ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F (ab)'.sub.2 dimer into a
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology.
[0126] The preparation of monoclonal or polyclonal antibodies is
well known in the art, and any of a large number of available
techniques can be used (see, e.g., Kohler & Milstein, Nature
256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy
(1985)). Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to
desired polypeptides. Also, transgenic mice, or other organisms
such as other mammals, may be used to express humanized, chimeric,
or similarly-modified antibodies. Alternatively, phage display
technology can be used to identify antibodies and heteromeric Fab
fragments that specifically bind to selected antigens (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990); Marks et al.,
Biotechnology 10:779-783 (1992)). For example, the repertoire may
be any (recombinant) repertoire of antibodies or fragments thereof,
optionally displayed by any suitable structure (e.g., phage,
bacteria, synthetic complex, etc.).
[0127] The step of immunizing a non-human mammal with an antigen
may be carried out in any manner well known in the art for (see,
for example, E. Harlow and D. Lane, Antibodies: A Laboratory
Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1988)). Generally, the immunogen is suspended or dissolved in
a buffer, optionally with an adjuvant, such as complete Freund's
adjuvant. Methods for determining the amount of immunogen, types of
buffers and amounts of adjuvant are well known to those of skill in
the art and are not limiting in any way on the present
invention.
[0128] Similarly, the location and frequency of immunization
sufficient to stimulate the production of antibodies is also well
known in the art. In a typical immunization protocol, the non-human
animals are injected intraperitoneally with antigen on day 1 and
again about a week later. This is followed by recall injections of
the antigen around day 20, optionally with adjuvant such as
incomplete Freund's adjuvant. The recall injections are performed
intravenously and may be repeated for several consecutive days.
This is followed by a booster injection at day 40, either
intravenously or intraperitoneally, typically without adjuvant.
This protocol results in the production of antigen-specific
antibody-producing B cells after about 40 days. Other protocols may
also be utilized as long as they result in the production of B
cells expressing an antibody directed to the antigen used in
immunization.
[0129] In another embodiment, lymphocytes from an unimmunized
non-human mammal are isolated, grown in vitro, and then exposed to
the immunogen in cell culture. The lymphocytes are then harvested
and the fusion step described below is carried out.
[0130] For monoclonal antibodies, which are preferred for the
purposes of the present invention, the next step is the isolation
of cells, e.g., lymphocytes, splenocytes, or B cells, from the
immunized non-human mammal and the subsequent fusion of those
splenocytes, or B cells, or lymphocytes, with an immortalized cell
in order to form an antibody-producing hybridoma. Accordingly, the
term "preparing antibodies from an immunized animal," as used
herein, includes obtaining B-cells/splenocytes/lymphocytes from an
immunized animal and using those cells to produce a hybridoma that
expresses antibodies, as well as obtaining antibodies directly from
the serum of an immunized animal. The isolation of splenocytes,
e.g., from a non-human mammal is well-known in the art and, e.g.,
involves removing the spleen from an anesthetized non-human mammal,
cutting it into small pieces and squeezing the splenocytes from the
splenic capsule and through a nylon mesh of a cell strainer into an
appropriate buffer so as to produce a single cell suspension. The
cells are washed, centrifuged and resuspended in a buffer that
lyses any red blood cells. The solution is again centrifuged and
remaining lymphocytes in the pellet are finally resuspended in
fresh buffer.
[0131] Once isolated and present in single cell suspension, the
antibody-producing cells are fused to an immortal cell line. This
is typically a mouse myeloma cell line, although many other
immortal cell lines useful for creating hybridomas are known in the
art. Preferred murine myeloma lines include, but are not limited
to, those derived from MOPC-21 and MPC-11 mouse tumors available
from the Salk Institute Cell Distribution Center, San Diego, Calif.
U.S.A., X63 Ag8653 and SP-2 cells available from the American Type
Culture Collection, Rockville, Md. U.S.A. The fusion is effected
using polyethylene glycol or the like. The resulting hybridomas are
then grown in selective media that contains one or more substances
that inhibit the growth or survival of the unfused, parental
myeloma cells. For example, if the parental myeloma cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0132] The hybridomas can be grown on a feeder layer of
macrophages. The macrophages are preferably from littermates of the
non-human mammal used to isolate splenocytes and are typically
primed with incomplete Freund's adjuvant or the like several days
before plating the hybridomas. Fusion methods are described, e.g.,
in (Goding, "Monoclonal Antibodies: Principles and Practice," pp.
59-103 (Academic Press, 1986)), the disclosure of which is herein
incorporated by reference.
[0133] The cells are allowed to grow in the selection media for
sufficient time for colony formation and antibody production. This
is usually between 7 and 14 days. The hybridoma colonies are then
assayed for the production of antibodies that specifically
recognize the desired substrate, e.g. a TLR3 protein. The assay is
typically a calorimetric ELISA-type assay, although any assay may
be employed that can be adapted to the wells that the hybridomas
are grown in. Other assays include immunoprecipitation and
radioimmunoassay. The wells positive for the desired antibody
production are examined to determine if one or more distinct
colonies are present. If more than one colony is present, the cells
may be re-cloned and grown to ensure that only a single cell has
given rise to the colony producing the desired antibody. Positive
wells with a single apparent colony are typically recloned and
re-assayed to ensure that only one monoclonal antibody is being
detected and produced.
[0134] Hybridomas that are confirmed to be producing a monoclonal
antibody of this invention are then grown up in larger amounts in
an appropriate medium, such as DMEM or RPMI-1640. Alternatively,
the hybridoma cells can be grown in vivo as ascites tumors in an
animal.
[0135] After sufficient growth to produce the desired monoclonal
antibody, the growth media containing monoclonal antibody (or the
ascites fluid) is separated away from the cells and the monoclonal
antibody present therein is purified. Purification is typically
achieved by gel electrophoresis, dialysis, chromatography using
protein A or protein G-Sepharose, or an anti-mouse Ig linked to a
solid support such as agarose or Sepharose beads (all described,
for example, in the Antibody Purification Handbook, Amersham
Biosciences, publication No. 18-1037-46, Edition AC, the disclosure
of which is hereby incorporated by reference). The bound antibody
is typically eluted from protein A/protein G columns by using low
pH buffers (glycine or acetate buffers of pH 3.0 or less) with
immediate neutralization of antibody-containing fractions. These
fractions are pooled, dialyzed, and concentrated as needed.
[0136] In preferred embodiments, the DNA encoding an antibody that
binds a determinant present on TLR3 is isolated from the hybridoma,
placed in an appropriate expression vector for transfection into an
appropriate host. The host is then used for the recombinant
production of the antibody, variants thereof, active fragments
thereof, or humanized or chimeric antibodies comprising the antigen
recognition portion of the antibody.
[0137] DNA encoding the monoclonal antibodies of the invention can
be readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Recombinant expression in bacteria of DNA encoding the
antibody is well known in the art (see, for example, Skerra et al.
(1993) Curr. Op. Immunol. 5:256; and Pluckthun (1992) Immunol.
Revs. 130:151. Antibodies may also be produced by selection of
combinatorial libraries of immunoglobulins, as disclosed for
instance in Ward et al. (1989) Nature 341:544.
[0138] Various anti-TLR3 antibodies are commercially available.
Examples include but are not limited to: Clone 40C1285, Imgenex
Corp, Biocarta US, San Diego, Calif.; Clone TLR3.7, Catalog no.
HM2096, HyCuit biotechnology B.V., The Netherlands; and Catalog no.
12-9039, Ebioscience Inc., San Diego, Calif.
Cancer and Therapeutic Methods
[0139] The present invention encompasses treatment protocols that
provide better prophylactic or therapeutic profiles than current
single agent therapies or combination therapies for cancer. In
particular, the invention encompasses the use of a TLR3 agonist for
the prevention, management, treatment or amelioration of cancer or
one or more symptoms thereof.
[0140] Examples of cancers that can be prevented, managed, treated
or ameliorated in accordance with the methods invention include,
but are not limited to solid tumors, and particularly cancers such
as cancer of the head, neck, eye, mouth, throat, esophagus, chest,
bone, lung, colon, rectum, stomach, prostate, breast, ovaries,
kidney, liver, pancreas, and brain.
[0141] The invention provides methods for preventing, managing,
treating or ameliorating cancer that has the potential to
metastasize or has metastasized to an organ or tissue (e.g., bone)
or one or more symptoms thereof, said methods comprising
administering to a subject in need thereof one or more doses of a
prophylactically or therapeutically amount of a TLR3 agonist.
Preferably, the TLR3 agonist is a dsRNA compound. Preferably, the
TLR3 agonist is administered more than once. Preferably, the TLR3
agonist is administered more than once. Optionally, the TLR3
agonist is administered at an interval of less than one month, less
than three weeks, less than two weeks, or less than one week.
Optionally, such treatment may be repeated, for example, every 1,
2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
[0142] The present invention provides methods for preventing,
managing, treating or ameliorating cancer or one or more symptoms
thereof, said methods comprising administering to a subject in need
thereof a dosage of a prophylactically or therapeutically effective
amount of a TLR3 agonist in combination with the administration of
a dosage of a prophylactically or therapeutically effective amount
of one or more other agents useful for cancer therapy. Preferably,
the agonist is a dsRNA compound. Preferably, the TLR3 agonist is
administered more than once. Optionally, the TLR3 agonist is
administered at an interval of less than one month, less than three
weeks, less than two weeks, or less than one week. Optionally, such
treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or
7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, or 12 months.
[0143] The invention provides methods for preventing, managing,
treating or ameliorating cancer that has is refractory to one or
more therapeutic agents or therapies, said methods comprising
administering to a subject in need thereof one or more doses of a
prophylactically or therapeutically amount of a TLR3 agonist.
Preferably, the TLR3 agonist is a dsRNA compound. Preferably, the
doses are administered at an interval of less than one month, less
than three weeks, less than two weeks, or less than one week.
Optionally, such treatment may be repeated, for example, every 1,
2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
[0144] The present invention provides methods for preventing,
treating, managing or ameliorating cancer or one or more symptoms
thereof, said methods comprising administering to a subject in need
thereof a TLR3 agonist alone or in combination with one or more
other therapies (e.g., one or more other prophylactic or
therapeutic agents) useful in the prevention, treatment, management
or amelioration of cancer or one or more symptoms thereof.
Preferably, the TLR3 agonist is a dsRNA compound.
[0145] In one embodiment, a TLR3 agonist (preferably, a dsRNA) is
administered to a subject using a dosing regimen that maintains the
plasma concentration of the agonist at a desirable level. In a
specific embodiment, the plasma concentration of the dsRNA is
maintained at 10 .mu.g/ml, 15 .mu.g/ml, 20 .mu.g/ml, 25 .mu.g/ml,
30 .mu.g/ml, 35 .mu.g/ml, 40 .mu.g/ml, 45 .mu.g/ml or 50 .mu.g/ml.
The plasma concentration that is desirable in a subject will vary
depending on several factors including, but not limited to, the
nature of the cancer, the severity of the cancer, and the
circulation half-life (stability) and binding affinity of the TLR3
agonist.
[0146] The dosage amounts and frequencies of administration
provided herein are encompassed by the terms therapeutically
effective and prophylactically effective. The dosage and frequency
further will typically vary according to factors specific for each
patient depending on the specific therapeutic or prophylactic
agents administered, the severity and type of cancer, the route of
administration, as well as age, body weight.
[0147] Preferably, a therapeutically effective amount of a TLR3
agonist (optionally in combination with another therapeutic agent
or therapeutic protocol) reduces the size of a tumor or the spread
of a tumor in a subject by at least 5%, preferably at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95% or at least 99% relative to a
control such as PBS.
[0148] As used herein, the terms "non-responsive" and "refractory"
describe patients treated with a currently available cancer therapy
(e.g., chemotherapy, radiation therapy, surgery, hormonal therapy
and/or biological therapy/immunotherapy), which is not clinically
adequate to treat or relieve one or more symptoms associated with
cancer. Typically, such patients suffer from severe, persistently
active disease and require additional therapy to ameliorate the
symptoms associated with their cancer. The phrase can also describe
patients who respond to therapy yet suffer from side effects,
relapse, develop resistance, etc.
[0149] In various embodiments, "non-responsive/refractory" means
that at least some significant portion of the cancer cells are not
killed or their cell division arrested. The determination of
whether the cancer cells are "non-responsive/refractory" can be
made either in vivo or in vitro by any method known in the art for
assaying the effectiveness of treatment on cancer cells, using the
art-accepted meanings of "refractory" in such a context. In various
embodiments, a cancer is "non-responsive/refractory" when the
number of cancer cells has not been significantly reduced, or has
increased.
Types of Cancers
[0150] In various embodiments, the present invention provides
methods for determining treatment regimens for cancer subjects. The
methods of the invention can be used to determine treatment
regimens of any cancer, or tumor, for example, but not limited to,
malignancies and related disorders include but are not limited to
the following.
[0151] Leukemias such as but not limited to, acute leukemia, acute
lymphocytic leukemia, acute myclocytic leukemias such as
myeloblastic, promyclocytic, myelomonocytic, monocytic,
erythroleukemia leukemias and myelodysplastic syndrome, chronic
leukemias such as but not limited to, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell
leukemia; polycythemia vera; lymphomas such as but not limited to
Hodgkin's disease, non-Hodgkin's disease; multiple myclomas such as
but not limited to smoldering multiple myeloma, nonsecretory
myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary
plasmacytoma and extramedullary plasmacytoma; Waldenstrom's
macroglobulinemia; monoclonal gammopathy of undetermined
significance; benign monoclonal gammopathy; heavy chain disease;
bone and connective tissue sarcomas such as but not limited to bone
sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
giant cell tumor, fibrosarcoma of bone, chordonia, periosteal
sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),
fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial
sarcoma; brain tumors such as but not limited to, glionia,
astrocytoma, brain stem glioma, ependymoma, oligodendroglioma,
nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningionia, pineocytoma, pineoblastoma, primary
brain lymphoma; breast cancer including but not limited to
adenocarcinoma, lobular (small cell) carcinoma, intraductal
carcinoma, medullary breast cancer, mucinous breast cancer, tubular
breast cancer, papillary breast cancer, Paget's disease, and
inflammatory breast cancer; adrenal cancer such as but not limited
to pheochromocytom and adrenocortical carcinoma; thyroid cancer
such as but not limited to papillary or follicular thyroid cancer,
medullary thyroid cancer and anaplastic thyroid cancer; pancreatic
cancer such as but not limited to, insulinoma, gastrinoma,
glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or
islet cell tumor; pituitary cancers such as but limited to
Cushing's disease, prolactin-secreting tumor, acromegaly, and
diabetes insipius; eye cancers such as but not limited to ocular
melanoma such as iris melanoma, choroidal melanoma, and cilliary
body melanoma, and retinoblastoma; vaginal cancers such as squamous
cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as
squamous cell carcinoma, melanoma, adenocarcinoma, basal cell
carcinoma, and sarcoma; cervical cancers such as but not limited
to, squamous cell carcinoma, and adenocarcinoma; uterine cancers
such as but not limited to endometrial carcinoma and uterine
sarcoma; ovarian cancers such as but not limited to, ovarian
epithelial carcinoma, borderline tumor, genn cell tumor, and
stromal. tumor; esophageal cancers such as but not limited to,
squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,
mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,
melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small
cell) carcinoma; stomach cancers such as but not limited to,
adenocarcinoma, fulminating (polypoid), ulcerating, superficial
spreading, diffusely spreading, malignant lymphoma, liposarcoma,
fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers;
liver cancers such as but not limited to hepatocellular carcinoma
and hepatoblastoma, gallbladder cancers such as adenocarcinoma;
cholangiocarcinomas such as but not limited to papillary, nodular,
and diffuse; lung cancers such as non-small cell lung cancer,
squamous cell carcinoma (epidemoid carcinoma), adenocarcinoma,
large-cell carcinoma and small-cell lung cancer; testicular cancers
such as but not limited to germinal tumor, seminoma, anaplastic,
classic (typical), spermatocytie, nonseminoma, embryonal carcinoma,
teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate
cancers such as but not limited to, adenocarcinoma, leiomyosarcoma,
and rhabdomyosarcoma; penal cancers; oral cancers such as but not
limited to squamous cell carcinoma; basal cancers; salivary gland
cancers such as but not limited to adenocareinoma, mucoepidermoid
carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but
not limited to squamous cell cancer, and cutaneous; skin cancers
such as but not limited to, basal cell carcinoma, squamous cell
carcinoma and melanoma, superficial spreading melanoma, nodular
melanoma, lentigo malignant melanoma, acral lentiginous melanoma;
kidney cancers such as but not limited to renal cell cancer,
adenocareinoma, hypernephroma, fibrosarcoma, transitional cell
cancer (renal pelvis and/or uterer); Wilins' tumor; bladder cancers
such as but not limited to transitional cell carcinoma, squamous
cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers
include inyxosarcoma, osteogenic sarcoma, endotheliosarcoma,
lyinphangioendotheliosarcoma, mesothelioma, synovionia,
hernangioblastoma, epithelial carcinoma, cystadenocarcinoma,
bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma and papillary adenocarcinomas (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997,
Informed Decisions: The Complete Book of Cancer Diagnosis,
Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A.,
Inc., United States of America).
[0152] Accordingly, the methods of the invention are also useful in
the treatment of a variety of cancers or other abnormal
proliferative diseases, including (but not limited to) the
following: carcinoma, including that of the bladder, breast, colon,
kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and
skin; including squamous cell carcinoma; hematopoietic tumors of
lymphoid lineage, including leukemia, acute lymphocytic leukemia,
acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma,
Berketts lymphoma; hematopoietic tumors of myeloid lineage,
including acute and chronic myelogenous leukemias and promyclocytic
leukemia; tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyoscarcoma; other tumors, including melanoma, seminoma,
tetratocarcilioma, neuroblastoma and glioma; tumors of the central
and peripheral nervous system, including astrocytoma,
neuroblastoma, glioma, and schwannomas; tumors of mesenchymal
origin, including fibrosafconia, rhabdomyosearama, and
osteosarcoma; and other tumors, including melanoma, xenoderma
pegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer
and teratocarcinoma. In specific embodiments, malignancy or
dysproliferative changes (such as metaplasias and dysplasias), or
hyperproliferative disorders, are treated in the ovary, bladder,
breast, colon, lung, skin, pancreas, or uterus. In other specific
embodiments, sarcoma, melanoma, or leukemia is treated.
[0153] In preferred embodiments, the methods of the invention are
used for TLR3 positive solid tumors. Example of tumors include
breast, colon, ovarian, lung, brain and prostate cancers and
melanoma. In a preferred embodiment, the methods of the invention
are directed at treating breast cancer.
Breast Cancer
[0154] Types of Breast Cancer. Most breast cancer develops in
glandular tissue and is classified as adenocarcinoma. The earliest
form of the disease, ductal carcinoma in situ (DCIS), develops
solely in the milk ducts. The most common type of breast cancer,
invasive ductal carcinoma (IDC), develops from DCIS, spreads
through the duct walls, and invades the breast tissue.
[0155] The term "premalignant lesion" as used herein is defined as
a collection of cells in a breast with histopathological
characteristics which suggest at least one of the cells has an
increased risk of becoming breast cancer. A skilled artisan
recognizes that the most important premalignant lesions recognized
today include unfolded lobules (UL; other names: blunt duct
adenosis, columnar alteration of lobules), usual ductal hyperplasia
(UDH; other names: proliferative disease without atypia,
epitheliosis, papillomatosis, benign proliferative disease),
atypical ductal hyperplasia (ADH), atypical lobular hyperplasia
(ALH), ductal carcinoma in situ (DCIS), and lobular carcinoma in
situ (LCIS). Other lesions which may have premalignant potential
include intraductal papillomas, sclerosisng adenosis, and
fibroadenomas (especially atypical fibroadenomas). In a specific
embodiment, the collection of cells is a lump, tumor, mass, bump,
bulge, swelling, and the like. Other terms in the art which are
interchangeable with "premalignant lesion" include premalignant
hyperplasia, premalignant neoplasia, and the like.
[0156] Invasive lobular carcinoma originates in the milk glands and
accounts for 10-15% of invasive breast cancers. Less common types
of breast cancer include the following:
[0157] Inflammatory (breast tissue is warm and appears red; tends
to spread quickly)
[0158] Medullary carcinoma (originates in central breast
tissue)
[0159] Mucinous carcinoma (invasive; usually occurs in
postmenopausal women)
[0160] Paget's disease of the nipple (originates in the milk ducts
and spreads to the skin of the nipples or areola)
[0161] Phyllodes tumor (tumor with a leaf-like appearance that
extends into the ducts; rarely metastasizes)
[0162] Tubular carcinoma (small tumor that is often undetectable by
palpation)
[0163] Rarely, sarcomas (cancer of the connective tissue) and
lymphomas (cancer of the lymph tissue) develop in the breasts.
[0164] Staging: The stage of a cancer is determined by the size and
location in the body of the primary tumor, and whether it has
spread to other areas of the body.
[0165] Staging involves using the letters T, N, and M to assess
tumors:
[0166] size of the primary tumor (T);
[0167] degree to which regional lymph nodes (N) are involved. Lymph
nodes are small organs located along the channels of the body's
lymphatic system which store special cells that fight infection and
other diseases); and
[0168] absence or presence of distant metastases (M)--cancer that
has spread from the original (primary) tumor to distant organs or
distant lymph nodes.
[0169] Each of these categories is further classified with a
number. Thus a T1-N1-M0 cancer would describe a T1 tumor, N1 lymph
node involvement, and no metastases.
[0170] Once the T, N, and M components are determined, a "stage" of
I, II, III or IV is assigned:
[0171] Stage I cancers are small, localized and usually
curable.
[0172] Stage II and III cancers typically are locally advanced
and/or have spread to local lymph nodes.
[0173] Stage IV cancers usually are metastatic (have spread to
distant parts of the body) and generally are considered
inoperable.
[0174] Details of this staging system are further provided in: The
International Union against Cancer details the TNM staging system
(Tumour/Nodes/Metastasis), (UICC-TNM Classification of malignant
tumours. Edited by L. H. Sobin and C. H. Wittekind. 5th Edition.
New York: Wiley-Liss; 1997)
[0175] The term "sample from a breast" as used herein is defined as
a specimen from any part or tissue of a breast. A skilled artisan
recognizes that the sample may be obtained by any method, such as
biopsy. In another specific embodiment, the sample is from
hyperplastic or malignant breast epithelium. In a specific
embodiment, the sample is from the epithelium. In another specific
embodiment, the sample is from a premalignant lesion.
[0176] In specific embodiments, a patient with breast cancer is
administered a prophylactically or therapeutically effective amount
of a TLR3 agonist. The patient may or may not have lymph node
involvement, may or may not have metastates to distant organs or
distant lymph nodes, may or may not have a recurrent cancer, or a
cancer that is refractory or non-refractory. The cancer may be a
stage 1, II, III or IV cancer, most preferably a stage II or III
cancer. The cancer may be a T1, T2 or T3 cancer. Optionally, the
cancer is a T1-3N0-3M0 cancer. Optionally, the patient has received
radiation therapy following surgery to remove breast cancer tissue.
In another aspect, the patient has not received radiation therapy
following surgery to remove breast cancer tissue.
[0177] Generally, although the therapeutic methods are not limited
thereto, patients will have received surgery to remove breast
cancer tissue. The TLR3 agonist may also be used a prophylactic
agent before surgery, or more preferably is used in combination
with surgery. The TLR3 agonist can advantageously be used soon or
immediately after surgery (e.g beginning of TLR3 agonist treatment
less than 8, 6, 4, 3, 2, 1 week following surgery), without
possible adverse effects on TLR3 agonist efficacy related to
immunosuppression that could be expected with immunomodulatory
compounds.
[0178] In other specific embodiments, a diagnostic assay is
performed on a sample from a breast to determine whether a
patient's tumor comprises TLR3-expression cells. Such assays are
described herein; for example antibody-based immunohistochemistry
assays can be used advantageously. Preferably a tumor biopsy is
performed, yielding a biological sample. A determination that said
biological sample comprises TLR3 expressing cells indicates that
the patient can benefit from the TLR3 agonist administration. The
patient is then treated with the TLR3 agonist.
[0179] In other specific embodiments, patients with breast cancer
are administered a prophylactically or therapeutically effective
amount of a TLR3 agonist in combination with a prophylactically or
therapeutically effective amount of one or more other therapies
useful for breast cancer treatment or management including, but not
limited to: doxorubicin, epirubicin, the combination of doxorubicin
and cyclophosphamide (AC), the combination of cyclophosphamide,
doxorubicin and 5-fluorouracil (CAF), the combination of
cyclophosphamide, epirubicin and 5-fluorouracil (CEF),
Herceptin.TM.), tamoxifen, or the combination of tamoxifen and
cytotoxic chemotherapy. In certain embodiments, patients with
metastatic breast cancer are administered a prophylactically or
therapeutically effective amount of a TLR3 agonist in combination
with the administration of a prophylactically or therapeutically
effective amount of taxanes such as docetaxel and paclitaxel. In
other embodiments, a patients with node-positive, localized breast
cancer are administered a prophylactically or therapeutically
effective amount of a TLR3 agonist in combination with the
administration of a prophylactically or therapeutically effective
amount of taxanes plus standard doxorubicin and cyclophosphamide
for adjuvant treatment of node-positive, localized breast
cancer.
Treatment of Colon Cancer
[0180] In specific embodiments, a patient with colon cancer is
administered a prophylactically or therapeutically effective amount
of a TLR3 agonist. The patient may or may not have metastates to
distant organs or distant lymph nodes, may or may not have a
recurrent cancer, or a cancer that is refractory or non-refractory.
Optionally, the patient has received radiation therapy following
surgery to remove cancerous tissue. In another aspect, the patient
has not received radiation therapy following surgery to remove
cancerous tissue.
[0181] Generally, although the therapeutic methods are not limited
thereto, patients will have received surgery to remove cancer
tissue. The TLR3 agonist may also be used a prophylactic agent
before surgery, or more preferably is used in combination with
surgery. The TLR3 agonist can advantageously be used soon or
immediately after surgery (e.g beginning of TLR3 agonist treatment
less than 8, 6, 4, 3, 2, 1 week following surgery), without
possible adverse effects on TLR3 agonist efficacy related to
immunosuppression.
[0182] In specific embodiments, patients with colon cancer are
administered a prophylactically or therapeutically effective amount
of a TLR3 agonist in combination with the administration of a
prophylactically or therapeutically effective amount of one or more
other therapies useful for colon cancer treatment or management
including but not limited to: the combination of 5-FU and
leucovorin, the combination of 5FU and levamisole, irinotecan
(CPT-11) or the combination of irinotecan, 5-FU and leucovorin
(IFL) or oxaliplatin.
Treatment of Prostate Cancer
[0183] In specific embodiments, patients with prostate cancer are
administered a prophylactically or therapeutically effective amount
of a TLR3 agonist in combination with the administration of a
prophylactically or therapeutically effective amount of one or more
other therapies useful for prostate cancer treatment or management
including but not limited to: external-beam radiation therapy,
interstitial implantation of radioisotopes (i.e., palladium, and
Iridium), leuprolide or other LHR agonists, nonsteroidal
antiandrogens (flutamide, nilutamide, and bicalutamide), steroidal
antiandrogens (cyproterone acetate), the combination of leuprolide
and flutamide, estrogens such as DES, chlorotrianisene, ethinyl
estradiol, conjugated estrogens U.S.P., DES-diphosphate,
radioisotopes, such as strontium-89, the combination of
external-beam radiation therapy and strontium-89, second-line
hormonal therapies such as aminoglutethimide, hydrocortisone,
flutamide withdrawal., progesterone, and ketoconazole, low-dose
prednisone, or other chemotherapy regimens reported to produce
subjective improvement in symptoms and reduction in PSA level
including docetaxcl, paclitaxel, estramustine/docetaxel,
estramustine/etoposide, estramustine/vinblastine, and
estramustine/paclitaxel.
Treatment of Melanoma
[0184] In specific embodiments, patients with melanoma are
administered a prophylactically or therapeutically effective amount
of a TLR3 agonist in combination with the administration of a
prophylactically or therapeutically effective amount of one or more
other therapies useful for melanoma cancer treatment or management
including but not limited to: dacarbazine (DTIC), nitrosoureas such
as carmustine (BCNU) and lomustine (CCNU), agents with modest
single agent activity including vinca alkaloids, platinum
compounds, and taxanes, the Dartmouth regimen (cisplatin, BCNU, and
DTIC), interferon alpha (IFN-.alpha.), and interleukin-2
(IL-2).
Treatment of Ovarian Cancer
[0185] In specific embodiments, patients with ovarian cancer are
administered a prophylactically or therapeutically effective amount
of a TLR3 agonist in combination with a prophylactically or
therapeutically effective amount of one or more other therapies
useful for ovarian cancer treatment or management including, but
not limited to: intraperitoneal radiation therapy, total abdominal
and pelvic radiation therapy, cisplatin, oxaliplatin, the
combination of paclitaxel (Taxol) or docetaxel (Taxotere) and
cisplatin or carboplatin, the combination of cyclophosphamide and
cisplatin, the combination of cyclophosphamide and carboplatin, the
combination of 5-fluorouracil (5FU) and leucovorin, etoposide,
liposomal doxorubicin, gerucitabine or topotecan. In a particular
embodiment, patients with ovarian cancer that is
platinum-refractory are administered a prophylactically or
therapeutically effective amount of a TLR3 agonist in combination
with the administration of a prophylactically or therapeutically
effective amount of Taxol. The invention encompasses the treatment
of patients with refractory ovarian cancer including administration
of--ifosfamide in patients with disease that is
platinum-refractory, hexamethylmelamine (HAM) as salvage
chemotherapy after failure of cisplatin-based combination regimens,
and tamoxifen in patients with detectable levels of cytoplasmic
estrogen receptor on their tumors.
Treatment of Lung Cancers
[0186] In specific embodiments, patients with small lung cell
cancer are administered a prophylactically or therapeutically
effective amount of a TLR3 agonist in combination with the
administration of a prophylactically or therapeutically effective
amount of one or more other therapies useful for lung cancer
treatment or management including but not limited to: thoracic
radiation therapy, cisplatin, vincristine, doxorubicin, and
etoposide, alone or in combination, the combination of
cyclophosphamide, doxorubicin, vincristine/etoposide, and cisplatin
(CAV/EP), local palliation with endobronchial laser therapy,
endobronchial stents, and/or brachytherapy.
[0187] In other specific embodiments, patients with non-small lung
cell cancer are administered a prophylactically or therapeutically
effective amount of a TLR3 agonist in combination with the
administration of a prophylactically or therapeutically effective
amount of one or more other therapies useful for lung cancer
treatment or management including but not limited to: palliative
radiation therapy, the combination of cisplatin, vinblastine and
mitomycin, the combination of cisplatin and vinorelbine, pa.
clitaxel, docetaxel or gemcitabine, the combination of carboplatin
and paclitaxel, interstitial radiation therapy for endobronchial
lesions or stereotactic radiosurgery
Combination with Chemotherapy, Preferred Examples
[0188] An adjunct therapy contemplated in the present invention is
chemotherapy. Adjunct chemotherapies may include, for example,
cisplatin (CDDP), carboplatin, oxaliplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding
agents, taxol, gemeitablen, navelbine, farnesyl-protein transferase
inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin
and methotrexate, or any analog or derivative variant of the
foregoing.
[0189] Taxol/Paclitaxel. Paclitaxel, also known as taxol is a
diterpene alkaloid thus it possesses a taxane skeleton in its
structure. Paclitaxel is extracted from the bark of the Pacific yew
(Taxus brevifolia) as a natural compound having anti-cancer
activity (Fuchs and Johnson, 1978). Paclitaxel works against cancer
by interfering with mitosis. Paclitaxel is a taxoid drug, widely
used as an effective treatment of primary and metastatic
cancers.
[0190] Paclitaxel (Taxol) is widely used in the treatment of
breast, ovarian, and other solid tumors. Randomized clinical trials
have shown a survival advantage among patients with primary breast
cancer who received paclitaxel in addition to
anthracycline-containing adjuvant chemotherapy (Eifel et al.,
2001). Furthermore, paclitaxel is effective for both metastatic
breast cancer (Holmes et al., 1991; Nabholtz et al., 1996; Bishop
et al., 1999) and advanced ovarian cancer (McGuire et al., 1996;
Piccart et al., 2000). The antitumor activity of paclitaxel is
unique because it promotes microtubule assembly and stabilizes the
microtubules, thus preventing mitosis (Huizing et al., 1995).
Paclitaxel does this by reversibly and specifically binding to the
B subunit of tubulin, forming microtubule polymers thereby
stabilizing them against depolymerization and thus leading to
growth arrest in the G2/M phase of the cell cycle (Gotaskie and
Andreassi, 1994). This makes taxol unique in comparison to
vincristine and vinblastine which cause microtubule disassembly
(Gatzenicier et al., 1995). Additionally, recent evidence indicates
that the microtubule system is essential to the release of various
cytokines and modulation of cytokine release may play a major role
in the drug's antitumor activity (Smith et al., 1995).
[0191] However, some patients are resistant to paclitaxel therapy,
and the characteristics of patients who will benefit from the drug
have not been well defined. Identification of molecular
characteristics predictive of paclitaxel sensitivity or resistance
could aid in selecting patients to receive this therapy. Thus, in
particular embodiments, the present invention relates to paclitaxel
sensitivity in a patient having cancer. Previous reports have
demonstrated that paclitaxel resistance is due to a variety of
mechanisms such as up-regulation of anti-apoptotic Bcl-2 family
members, such as Bel-2 and BCl-XL (Tang et al., 1994);
up-regulation of membrane transporters (e.g., mdr-1), resulting in
an increased drug efflux--(Huang et al., 1997); mutations in
beta-tubulin resulting in abolishment of paclitaxel binding
(Giannakakou et al., 1997); and up-regulation of ErbB2 (HER2)
through inhibition of cyclin-dependent kinase-1 (Cdkl), resulting
in delayed mitosis (Yu et al., 1998).
[0192] Due to the antimitotic activity of paclitaxel it is a useful
cytotoxic drug in treating several classic refractory tumors.
Paclitaxel has primarily been use to treat breast cancer and
ovarian cancer. It may also be used in treating head and neck
cancer, Kaposi's sarcoma and lung cancer, small cell and non-small
cell lung cancer. It may also slow the course of melanoma. Response
rates to taxol treatment varies among cancers. Advanced drug
refractory ovarian cancer is reported to respond at a 19-36% rate,
previously treated metastatic breast cancer at 27-62%, and various
lung cancers at 21-37%. Taxol has also been shown to produce
complete tumor remission in some cases (Guchelaar et al.,
1994).
[0193] Paclitaxel is given intravenously since it irritates skin
and mucous membranes on contact. It is typically administered
intravenously by a 3 to 24 hour infusion three times per week
(Guchelaar et al., 1994).
[0194] Doxorubicin. Doxorubicin hydrochloride,
5,12-Naphthacenedione,
(8s-ds)-10-[(3-amino-2,3,6-trideoxy-a-L-Iyxo-hexopyranosyl)oxy]-7,8,9,10--
tetrahydro-6,8,11-trihydroxy-g-(hydroxyacetyl)
1-methoxy-hydrochloride (hydroxydaunorubicin hydrochloride,
Adriamycin) is used in a wide antineoplastic spectrum. It binds to
DNA and inhibits nucleic acid synthesis and mitosis, and promotes
chromosomal aberrations.
[0195] Administered alone, it is the drug of first choice for the
treatment of thyroid adenoma and primary hepatocellular carcinoma.
It is a component of first-choice in combination with other agents
for the treatment of ovarian tumors, endometrial and breast tumors,
bronchogenic oat-cell carcinoma, non-small cell lung carcinoma,
gastric adenocarcinoma, retinoblastoma, neuroblastoma, mycosis
fungoides, pancreatic carcinoma, prostatic carcinoma, bladder
carcinoma, myeloma, diffuse histiocytic lymphoma, Wilms' tumor,
Hodgkin's disease, adrenal tumors, osteogenic sarcoma soft tissue
sarcoma, Ewing's sarcoma, rhabdomyosarcoma and acute lymphocytic
leukemia. It is an alternative drug for the treatment of islet
cell, cervical, testicular and adrenocortical cancers. It is also
an immunosuppressant.
[0196] Since doxorubicin is poorly absorbed it is administered
intravenously. The pharmacokinetics of this chemotherapeutic agent
are multicompartmental. Distribution phases have half-lives of 12
minutes and 3.3 hrs. The elimination half-life is about 30 hrs.
Forty to 50% is secreted into the bile. Most of the remainder is
metabolized in the liver, partly to an active metabolite
(doxorubicinol), but a few percent is excreted into the urine. In
the presence of liver impairment, the dose should be reduced.
[0197] Appropriate doses are, for an adult, administered
intravenously, are 60 to 75 mg/m.sup.2 at 21-day intervals, or 25
to 30 mg/m.sup.2 on each of 2 or 3 successive days repeated at 3-
or 4-wk intervals, or 20 mg/m.sup.2 once a week. The lowest dose
should be used in elderly patients, when there is prior bone-marrow
depression caused by prior chemotherapy or neoplastic marrow
invasion, or when the drug is combined with other myelopoietic
suppressant drugs. The dose should be reduced by 50% if the serum
bilirubin lies between 1.2 and 3 mg/dL and by 75% if above 3 mg/dL.
The lifetime total dose should not exceed 550 mg/m.sup.2 in
patients with normal heart function and 400 mg/m.sup.2 in persons
having received mediastinal irradiation. Alternatively, 30
mg/m.sup.2 on each of 3 consecutive days, repeated every 4 wk may
be administered. Exemplary doses may be 10 mg/m.sup.2, 20
mg/m.sup.2, 30 mg/m.sup.2, 50 mg/m.sup.2, 100 mg/m.sup.2, 150
mg/m.sup.2, 175 mg/m.sup.2, 200 mg/m.sup.2, 225 mg/m.sup.2, 250
mg/m.sup.2, 275 mg/m.sup.2, 300 mg/m.sup.2, 350 mg/m.sup.2, 400
mg/m.sup.2, 425 mg/m.sup.2, 450 mg/m.sup.2, 475 mg/m.sup.2, 500
mg/m.sup.2. Of course, all of these dosages are exemplary, and any
dosage in-between these points is also expected to be of use in the
present invention.
Combination with Radiotherapy
[0198] Radiotherapy, also called radiation therapy, involves the
use of ionizing radiation to treat cancers and other diseases.
Ionizing radiation deposits energy that injures or destroys cells
in the area being treated (the "target tissue") by damaging their
genetic material, and thereby inhibiting cell proliferation.
Ionizing radiation induces the formation of hydroxyl radicals,
placing the cells under oxidative stress. These radicals damage
DNA, which causes cytotoxicity.
[0199] Radiotherapeutic agents that cause DNA damage are well known
in the art and have been extensively used. Radiotherapeutic agents,
through the production of oxygen-related free radicals and DNA
damage, may lead to cell death or apoptosis. These agents may
include, but are not limited to, 7-rays, X-rays, and/or the
directed delivery of radioisotopes to tumor cells (known as
internal radiotherapy). Internal radiotherapy may further include
but is not limited to, brachytherapy, interstitial irradiation, and
intracavitary irradiation. Other radiotherapeutic agents that are
DNA damaging factors include microwaves and UV-irradiation. These
factors effect a broad range of damage on DNA, on the precursors of
DNA, on the replication and repair of DNA, and on the assembly and
maintenance of chromosomes. Other approaches to radiation therapy
are also contemplated in the present invention.
[0200] Such techniques may comprise intraoperative irradiation, in
which a large dose of external radiation is directed at the tumor
and surrounding tissue during surgery; and particle beam radiation
therapy which involves the use of fast-moving subatomic particles
to treat localized cancers. Radiotherapy may further involve the
use of radiosensitizers and/or radioprotectors to increase the
effectiveness of radiation therapy. Radiolabeled antibodies may
also be used to deliver doses of radiation directly to the cancer
site, this is known as radio immunotherapy.
[0201] Dosage ranges for X-rays range from daily doses of 50 to 200
roentgens for prolonged periods of time (3 to 4 wk), to single
doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes
vary widely, and depend on the half-life of the isotope, the
strength and type of radiation emitted, and the uptake by the
neoplastic cells.
Surgery
[0202] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery includes
resection in which all or part of cancerous tissue is physically
removed, excised, and/or destroyed. Tumor resection refers to
physical removal of at least part of a tumor. In addition to tumor
resection, treatment by surgery includes laser surgery,
cryosurgery, electrosurgery, and microscopically controlled
surgery. It is further contemplated that the present invention may
be used in conjunction with removal of superficial cancers,
precancers, or incidental amounts of normal tissue.
[0203] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by administration of an additional anti-cancer
therapy, more particularly a TLR3 agonist. Such treatment may be
repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every
1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
or 12 months. These treatments may be of varying dosages as
well.
Hormonal Therapy
[0204] Hormonal therapy may also be used in conjunction with the
present invention or in combination with any other cancer therapy
previously described. The use of hormones may be employed in the
treatment of certain cancers such as breast, prostate, ovarian, or
cervical cancer to lower the level or block the effects of certain
hormones such as testosterone or estrogen. This treatment is often
used in combination with at least one other cancer therapy as a
treatment option or to reduce the risk of metastases.
Other Agents
[0205] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, or inhibitors of cell adhesion.
Immunomodulatory agents include tumor necrosis factor; interferon
alpha, beta, and gamma; IL-2, IL-12, IL-15, IL-21 and other
cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1 beta,
MCP-1, RANTES, and other chemokines.
[0206] The treatment with a TLR3 agonist, and more particularly
dsRNA molecule, may be accomplished as disclosed in the literature
cited above. Furthermore, the treatment may be performed either
alone or in combination with other drugs or treatments. The
treatment may include a reduction in tumor size, a reduction or
delay in tumor growth, development or metastasis, or a regression
of cancer.
[0207] Thus, various non-limiting embodiments as set forth herein
include:
[0208] 1. The use of a TLR3 agonist for the manufacture of a
medicament for treating cancer in a subject, wherein said cancer in
said subject comprises cancer cells expressing a TLR3 receptor;
[0209] 2. A method for assessing the response of a subject having
cancer to a treatment using a TLR3 agonist, the method comprising
determining whether cancer cells in said subject express a TLR3
receptor, the expression of a TLR3 receptor being indicative of a
responder subject;
[0210] 3. A method for selecting subjects having a cancer that
respond to a treatment using a TLR3 agonist, the method comprising
determining whether cancer cells in said subject express a TLR3
receptor, the expression of a TLR3 receptor being indicative of a
responder subject:
[0211] 4. A method for treating a subject having a cancer, the
method comprising determining whether cancer cells in said subject
express a TLR3 receptor, the expression of a TLR3 receptor being
indicative of a subject responding to a TLR3 agonist, and treating
said subject whose cancer cells express a TLR3 receptor with a
double-stranded RNA molecule;
[0212] 5. A use or method of any one of the preceding embodiments,
wherein the subject is a human subject;
[0213] 6. A use or method of any one of the preceding embodiments,
wherein the cancer is a solid tumor and carcinoma;
[0214] 7. The use or method of embodiment 6, wherein the solid
tumor is selected from breast cancer, colon cancer, lung cancer,
renal cancer, metastatic or invasive malignant melanoma, prostate
cancer, brain tumor, bladder cancer and liver cancer;
[0215] 8. The use or method of any one of the preceding
embodiments, wherein the expression of a TLR3 receptor in said
cancer cell is determined using a TLR3-specific ligand;
[0216] 9. The use or method of embodiment 8, wherein the ligand is
an antibody, or a fragment or derivative thereof;
[0217] 10. The use or method of any one of embodiments 1 to 7,
wherein the expression of a TLR3 receptor in said cancer cell is
determined using a TLR3-specific primer or probe;
[0218] 11. The use or method of any one of the preceding
embodiments, wherein the expression of a TLR3 receptor in said
cancer cell is determined in vitro or ex vivo;
[0219] 12. The use or method of any one of the preceding
embodiments, wherein the TLR3 agonist is a double-stranded RNA
molecule;
[0220] 13. The use or method of embodiment 12, wherein the
double-stranded RNA molecule is a polyA/polyU molecule;
[0221] 14. The use or method of embodiment 12, wherein the
double-stranded RNA molecule is a polyI/polyC molecule;
[0222] 15. The use of a double-stranded polyA/polyU RNA molecule
for the manufacture of a medicament for treating breast cancer in a
subject, wherein said breast cancer in said subject comprises
cancer cells expressing a TLR3 receptor;
[0223] 16. A kit for selecting subjects that respond to a treatment
using a TLR3 agonist, the kit comprising reagents for determining
the expression of a TLR3 receptor in a cancer cell in a sample;
or
[0224] 17. The kit of embodiment 16, wherein the TLR3 agonist is a
double-stranded RNA molecule.
[0225] Further aspects and advantages of this invention will be
disclosed in the following examples, which should be regarded as
illustrative and not limiting the scope of this invention.
EXAMPLES
[0226] Toll like receptor 3 (TLR3) is known to be expressed by
myeloid dendritic cells (DC) and to induce their maturation
following binding with double stranded RNA (dsRNA) or its synthetic
homologues polyAU and polyI:C. Several clinical trials have
reported that injection of dsRNA is associated with survival
benefit in cancer patients. In the present study, the inventors
have asked whether dsRNA could act directly on tumor cells through
TLR3. Patients and methods: 300 patients with early breast cancer
have been included from 1972 to 1979 in a randomized trial
comparing post-operative administration of polyAU with no
treatment. Results have been reported that showed a trend for a
survival benefit in patients with involved auxiliary lymph nodes
(n=200).
[0227] Tumor biopsies from these patients were stained with
TLR3-specific mAb and correlation between TLR3 expression and
polyAU efficacy was determined.
[0228] To investigate directly the effects of dsRNA, both freshly
isolated breast tumor cells and cancer cell lines were cultured
with polyI: C, and apoptosis was measured. The involvement of TLR3
in cell response was established by TLR3RNA interference.
[0229] Results: 182 tumor samples (91%) were available from the 200
pT.times.N+M0 patients included in this randomized trial. TLR3 was
strongly expressed by tumor cells in 18 patients (10%). Table 1
reports the 20-year survival rates according to treatment and TLR3
expression.
Targeting Toll Like Receptor 3 in Breast Cancer: Results of
Randomized Trial and In Vitro Studies
Material and Methods:
Patients:
[0230] 200 patients were included in the present study. All
patients had been previously included in a prospective randomized
trial that compared double stranded RNA (polyAU) to placebo. This
trial has already been reported elsewhere. Briefly, this randomized
trial included patients with T1-3N0-3M0 breast cancer treated with
surgery. Treatment consisted in weekly iv injection of polyAU
(Beaufour Ipsen). A total of 6 injections were performed. PolyAU
was administered at a fixed dose of 60 mg/injection. This trial
initially included 300 patients. Since initial results of the trial
reported a trend for benefit only in patients with auxiliary lymph
node involvement, only the 200 patients with auxiliary node
involvement were included in the present study.
Immunostainings:
[0231] Tumor blocks were available in 182 out of 200 patients
included in the present study. Paraffin-embedded, 5 um-thick tissue
section from all 182 tumors were stained with either polyclonal
antiTLR3 (gift from Dr Pobolslky, Massachusetts General Hospital,
Boston) or rabbit preimmune serum. A mouse monoclonal anti-rabbit
IgG was used as secondary antibody. Immunostainings were assessed
by 2 pathologists who were blinded for clinical files. The TLR3
expression was classified according to the percentage of tumor
cells stained and the intensity of staining. A tumor was classified
as positive when more than 10% of tumor cells were strongly stained
with the anti-TLR3 antibody.
Statistics:
[0232] Survival curves were determined according to Kaplan-Meier
method. Survival curves were compared using Khi2 test.
Results:
Patients Characteristics
[0233] One hundred eighty two tumors were processed. The
immunostaining could not be interpreted in 7 patients (absence of
tumor cells in 4 patients, artefact in 3 patients). The analysis
was therefore performed on 175 patients. This represents 87% of the
patients included in the randomized trial. The median follow-up of
living patients was 23 years (12 to 26 years). The patients
characteristics are reported in Table 1. Briefly, the median age is
50, the median number of lymph node involved was 4 (1-31), 26% of
tumor were staged pT3 and 35% were classified as grade III
according to Scarf and Bloom Richardson.
TABLE-US-00001 TABLE 1 Patients characteristics TLR3- tumors TLR3+
tumors (n = 157) (n = 18) Obser- Obser- Character- vation Poly AU
vation Poly AU Total istics (n = 77) (n = 80) (n = 10) (n = 8) (n =
175) Age (median) 50 50 52 49 50 Nb lymph node 5 (1-31) 4 (1-27) 2
(1-8) 4 (1-9) 4 (1-31) involved (median) pT pT1 8 1 0 0 9 pT2 56 54
6 6 122 pT3 13 27 4 2 46 Tumor grade I 11 9 1 2 23 II 34 49 6 3 92
III 32 24 3 3 62 Post- operative radiotherapy Yes 74 77 9 8 168 No
3 3 1 0 7
Immunostainings
[0234] TLR3 was strongly expressed by tumor cells in 18 samples
(10.4% of assessable tumors). Immunostainings are shown in FIG. 1.
TLR3 was mainly expressed on the cell surface and cytoplasm of
tumor cells. In situ carcinoma and normal breast tissues were
stained by anti-TLR3 in most cases. The patients characteristics of
the TLR3+ tumors did not differ to that of TLR3-tumors (Table
1).
Correlation Between TLR3 Expression and Survival After Treatment
with polyAU
[0235] The 20 year OS of patients treated or not with polyAU were
42% and 35% respectively (p=0.09). When only patients with
TLR3-tumors were considered, the 20 year OS were 41% for patients
treated with polyAU, and 37% for those assigned to observation arm
(p=0.52) (FIG. 2a). When only patients presenting TLR3+ tumors were
considered, the 20 year OS were 88% for patients treated with
polyAU, and 22% for patients assigned to the observation arm
(p=0.01) (FIG. 2b).
CONCLUSION
[0236] TLR3 is overexpressed by tumor cells in around 10% of cancer
cases.
[0237] TLR3 expression correlates with the benefit of adjuvant
therapy with polyAU in patients with lymph node positive breast
cancer.
Sequence CWU 1
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480Ser Leu Gln Arg Leu Met Leu Arg Arg Val Ala Leu Lys Asn Val Asp
485 490 495Ser Ser Pro Ser Pro Phe Gln Pro Leu Arg Asn Leu Thr Ile
Leu Asp 500 505 510Leu Ser Asn Asn Asn Ile Ala Asn Ile Asn Asp Asp
Met Leu Glu Gly 515 520 525Leu Glu Lys Leu Glu Ile Leu Asp Leu Gln
His Asn Asn Leu Ala Arg 530 535 540Leu Trp Lys His Ala Asn Pro Gly
Gly Pro Ile Tyr Phe Leu Lys Gly545 550 555 560Leu Ser His Leu His
Ile Leu Asn Leu Glu Ser Asn Gly Phe Asp Glu 565 570 575Ile Pro Val
Glu Val Phe Lys Asp Leu Phe Glu Leu Lys Ile Ile Asp 580 585 590Leu
Gly Leu Asn Asn Leu Asn Thr Leu Pro Ala Ser Val Phe Asn Asn 595 600
605Gln Val Ser Leu Lys Ser Leu Asn Leu Gln Lys Asn Leu Ile Thr Ser
610 615 620Val Glu Lys Lys Val Phe Gly Pro Ala Phe Arg Asn Leu Thr
Glu Leu625 630 635 640Asp Met Arg Phe Asn Pro Phe Asp Cys Thr Cys
Glu Ser Ile Ala Trp 645 650 655Phe Val Asn Trp Ile Asn Glu Thr His
Thr Asn Ile Pro Glu Leu Ser 660 665 670Ser His Tyr Leu Cys Asn Thr
Pro Pro His Tyr His Gly Phe Pro Val 675 680 685Arg Leu Phe Asp Thr
Ser Ser Cys Lys Asp Ser Ala Pro Phe Glu Leu 690 695 700Phe Phe Met
Ile Asn Thr Ser Ile Leu Leu Ile Phe Ile Phe Ile Val705 710 715
720Leu Leu Ile His Phe Glu Gly Trp Arg Ile Ser Phe Tyr Trp Asn Val
725 730 735Ser Val His Arg Val Leu Gly Phe Lys Glu Ile Asp Arg Gln
Thr Glu 740 745 750Gln Phe Glu Tyr Ala Ala Tyr Ile Ile His Ala Tyr
Lys Asp Lys Asp 755 760 765Trp Val Trp Glu His Phe Ser Ser Met Glu
Lys Glu Asp Gln Ser Leu 770 775 780Lys Phe Cys Leu Glu Glu Arg Asp
Phe Glu Ala Gly Val Phe Glu Leu785 790 795 800Glu Ala Ile Val Asn
Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val 805 810 815Ile Thr His
His Leu Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val 820 825 830His
His Ala Val Gln Gln Ala Ile Glu Gln Asn Leu Asp Ser Ile Ile 835 840
845Leu Val Phe Leu Glu Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Leu
850 855 860Cys Leu Arg Arg Gly Met Phe Lys Ser His Cys Ile Leu Asn
Trp Pro865 870 875 880Val Gln Lys Glu Arg Ile Gly Ala Phe Arg His
Lys Leu Gln Val Ala 885 890 895Leu Gly Ser Lys Asn Ser Val His
900321PRTArtificial SequencePrimer 3Cys Thr Cys Ala Gly Ala Ala Gly
Ala Thr Thr Ala Cys Cys Ala Gly1 5 10 15Cys Cys Gly Cys Cys
20422DNAArtificial
SequencePrimer 4ccattatgag acagatctaa tg 22
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