# Patent

## Title
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.

## Background
TECHNICAL FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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.
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.
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, inummosuppression, 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.
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).
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.
Therefore, a method allowing to select responding patient would greatly enhance the therapeutic efficacy of double-stranded therapies.
SUMMARY OF THE INVENTION
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, a nucleotide-like compound, a nucleotide analog, or a 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 and other 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.
Furthermore, the finding that TLR3 agonist therapy can increase survival will peimit therapeutic strategies that are expected to be efficacious and a suitable treatment for tumors, including particularly breast tumors, whether they involve axillary lymph nodes or not, that is beyond only breast cancers presenting axillary lymph node involvement. Likewise, the TLR3 agonists may find use in breast (and other) cancers that are metastatic, recurring and/or refractory.
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.
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. 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. 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 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.
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.
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. In a related embodiment, the method for treating a subject having a cancer comprises the additional step of surgically removing a portion of the cancer prior to treating said subject with a TLR3 agonist. In yet another embodiment, the method comprises the additional step of treating said patient with radiotherapy in combination with a TLR3 agonist.
In another embodiment, the invention provides a method for treating a subject having a cancer, comprising the steps of: administering to said subject an agent that causes increased expression of a TLR3 receptor in cancer cells; and administering to said patient a TLR3 agonist. In a related embodiment, this method of treatment additionally comprises the step of determining whether cancer cells in said subject express a TLR3 receptor. This determination step may be performed subsequent to and/or following the treatment with the agent that causes increased expression of TLR3 receptor, but prior to treating said subject with a TLR3 agonist. In another related embodiment, this method comprises the additional step of surgically removing a portion of the cancer prior to administering the agent that causes increased expression of a TLR3 receptor. In yet another embodiment, the method comprises the additional step of treating said patient with radiotherapy in combination with a TLR3 agonist.
Moreover, the inventors have surprisingly found in the follow up study to the 300 patient clinical trial 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.
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. In yet another preferred embodiment the breast cancer involved 1 to 3 lymph nodes.
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. In another preferred embodiment, the subject is administered an intravenous dose of a TLR3 agonist about once a week for a period of six weeks. In another preferred embodiment, the TLR3 agonist is a double-stranded RNA molecule. In another preferred embodiment, the TLR3 agonist is selected from polyA:polyU or polyI:polyC. In a more preferred embodiment, the dose of polyA:polyU or polyI:polyC is between about 10mg and 100 mg.
In a preferred embodiment of the methods and uses according to the present invention, the subject is a human subject.
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. 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, bladder 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, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, Burkett's lymphoma, acute and chronic myelogenous leukemias and promyelocytic leukemia. The present invention is also relevant for the treatment of metastasis.
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.
In an alternative embodiment, the expression of a TLR3 receptor in said cancer cell is determined using a TLR3-specific primer or probe.
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.
In a preferred embodiment of the methods and uses according to the present invention, the double-stranded RNA molecule is a polyA/polyU (“polyAU”) 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.
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. In yet another aspect, the invention relates to compositions of matter and therapeutic and screening methods that take advantage of the presence of TLR3 on a cancer cell surface. In one embodiment, the invention provides a complex comprising an agent that binds to TLR3 in association with a tumoricidal or cytotoxic agent. In another embodiment, the invention provides a method of treating a subject with a cancer characterized by the presence of TLR3 on a cancer cell surface comprising the step of administering to said patient such a complex. In another embodiment, the invention provides 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. In a preferred embodiment, the method of determining if a test compound is useful for the treatment of cancer comprises the steps of: contacting said test compound with a cancer cell characterized by the presence of TLR3 on its cell surface; and determining if said test compound induces a TLR3-mediated biological effect. Such a method is useful for screening proteins, small molecules, nucleic acids and other biomolecules.



DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the TLR3 expression by primary tumor. TLR3 is overexpressed by tumor cells in 10% of samples (n=18).
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
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.
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.
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 monoclonal antibody (“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.
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 will 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, expression of TLR3 is determined using an immunohistochemical assay, as used in the Examples. Preferably TLR3 expression is determined using an antibody that specifically binds TLR3.
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
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.
Where “comprising” is used, this can preferably be replaced by “consisting essentially of”, more preferably by “consisting of”.
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).
The term “breast cancer” as used herein is defined as cancer which originates in the breast. In a specific embodiment, the breast cancer has spread to other organs, such as lymph nodes. In a specific embodiment, the breast cancer is invasive and may be metastatic.
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 has spread out of a duct into surrounding breast epithelium. In a specific embodiment, “metastatic” breast cancer is within the scope of “invasive.”
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.
“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”.
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.
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.
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.
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 12-72 hours. In this case, the agents may be administered in either order. In a preferred embodiment of the instant invention, the two agents are co-administered in a single formulation, or are co-administered simultaneously.
As used herein, the terms “prevent”, “preventing” and “prevention” refer to the inhibition of the development or onset of cancer (particularly, a breast cancer) or the prevention, recurrence, onset, or development of one or more symptoms of cancer, particularly a breast cancer, 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).
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
A used herein, a “protocol” includes dosing schedules and dosing regimens. The protocols herein are methods of use and include prophylactic and therapeutic protocols.
As used herein, a “prophylactic protocol” refers to a regimen for dosing and timing the administration of one or more prophylactic agents.
As used herein, the term “therapeutic protocol” refers to a regimen for dosing and timing the administration of one or more therapeutic agents.
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.
As used herein, the term “small molecules” includes, but is 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.
As used herein, the terms “subject” and “patient” are used interchangeably.
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 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.
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.
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.
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 Preferably, a therapeutic agent is an agent which is known 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 A single therapeutic agent may be characterized as two or more different types of agents based upon one or more effects the agent has in vivo and/or in vitro.
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).
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).
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).
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).
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.
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.
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 signaling, 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.
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). An “antibody agonist” refers to the situation where the affinity agent is an antibody.
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 phosphoramidates 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, uracil, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide 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 polynucleotide, or a solid support.
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.
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×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.
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× sodium chloride/sodium citrate (“SSC”) at about 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at about 68° C. In a preferred, non-limiting example stringent hybridization conditions are hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. (i.e., one or more washes at 50° C., 55° C., 60° C. or 65° 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 hybridization conditions include temperatures above 30° C., preferably above 35° C., more preferably in excess of 42° 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
“TLR3”, “TLR3 protein” 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—003256, the disclosure of which is incorporated herein by reference). 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-κB 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—003265, the sequence of which is shown in SEQ ID No 1. TLR3 is described in WO 98/50547 (the disclosure of which is incorporated herein by reference).
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 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—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 003265 sequence. A particular example of a TLR3 gene comprises NM 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. Genetic polymorphisms of the human TLR3 DNA sequence are known, for example allelic variations in the cytoplasmic region of TLR3 gene and in the immediate 5′ sequence of the TLR3 gene (see Piriel et al. (2005) Tissue Antigens 66(2): 125, the disclosure of which is incorporated herein by reference), for example the C/T polymorphism at position 2593, the C/A polymorphism at position 2642 and the A/G polymorphism at position 2690 in the TLR3 gene.
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.
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.
A “TLR3 polypeptide” designates any molecule comprising a stretch of at least six consecutive amino acids present in a TLR3 protein. A TLR3 polypeptide may be encoded by a fragment of a TLR3 gene, may be synthetically produced or may be produced through proteolytic digestion of a TLR3 protein or another TLR3 polypeptide. A TLR3 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_003256 sequence.
TLR3 Agonists
1. Assaying TLR3 Agonist Activity
A TLR3 agonist according to the present invention can be selected from any suitable agent that activates TLR3 and/or the subsequent cascade of biochemical events associated with TLR3 activation in vivo. Assays for detecting TLR3 agonist activity are known in the art and include, for example, the detection of luciferase (luc) production from an NF-κB reporter plasmid, or the induction of endogenous IL-8 (K. Kariko et al., J. Immunol. 2004,172: 6545-49, the disclosures of which is incorporated herein by reference). Assays for detecting TLR3 agonism of test compounds are also 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.
Regardless of the particular assay employed, a compound can be identified as an agonist of TLR3 if performing the assay with that compound results in at least a threshold increase of some biological activity known to he 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, 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. However, regardless of the particular assay employed, a compound can generally be identified as an agonist of TLR3 if perfoiming the assay with a compound results in at least a threshold increase of some biological activity mediated by TLR3.
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 μM to about 10 μM 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.
2. TLR3 Agonistic Antibodies
A TLR3 agonist of this invention may be an agonistic antibody. an agonistic fragment of such antibodies, a chimeric version of such antibodies or fragment, or another active antibody derivative. TLR3 agonist antibodies useful in 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 or TLR3 peptide. The immunogen may comprise intact TLR3-expressing tumor cells, cell membranes from TLR-3-expressing cells, the full length sequence of the TLR3 protein (produced recombinantly or isolated from a natural source), 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.
An immunogenic fragment typically contains at least 7 consecutive amino acids present in the full-length TLR3 amino acid sequence, even more preferably at least 10 consecutive amino acids thereof. The amino acid sequence of the fragment is 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.
In a most preferred embodiment, the immunogen comprises a wild-type human TLR3 polypeptide in a lipid membrane, typically derived from a membrane fraction of a TLR-3 expressing cell. In a specific embodiment, the immunogen comprises whole TLR3 expressing tumor cells, intact or optionally chemically or physically lysed.
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)). The step of immunizing a non-human mammal with an immunogen 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.
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.
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.
For monoclonal antibodies, which are preferred for producing agonistic antibodies for use in 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. 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.
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.
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.
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 supernatants from the hybridoma colonies are then assayed for the production of antibodies that specifically activate the TLR3 protein. 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.
Hybridomas that are confirmed to be producing a TLR3 agonistic monoclonal antibody 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.
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.
In certain embodiments, the DNA encoding an antibody that agonizes TLR3 is isolated from the hybridoma, and 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.
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.
The TLR3 agonist antibodies can be full length antibodies or antibody fragments or derivatives. Examples of antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)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)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into an 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.
3. Small Molecule TLR3 Agonists
A small molecule TLR3 agonist is preferably an organic molecule of less than about 1500 daltons. The design and selection (e.g., from a combinatorial library) or synthesis of a small molecule TLR3 agonist may be achieved through the use of the known crystal structure of TLR3 (Choe et al., Science, 309, pp. 581-85 (2005), the disclosure of which is herein incorporated by reference). This design or selection may begin with selection of the various moieties which fill the putative binding pocket(s) in which known double-stranded RNA agonists bind. There are a number of ways to select moieties to fill individual binding pockets. These include visual inspection of a physical model or computer model of the active site elucidated from the crystal structure and manual docking of models of selected moieties into various binding pockets.
Modeling software that is well known and available in the art may be used. These include QUANTA [Molecular Simulations, Inc., Burlington, Mass., 1992], and SYBYL [Molecular Modeling Software, Tripos Associates, Inc., St. Louis, Mo., 1992]. This modeling step may be followed by energy minimization with standard molecular mechanics force fields such as CHARMM and AMBER. [AMBER: (S. J. Weiner, P. A. Koliman, D. A. Case, U. C. Singh, C. Ghio, G. Alagona, and P. Weiner, J. Am. Chem. Soc., 1984, 106, 765); CHARMM: (B. R. Brooks, R. E. Bruccoleri, B. D. Olafson, D. J. States, S Swaminathan, and M. Karplus, J. Comp. Chem. 1983, 4, 187). In addition, there are a number of more specialized computer programs to assist in the process of optimally placing either complete molecules or molecular fragments into the TLR3 agonist binding site. These include: GRID (Goodford, P. J. A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules. J. Med. Chem. 1985, 28, 849-857). GRID is available from Oxford University, Oxford, UK; MCSS (Mariner, A.; Karplus, M. Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method. Proteins: Structure, Function and Genetics 1991, 11, 29-34). MCSS is available from Molecular Simulations, Burlington, Mass.; and DOCK (Kuntz, I. D.; Blaney, J. M.; Oatley, S. J.; Langridge, R.; Ferrin, T. E. A Geometric Approach to Macromolecule-Ligand Interactions. J. Mol. Biol. 1982, 161, 269-288). DOCK is available from the University of California, San Francisco, Calif.
Once suitable binding orientations have been selected, complete molecules can be chosen for biological evaluation. In the case of molecular fragments, they can be assembled into a single agonist. This assembly may be accomplished by connecting the various moieties to a central scaffold. The assembly process may, for example, be done by visual inspection followed by manual model building, again using software such as Quanta or Sybyl. A number of other programs may also be used to help select ways to connect the various moieties. These include: CAVEAT (Bartlett, P. A.; Shea, G. T.; Telfer, S. J.; Waterman, S. CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules. In “Molecular Recognition in Chemical and Biological Problems,” Special Pub., Royal Chem. Soc. 1989, 78, 182-196). CAVEAT is available from the University of California, Berkeley, Calif.; 3D Database systems, such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area has been reviewed by Martin (Martin, Y. C. 3D Database Searching in Drug Design. J. Med. Chem. 1992, 35, 2145); and HOOK (available from Molecular Simulations, Burlington, Mass.).
In addition to the above computer assisted modeling of agonist compounds, a TLR3 agonist may be constructed “de novo” using either an empty agonist binding site of TLR3 or optionally including some portions of a known agonist. Such methods are well known in the art. They include, for example: LUDI (Bohm, H. J. The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors. J. Comp. Aid. Molec. Design. 1992, 6, 61-78). LUDI is available from Biosym Technologies, San Diego, Calif.; LEGEND (Nishibata, Y., Itai, A., Tetrahedron, 1991, 47, 8985). LEGEND is available from Molecular Simulations, Burlington, Mass.; and LeapFrog (available from Tripos Associates, St. Louis, Mo.).
A number of techniques commonly used for modeling drugs may be employed (For a review, see: Cohen, N. C.; Blaney, J. M.; Humblet, C.; Gund, P.; Barry, D. C., “Molecular Modeling Software and Methods for Medicinal Chemistry”, J. Med. Chem., 1990, 33, 883). There are likewise a number of examples in the chemical literature of techniques that can be applied to specific drug design projects. For a review, see: Navia, M. A. and Murcko, M. A., Current Opinions in Structural Biology, 1992, 2, 202. Some examples of these specific applications include: Baldwin, J. J. et al., J. Med. Chem., 1989, 32, 2510; Appelt, K. et al., J. Med. Chem., 1991, 34, 1925; and Ealick, S. E. et al., Proc. Nat. Acad. Sci. USA, 1991, 88, 11540.
An alternative to designing or modelling small molecule TLR3 agonists is to screen existing small molecule chemical libraries for a TLR3 agonist. Such libraries can be screened by any TLR3 agonist assay known in the art, including those described herein. The compound libraries may be initially screened using a higher throughput assay, such as a competition assay with a known, labeled TLR3 agonist, such as the double-stranded RNA molecules polyI:polyC or polyA:polyU. Compounds that are positive in a competition assay are then further assayed for their ability to activate TLR3 and cause the subsequent cascade of biochemical events.
Thus, according to one embodiment, the invention provides a kit for determining if a test compound is useful for the treatment of cancer comprising a cancer cell characterized by the presence of TLR3 on its cell surface; and a reagent for detecting apoptosis or cell death. Such a kit optionally comprises a reagent that detects agonism of TLR3 by said test compound. This optional component may be used to confirm that any apopotisos or cell death of the cancer cell caused by said test compound is TLR3-mediated.
4. Nucleic Acid-based TLR3 Agonists
Nucleic acid-based TLR3 agonists useful in this invention comprise a region of double-stranded ribonucleic acids. The term “double-stranded” means a portion of the agonist where ribonucleotides are hydrogen bonded (base-paired) to complementary ribonucleotides to form a double-stranded structure. Preferably the entire nucleic acid-based TLR3 agonist consists of ribonucleotides and chemically modified ribonucleotides (“dsRNA TLR3 agonist”). More preferably, at least 50% of the dsRNA TLR3 agonist is in a double-stranded conformation under in vivo conditions. Even more preferred is if at least 60%, 70%, 80%, 90%, 95%, or 97% of the dsRNA TLR3 agonist is in a double-stranded conformation under in vivo conditions. The determination of what percentage of the dsRNA TLR3 agonist is in a double-stranded conformation is achieved by dividing the number of nucleotides that are base-paired by the total number of nucleotides in a molecule. Thus, a 21 base-paired molecule containing 2 nucleotide overhangs at both the 3′ and 5′ end would have 42 nucleotides that are base-paired and 4 nucleotides that are not base-paired, making it 42/46 or 91.3% double-stranded. Similarly, a molecule comprised of two 21 nucleotide strands that are complementary to one another at all nucleotides except for two nucleotides within the middle of each strand would have 38 (19+19) nucleotides that were base-paired and 4 (2+2) that were not base-paired. Such a molecule would be 38/42 or 90.5% double-stranded.
A double-stranded region of a dsRNA TLR3 agonist can be formed by a self-complementary region of a single RNA molecule (e.g., a stem and loop structure, such as hairpin RNA and shRNA), by two molecules of RNA that hybridize with one another in whole or in part (as in double-stranded RNA), or a mixture of both (e.g., a partially self-complementary molecule of RNA and a second RNA molecule that hybridizes to regions in the former in that remain single-stranded after the formation of the hairpin). The dsRNA TLR3 agonist of this invention may also comprise single-stranded regions, such as 3′ and/or 5′ overhangs at either end of the agonist, and/or “mismatched” or “loop-out” structures within the agonist.
In the case of shRNA, the dsRNA TLR3 agonist is encoded by a DNA sequence present on an expression vector. The expression vector is the molecule that is administered to the subject. The expression vector typically comprises a promoter that is activated by RNA polymerase II or III and terminator sequences, each of which is operably linked to the shRNA coding sequence to ensure its proper transcription. Promoters activated by RNA polymerase II include, but are not limited to, U6, tRNAval, H1, and modified versions of the foregoing. Promoters activated by RNA polymerase III include, but are not limited to, CMV and EF1α. In one preferred embodiment, the promoter is an inducible promoter or a tumor cell-specific promoter.
Within the context of the present invention, the term “dsRNA TLR3 agonist” designates any therapeutically or prophylactically effective RNA compound that comprises a double-stranded region. Such compounds are typically active per se, i.e., they do not encode a polypeptide or do not require translation to be active. A dsRNA TLR3 agonist can be of any length. Preferably, a dsRNA 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 TLR3 agonist 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 TLR3 agonist 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 TLR3 agonist is a long dsRNA having a chain length of greater than 1000 bp.
In one aspect, a dsRNA TLR3 agonist is a composition that comprises a heterogeneous mixture of dsRNA molecules, wherein a plurality of molecules have differing lengths. Preferably the dsRNA molecules in such a composition 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 TLR3 agonist 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 per strand. In one preferred example, the dsRNA is a short dsRNA having between 10 and 30, more preferably between 20 and 30 bp per strand. In a preferred embodiment a dsRNA TLR3 agonist composition has a substantially homogenous mixture of dsRNA molecules, where substantially all the molecules on each strand do not differ in chain length by more than 30 bp, 50 bp, 80 bp, 100 bp or 200 bp. Average chain length of dsRNA TLR3 agonists in a composition can be determined easily, for example, by gel permeation chromatography. One or more of the dsRNA molecules within such a compositions is optionally a siRNA molecule targeted against a cancer antigen.
The ribonucleotides in the dsRNA TLR3 agonist of this invention can be natural or synthetic, and may be chemically modified derivatives or analogs of natural nucleotides. Preferred modification are stabilizing modifications, and thus can include at least one modification in the phosphodiester linkage and/or on the sugar, and/or on the base. For example, one or both strands of the dsRNA can independently include one or more phosphorothioate linkages, phosphorodithioate linkages, and/or methylphosphonate linkages; modifications at the 2′-position of the sugar, such as 2′-O-methyl modifications, 2′-O-methoxyethyl modifications, 2′-amino modifications, 2′-deoxy modifications, 2′-halo modifications such as 2′-fluoro; combinations of the above, such as 2′-deoxy-2′-fluoro, modifications; acyclic nucleotide analogs, and can also include at least one phosphodiester linkage.
Oligonucleotides used in the dsRNA TLR3 agonist of this invention may also include base modifications or substitutions. Modified bases include other synthetic and naturally-occurring bases such as 5-methylcytosine (5-Me—C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and inosine, 2-propyl and other alkyl derivatives of adenine and inosine, 2-thiouracil and 2-thiocytosine, 5-halouracil and cytosine, 5-propynl(—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil and cytosine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, S-thioalkyl, 8-hydroxyl and other 8-substituted adenines and inosines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylinosine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azainosine and 8-azaadenine, 7-deazainosine and 7-deazaadenine and 3-deazainosine and 3-deazaadenine.
Other modifications include a 3′- and/or 5′-teiminal cap, a terminal 3′-5′ linkage, and a 5′-terminal phosphate group or modified phosphate group. Examples of terminal cap moieties include, but are not limited to, an inverted deoxy abasic moiety, an inverted deoxynucleotides, or a glyceryl moiety. In addition one or both strands (in a two-stranded dsRNA TLR3 agonist) may be or include a concatemer consisting of two or more oligonucleotide sequences joined by a linker(s). All of these modifications are well known in the art (see, for example, Kandimalla et al. ((2003) Nucl. Acid. Res. 31(9): 2393-2400).
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.
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 AmpligenTM appear to retain their TLR3 agonist activity.
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 comprises a homopolyRNA region.
The base in such homopolyRNA strands 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 RNA 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. The term “analogue” as used herein means any of the nucleotide modifications described above.
It will be appreciated that dsRNA TLR3 agonists can comprise any combination of bases and be designed using any suitable method. Preferably, the basic requirement of a region of double-strandedness, stability and resistance to nuclease attack and the preferences for chain length are taken into account. These properties, as well as relative TLR3 agonistic activity of any dsRNA TLR3 agonist can be tested and assessed with reference to the a rAn:rUn or rIn:rCn complex for example. Measures can be taken to increase stability and resistance to nucleases, or to increase or optionally decrease interferon-inducing action.
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 poylnucleotides, 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.
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).
One example of a TLR3 agonist is AmpligenTM (Hemispherx, Inc., of Rockville, Md., U.S.A.), a dsRNA formed by complexes of polyriboinosinic and polyribocytidylic/uridylic acid, such as rIn:r(CX,U or G)n where x has a value from 4 to 29, e.g., rIn:r(C12 U)n. Many mismatched dsRNA polymers which behave similarly to Ampligen have been studied; mismatched dsRNA based on poly I:C have 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 is a reported reduction in toxicity over compounds such as those described in Lampson et al. in U.S. Pat. No. 3,666,646.
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).
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.
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.
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, as well as any other dsRNA as herein described.
In a more preferred embodiment, the dsRNA is a fully double stranded (e.g., blunt-ended; no overhangs) polyA:polyU molecule consisting of between 19 and 30 base pairs (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs) and comprising between 1 and 30 stabilizing modifications and/or a 3′ and/or a 5′ cap. Stabilizing modification and caps are described above and are well-known in the art. The stabilizing modifications and/or the presence of a cap make the dsRNA more resistant to serum degradation. Preferred stabilizing modifications include phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications are known to dramatically increase the serum stability of dsRNA compounds. One example of a stabilized dsRNA is StealthTM RNAi (commercially available from Invitrogen, Carlsbad, Calif. USA).
In another preferred embodiment the dsRNA TLR3 agonist is a siRNA molecule or a shRNA molecule that is designed to specifically hybridize with the mRNA coding a tumor cell antigen or another protein involved in tumor proliferation. In this embodiment, the dsRNA molecule plays a dual role in the treatment of cancer. It is both an agonist of TLR3 and a suppressor of a specific tumor antigen expression. It has been demonstrated that siRNA molecules and shRNA molecules targeted against cellular proteins exhibit both sequence-dependent gene suppression and sequence-independent effects mediated through TLR3 (K. Kariko et al., J. Immunol. , 2004, 172: 6545-6549). Thus, it is expected that tumor antigen- or tumor-proliferation specific siRNA and shRNA molecules will also be agonists of TLR3 in cancer cells.
The choice of ribonucleotide sequences to use for such dsRNA TLR3 agonists will depend upon the specific tumor antigen or tumor proliferative protein to be suppressed, as well as the specific type of cancer to be treated. By way of example, the dsRNA TLR3 agonist may have a ribonucleotide sequence designed to hybridize to a mRNA encoding VEGF, VEGF receptors, A-Raf, B-Raf, C-Raf, Raf-1, HSP70, HSP90, PDGF, TGF-alpha, EGF, EGF receptor, a member of the human EGF-like receptor family such as HER-2/neu, HER-3, HER-4 or a heterodimeric receptor comprised of at least one HER subunit, carcinoembryonic antigen, gastrin releasing peptide receptor antigen, Muc-1, CA125, αvβ3 integrins, α5β1 integrins, αIIb β3-integrins, CTLA-4, CD20, CD22, CD30, CD33, CD52, CD56, CD80, PDGF beta receptor, Sre, VE-cadherin, IL-8, hCG, IL-6, IL-6 receptor, IL-15, or an mRNA encoding any additional protein targets set forth in http://oncologyknowledaebase.com/oksite/TargetedTherapeutics/TTOExhibit2.pdf and http://oncologyknowledgebase.com/oksite/TargetedTherapeutics/TTOExhibit3.pdf, the disclosures of which are herein incorporated by reference. The gene sequences encoding these proteins are known. Thus, siRNA and shRNA intended to suppress the expression of these proteins can therefore be designed using well-known software and algorithims (see, e.g., https://rnaidesigner.invitrogen.com/rnaiexpress/).
Detection of Cancer Cells expressing TLR3
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.
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.
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 arc 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.
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.
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. It will be appreciated that detecting TLR3 can be carried by detecting any known TLR3 allelic variant, including but not limited to allelic variations in the cytoplasmic region of TLR3 gene and in the immediate 5′ sequence of the TLR3 gene (see Piriel et al. (2005) Tissue Antigens 66(2): 125, the disclosure of which is incorporated herein by reference), for example a variant at the C/T polymorphism at position 2593, the C/A polymorphism at position 2642 and the A/G polymorphism at position 2690 in the TLR3 gene.
Various techniques known in the art may be used to detect or quantify TLR3 at the nucleotide level, including sequencing, hybridization, and amplification. Suitable methods include Southern blot (for DNAs), Northern blot (for RNAs), and fluorescent in situ hybridization (FISH). The detection or quantification of TLR3 at the protein level may be achieved by well-known methods including, but not limited to, gel migration, ELISA, radio-immunoassays (RIA) immuno-enzymatic assays (IEMA), Western Blot or another method for detecting a bound ligand. Protein detection methods require the use of a ligand specific for the TLR3 protein, preferably a specific antibody or a double-stranded RNA molecule. The binding of the antibody or dsRNA molecule may be directly detected if the ligand is labeled with, for example, a fluorescent dye, a radioisotope or another detectable moiety. Alternatively, the binding of the ligand to TLR3 may be indirectly detected through the use of a labeled second reagent that binds to the ligand. Such reagents include labeled anti-immunoglobulin antibodies and enzyme-linked anti-immunoglobulin antibodies that bind to the anti-TLR3 antibody, and labeled nucleic acids that can bind to one or both strands of a dsRNA molecule.
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, eBioscience, San Diego, Calif. USA; polyclonal anti TLR3, Ref ab13555, Abeam, UK; Clone 40C1285, Imgenex Corp, Biocarta US, San Diego, Calif.; and Clone TLR3.7, catalog no. HM2096, HyCuit biotechnology B.V., The Netherlands.
The antibody may be an agonistic antibody (as described above), an antagonistic antibody or any other antibody that binds to TLR3. Methods for making any of such antibodies are well-known in the art, as well as described in detail above for agonistic antibodies (with the exception of the screening step which for a diagnostic antibody, would be based on affinity for TLR3 rather than activation of that receptor).
In a specific embodiment, the method comprises contacting a sample from the subject with an antibody specific for the TLR3 protein, and determining the presence of an immune complex.
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 hybridize with a TLR3 gene, under suitable hybridization 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 μg) is reverse-transcribed by using random primers with RNaseH-free 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).
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).
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.
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.
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.
Upregulation of TLR3 Expression in Tumor Cells
Because the efficacy of the methods of treatment described herein depends upon the expression of TLR3 by the cells of the cancer to be treated, it may be advantageous to stimulate TLR3 expression prior to administering a TLR3 agonist. Thus, according to one embodiment, the invention provides a method of treating a subject suffering from cancer comprising the steps of a) administering to said patient an agent that causes increased expression of TLR3 in a cancer cell; and b) administering to said patient a TLR3 agonist.
Agents that cause upregulation of TLR3 expression are known in that art and include, but are not limited to, inflammatory cytokines such as type I interferons, respiratory syncytial virus and attenuated form thereof, and double-stranded RNA molecules. In addition, TLR3 expression can be upregulated by gene therapy techniques that result in in vivo transformation of cancer cells with an expression vector that encodes TLR3. Preferably, such techniques make use of delivery systems, such as viral vector systems or liposome-based systems that are modified to target cancer cells specifically. Targeting is typically achieved by modifying the outer surface of the delivery system to include a ligand or an antibody specific for the cancer cell surface.
The ability of an agent to cause upregulation of TLR3 expression, particularly in the cells of the cancer to be treated by a TLR3 agonist, may be assayed prior to administration of said agent to a subject. Such an assay is preferably carried out in vitro using a culture of cells derived from the cancer to be treated. Such a culture may be an established cell culture line or a culture of cells derived from a sample of the cancer obtained from the subject.
According to a related embodiment, the invention provides a composition of matter for treating a subject having a cancer comprising: an agent that causes upregulation of TLR3 expression; and a TLR3 agonist, wherein said agent and said agonist are present as separate dosage forms, but are associated with one another. The term “associated with one another” as used herein means that the separate dosage forms are packaged together or otherwise attached to one another such that it is readily apparent that the separate dosage forms are intended to be sold and administered as part of the same regimen. The agent and the TLR3 agonist are preferably packaged together in a blister pack or other multi-chamber package, or as connected, separately sealed containers (such as foil pouches or the like) that can be separated by the user (e.g., by tearing on score lines between the two containers).
According to another related embodiment, the invention provides a kit for determining if an agent is capable of upregulating TLR3 expression in a cancer cell, wherein the kit comprises a cancer cell or cancer cell line which can express TLR3; a TLR3-specific ligand; and a reagent for detecting the binding of said TLR3-specific ligand to a TLR3 expressed by said cancer cell.
Cytotoxic and Tumoricidal TLR3 Ligand Complexes
In one embodiment, the invention provides a composition comprising a TLR3 ligand in association with a cytotoxic agent. The term “cytotoxic agent” as used herein is a molecule that is capable of killing or inhibiting the proliferation of a cancer cell. Such complexes take advantage of the fact that TLR3 is expressed endosomally by dendritic cells, but is present on the surface of cancer cells. Thus, the TLR3 ligand portion of the complex targets the cytotoxic agent specifically to cancer cells with minimal affect on cells of the immune system. The term “in association with,” as used herein, means that the two agents are either hound to each other through a covalent and/or non-covalent bond; conjugated to one another (e.g., tethered or otherwise connected to one another directly of through a linking moiety); or are separate from one another, but present in the same carrier, such as a liposome or other carrier.
The TLR3 ligand component of such compositions may be selected from any TLR3 agonist that directly binds TLR3, any TLR antagonist that directly bind TLR3 or any other molecule that directly binds TLR3. Antibodies to TLR3, double-stranded RNA molecule and small molecule ligands of TLR3 are all potential TLR3 ligands that can be used in such compositions. Non-agonistic antibodies to TLR3 are the preferred ligand component and, as described herein, are commercially available.
The cytotoxic agent in such compositions may be selected from any agent known or being developed for the treatment of cancer, including general anti-proliferative agents, chemotherapy agents, tumor-specific anti-proliferative agents, cytotoxic agents, cytoinhibitory agents, etc. Examples include, but are not limited to, radioisotopes, toxic proteins, toxic small molecules, such as drugs, toxins, immunomodulators, hormones, hormone antagonists, enzymes, oligonucleotides, enzyme inhibitors, therapeutic radionuclides, angiogenesis inhibitors, chemotherapeutic drugs, vinca alkaloids, anthracyclines, epi dophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, COX-2 inhibitors, SN-38, antimitotics, antiangiogenic and apoptotic agents, particularly doxorubicin. methotrexate, taxol, CPT-11, camptothecans, nitrogen mustards, gemcitabine, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, Pseudomonas exotoxin, ricin, abrin, 5-fluorouridine, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonas endotoxin and others (see, e.g., Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995); Goodman and Gilman's The Pharmacological Basis of Therapeutics (McGraw Hill, 2001); Pastan et al. (1986) Cell 47:641; Goldenberg (1994) Cancer Journal for Clinicians 44:43; U.S. Pat. No. 6,077,499; http://oncologyknowledgebase.com/oksite/TargetedTherapeuties/TTOExhibit4.pdf, and http://oncologyknowledgebase.com/oksite/TargetedTherapeutics/TTOExhibit5.pdf, the entire disclosures of which are herein incorporated by reference). It will be appreciated that a toxin can be of animal, plant, fungal, or microbial origin, or can be created de novo by chemical synthesis.
If required, the toxins or other compounds can be linked to the TLR3 ligand directly or indirectly, using any of a large number of available methods depending upon the nature of the TLR3 ligand.
For example, an agent can be attached at the hinge region of a TLR3 antibody component via disulfide bond formation, using cross-linkers such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP), or via a carbohydrate moiety in the Fc region of the antibody (see, e.g., Yu et al. (1994) Int. J. Cancer 56: 244; Wong, “Chemistry of Protein Conjugation and Cross-linking,” (CRC Press 1991); Upeslacis et al., “Modification of Antibodies by Chemical Methods,” in Monoclonal Antibodies: Principles And Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies: Production, Engineering And Clinical Application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995); Cattel et al. (1989) Chemistry Today 7:51-58; Delprino et al. (1993) J. Pharm. Sci 82:699-704; Arpicco et al. (1997) Bioconjugate Chemistry 8:3; Reisfeld et al. (1989) Antibody, Immunoconj. Radiopharm. 2:217; the entire disclosures of each of which are herein incorporated by reference).
A toxin or other compound can be conjugated to a dsRNA molecule using a wide variety of well-known techniques. Exemplary U.S. patents that describe the preparation of oligonucleotide conjugates include, for example, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,508,046; 4,587,044; 4,605,735; 4,667,025; 4,752,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,482,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,51 0,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is incorporated by reference herein in its entirety.
When a TLR3 ligand and a cytotoxic agent are present as separate agents in the same vehicle, it is necessary that the TLR3 ligand be at least partially exposed on the surface of the vehicle so that the vehicle will be targeted to the TLR3-expressing tumor cell. Conjugation of antibodies to liposomes to make a delivery vehicle (“immunoliposomes”) is well known in the art, e.g., described by Torchilin et al., 1992, FASEB J. 6:2716-2719; Bondas et al., 1999, International J. of Pharmaceutics 181:79-93; Tana et al., 1998, Japanese J. of Cancer Res. 89:1201-1211; or Koning et al., 1999, Biochimica et Biophysica Acta 1420:153-167. Conjugation of other TLR3 ligands to liposomes may be carried out in a similar manner.
TLR3 Agonist Formulations and Delivery Systems
The methods of treatment according to this invention comprise the step of administering to a subject a TLR3 agonist. Preferably, the TLR3 agonist is formulated into a pharmaceutically acceptable composition. The pharmaceutically acceptable compositions useful in the invention additionally comprise a pharmaceutically acceptable carriers, adjuvants and vehicles. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions useful in this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, poly acrylates, waxes, polyethyl ene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The pharmaceutical compositions of the invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. In certain embodiments, the compound of the formulae herein is administered transdermally (e.g., using a transdermal patch or iontophoretic techniques). Other formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA (17th ed. 1985).
Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.
In certain preferred embodiments, the compound is administered orally. Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative. surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets optionally may be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. Methods of formulating such slow or controlled release compositions of pharmaceutically active ingredients, such as those herein and other compounds known in the art, are known in the art and described in several issued US Patents, some of which include, but are not limited to, U.S. Pat. Nos. 4,369,172; and 4,842,866, and references cited therein. Coatings can be used for delivery of compounds to the intestine (see, e.g., U.S. Pat. Nos. 6,638,534, 5,217,720, and 6,569,457, 6,461,631, 6,528,080, 6,800,663, and references cited therein).
In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. Surfactants such as sodium lauryl sulfate may be useful to enhance dissolution and absorption.
Compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.
Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
Such injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.
The pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal or vaginal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition will be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches and iontophoretic administration are also included in this invention.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Aerosol formulations that may be utilized in the methods of this invention also include those described in U.S. Pat. No. 6,811,767, the disclosure of which is herein incorporated by reference.
For dsRNA TLR3 agonists, particularly those dsRNA TLR3 agonists that are also siRNA molecules, effective delivery requires that at least a portion of the administered TLR3 agonist enter into the tumor cell. Naked siRNA is known to slowly enter cells by a mechanism that has not yet been elucidated. However, naked siRNA is not very stable in serum and therefore it is preferred that the siRNA compositions useful in this invention be formulated in a delivery vehicle. Preferably the delivery vehicle for a dsRNA TLR3 agonist that is also a siRNA is a liposome.
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged dsRNA molecules to form a stable complex. The positively charged dsRNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et at., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap dsRNA rather than complex with it. Since both the dsRNA and the lipid are similarly charged, repulsion rather than complex formation occurs. The dsRNA is thus entrapped in the aqueous interior of these liposomes. pH-sensitive liposomes have been used, for example, to deliver dsRNA encoding the thymidine kinase gene to cell monolayers in culture (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Liposomes that include nucleic acids have been described, for example, in Thierry et al., WO 96/40062 (methods for encapsulating high molecular weight nucleic acids in liposomes); Tagawa et al., U.S. Pat. No. 5,264,221 (protein-bonded liposomes containing RNA); Rahman et al., U.S. Pat. No. 5,665,710 (methods of encapsulating oligodeoxynucleotides in liposomes); Love et al., WO 97/04787 (liposomes that include antisense oligonucleotides).
Another type of liposome, transfersomes are highly deformable lipid aggregates which are attractive for drug delivery vehicles. (Cevc et al., 1998, Biochim Biophys Acta. 1368(2): 201-15.) Transfersomes may be described as lipid droplets which are so highly deformable that they can penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, for example, they are shape adaptive, self-repairing, frequently reach their targets without fragmenting. and often self-loading. Transfersomes can be made, for example, by adding surface edge-activators, usually surfactants, to a standard liposomal composition.
TLR3 agonists that are shRNA also require entry into the cell because the agent that is administered to the subject is a DNA expression vector that encodes the shRNA. That DNA molecule must enter the cell in order to be transcribed. It is to be understood that the term “TLR3 agonist” includes both a DNA molecule that codes upon transcription for an shRNA that is a dsRNA TLR3 agonist and the transcribed shRNA. The liposomal delivery systems described above are one way in which the DNA encoding a shRNA TLR3 agonist can enter the cell. Alternatively, the DNA encoding a shRNA TLR3 agonist can be prepared in a viral vector system that has the capability of entering into cells. These are well-known in the art and include Madzak et al., J. Gen. Virol., 73: 1533-36 (1992) (papovavirus SV40); Berkner et al., Curr. Top. Microbiol. Immunol., 158: 39-61 (1992) (adenovirus); Moss et al., Curr. Top. Microbiol. Immunol., 158: 25-38 (1992) (vaccinia virus); Muzyczka, Curr. Top. Microbiol. Immunol., 158: 97-123 (1992) (adeno-associated virus); Margulskee, Curr. Top. Microbiol. Immunol., 158: 67-93 (1992) (herpes simplex virus (HSV) and Epstein-Barr virus (HBV)); Miller, Curr. Top. Microbial. Immunol., 158: 1-24 (1992) (retrovirus); Brandyopadhyay et al., Mol. Cell. Biol., 4: 749-754 (1984) (retrovirus); Miller et al., Nature, 357: 455-450 (1992) (retrovirus); Anderson, Science, 256: 808-813 (1992) (retrovirus); C. Hofmann et al., Proc. Natl. Acad. Sci. USA, 1995; 92, pp. 10099-10103 (baculovirus).
The liposomal and virus-based delivery systems discussed above may include a targeting molecule to direct delivery of the TLR3 agonist to the cancer. Such targeting molecules include ligands and/or antibodies (or binding fragments thereof) that recognize a molecule on the tumor cell surface. One particular tumor cell surface antigen that can be targeted by such delivery systems is TLR3 itself. Preferably, the ligand binds to as site on TLR3 that is separate from and does not interfere with the TLR3 agonist binding site.
In certain embodiments of the invention it is desirable that a dsRNA TLR3 agonist not enter into a cell. Without being bound by theory, the inventors believe that in certain instances agonizing TLR3 expressed on the surface of a cancer cell is sufficient to kill the cancer cells without the need to agonize cytoplasmic TLR3. Many other cell types and in particular dendritic cells, express TLR3 only internally, typically in the endosomal membrane. In order to distinguish between different cellular locations for TLR3, the agonist must be prevented from entering into the cells of the subject.
One way to achieve this is to administer the dsRNA TLR3 agonist without a vehicle, such as a liposome, that enhances the ability of the agonist to penetrate cells. The ability of naked dsRNA to enter cells appears to be relatively limited. The mechanism by which naked dsRNA enters cells is unknown, but it is a relatively inefficient process and seems to preferentially occur in tissues such as adipose, liver and parts of the kidney. Thus, administering a dsRNA TLR3 agonist that is not formulated into a liposome or other vehicle that enhances cell penetration will be sufficient to prevent agonism of cytoplasmic TLR3 in many instances.
The treatment of cancer typically is performed in conjunction with a surgical procedure to remove most of all of the cancer from the subject. One advantage of such a surgical procedure is the ability to either implant a device that releases a therapeutic agent directly at the site of the cancer or to administer a therapeutic agent locally to the cancer. Various techniques can be used for providing the subject compositions at the site of interest, such as injection, use of catheters, trocars, projectiles, pluronic gel, stents, sustained drug release polymers or other device which provides for internal access.
Thus, according to another embodiment, the invention provides a method of impregnating or filling an implantable drug release device comprising the step of contacting said drug release device with a TLR3 agonist or composition comprising a TLR3 agonist. Implantable drug release devices include, but are not limited to, biodegradable polymer capsules or bullets, non-degradable, diffusible polymer capsules and biodegradable polymer wafers.
According to another embodiment, the invention provides 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.
The invention also provides a method of treating a patient suffering from cancer comprising the steps of: surgically removing at least a portion of said cancer; and implanting a drug release device impregnated with or comprising a TLR3 agonist at the site of said cancer. This method optionally includes the step of determining if the cells of said cancer express TLR3 prior to the step of implanting said drug release device.
TLR3 Agonist Combination Compositions
Treatment with a TLR3 agonist as described herein can optionally advantageously be combined with one or more other therapeutic agents useful in the treatment of cancer. Thus, the TLR3 agonists described herein can be used conjointly, or in combination with, another therapeutic agent useful in the treatment of cancer. The TLR3 agonist compositions described above may thus additionally include other therapeutic agents useful in the treatment of cancer. The other therapeutic agent may be in the same or preferably in a separate container. Such agents include other TLR3 agonists of different molecular composition (i.e., either a different type of molecule or the same type of molecule with a different amino acid or nucleotide sequence or a different chemical formula); cytotoxins, including but not limited to those recited above for use in cytotoxic and tumoricidal TLR3 ligand complexes; cytotoxic and tumoricidal TLR3 ligand complexes; agents that target a tumor antigen or a tumor proliferative protein, such as those disclosed above as targets for siRNA; chemotherapy agents including, but not limited to, cisplatin (CDDP), carboplatin, oxaliplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan. nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tarnoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitablen, navelbine, famesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing; therapeutic agents and combination of therapeutic agents for treatment of specific cancers, such as for breast cancer: 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),HerceptinTM), tamoxifen, the combination of tamoxifen and a cytotoxin, taxanes including docetaxel and Paclitaxel, the combination of a taxane plus doxorubicin and cyclophophamide; for colon cancer: 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; for prostate cancer: a radioisotope (i.e., palladium, strontium-89 and. Iridium), leuprolide or other LHR agonists, nonsteroidal antiandrogens (flutamide, nilutamide, and bicalutamide), steroidal antiandrogens (cyproterone acetate), the combination of leuprolide and flutainide, estrogens such as DES, chlorotrianisene, ethinyl estradiol, conjugated estrogens U.S.P., DES-diphosphate, second-line hormonal therapies such as aminoglutethimide, hydrocortisone, flutamide withdrawal, progesterone, and ketoconazole, low-dose prednisone, or other chemotherapy agents or combination of agent reported to produce subjective improvement in symptoms and reduction in PSA level including docetaxcl, paclitaxel, estramustine/docetaxel, estramustine/etoposide, estramustine/vinblastine, and estramustine/Paclitaxel; for melanoma: 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-A), and interleukin-2 (IL-2);for ovarian cancer: Paclitaxel, docetaxel, cisplatin, oxaliplatin, hexamethylmelamine, tamoxifen, ifosfamide, 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; for lung cancer: cisplatin, vincristine. vinblastine, mitomycin, doxorubicin, and etoposide, alone or in combination, the combination of cyclophosphamide, doxorubicin, vincristine/etoposide, and cisplatin (CAV/EP), the combination of cisplatin and vinorelbine, paclitaxel, docetaxel or gemcitabine, and the combination of carboplatin and paclitaxel.
The methods of using a TLR3 agonist composition described herein may also comprises combination treatment with an anti-angiogenic agent. The TLR3 agonist compositions described above may thus also include an anti-angiogenic agent. New blood vessel formation (angiogenesis) is a fundamental event in the process of tumor growth and metastatic dissemination. The vascular endothelial growth factor (VEGF) pathway is well established as one of the key regulators of this process. The VEGF/VEGF-receptor axis is composed of multiple ligands and receptors with overlapping and distinct ligand-receptor binding specificities, cell-type expression, and function. Activation of the VEGF-receptor pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature. In addition, VEGF mediates vessel permeability, and has been associated with malignant effusions. The VEGF-related gene family comprises six secreted glycoproteins referred to as VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PIGF)-1 and -2. A number of exemplary anti-angiogenic agents acting of the VEGR pathway are known, any of which can be used in accordance with the invention, including small molecule inhibitor, neutralizing antibodies antisense strategies, RNA aptamers and ribozymes against VEGF-related gene family (e.g. the VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E proteins). Variants of VEGF with antagonistic properties may also be employed, as described in WO 98/16551, specifically incorporated herein by reference. Further exemplary anti-angiogenic agents that are useful in connection with combined therapy are listed in Table D of U.S. Pat. No. 6,524,583, the disclosure of which agents and indications are specifically incorporated herein by reference. Particularly preferred anti-angiogenic agents inhibit signaling by a receptor tyrosine kinase including but not limited to VEGFR1, VEGFR-2,3 PDGFR-beta, Flt-3, c-Kit, p38 alpha and FGFR-1. Further anti-angiogenic agent may include agents that inhibit one or more of the various regulators of VEGF expression and production, such as EGFR, HER-2, COX-2, or HIF-1. Another preferred class of agents includes thalidomide or the analogue CC-5013.
Selected VEGF/VEGFR inhibitors of the monoclonal antibody (mAb) and tyrosine kinase inhibitor (TKI) class include bevacuzimab (Avastin) (mAb, inhibiting VEGF-A, Genentech); IMC-1121B (mAb, inhibiting VEGFR-2, ImClone Systems); CDP-791 (Pegylated VEGFR-2, Celltech); 2C3 (mAb, VEGF-A, Peregrine Pharmaceuticals); PTK-787 (TKI, VEGFR-1, -2, Novartis); AEE788 (TKI, VEGFR-2 and EGFR, Novartis); ZD6474 (TKI, VEGFR-1, -2, -3, EGFR AstraZeneca); AZD2171 (TKI, VEGFR-1, -2, AstraZeneca), SU11248 (TKI, VEGFR-1, -2, PDGFR Pfizer); AG 13925 (TKI, VEGFR-1, -2, Pfizer); AG013736 (TKI, VEGFR-1, -2, Pfizer); CEP-7055 (TKI, VEGFR-1, -2, -3, Cephalon); CP-547,632 (TKI, VEGFR-1, -2, Pfizer); VEGF-trap (Soluble hybrid receptor VEGF-A, PIGF (placenta growth factor) Aventis/Regeneron); GW786024 (TKI, VEGFR-1, -2, -3, GlaxoSmithKline); Bay 93-4006 (TKI, VEGFR-1, -2, PDGFR Bayer/Onyx): and AMG706 (TKI, VEGFR-1, -2, -3, Amgen). Most preferred are tyrosine kinase inhibitors that inhibit one or more receptor tyrosine kinases selected from the group consisting of VEGFR1, VEGFR-2,3 PDGFR-beta, Flt-3, c-Kit, p38 alpha and FGFR-1. Preferred examples include SU11248 (Pfizer) and Bay 93-4006 (sorefanib, Bayer).
The methods of using a TLR3 agonist composition described herein may also comprises combination treatment with a pro-apoptotic agent. The TLR3 agonist compositions described above may thus also include a pro-apoptotic agent. A number of proteins useful as targets for modulation by pharmaceutical agents are known in the art, including any of those reviewed in Green and Kroemer, J. (2005) Clin. Investig. 115(10):2610-2617, the disclosure of which is incorporated by reference in its entirety. Examples of pro-apoptotic pharmaceutical agents include:

    
    
        drugs that induce mitochondrial outer membrane permeabilization such as oblimirsen (Bcl-2 antisense oligonucleotide, Genta), EGCG (small molecule targeting Bcl-2, Burnham Inst./Mayo Clinic), Gossypol (small molecule targeting Bcl-2, Univ. Michigan), LY2181308 (antisense oligonucleotide targeting surviving (Eli Lilly/Isis), and arsenic trioxide;
        drugs that regulate p53 activity such as Advexin INGN201 (adenovirus modulating p53, Introgen), SCH58500 (adcnovirus modulating p53, Schering-Plough), ONYX-015 (E1B mutated adenovirus modulating p53, Onyx Pharma.); drugs that modulate caspases and/or endogenous inhibitors or caspases such as AEG35156 (antisense oligonucleotide targeting XIAP, Aegara/Hybridon); drugs that modulate death receptors and/or their ligands, such as TNF-alpha polypeptides, HGS-ETR1 (agonistic mAb targeting TRAIL-R1, Human Genome Sciences), HGS-ETR2 and HGS-TR2J ((two agonistic mAbs targeting TRAIL-R2, Human Genome Sciences); PRO1764 (soluble TRAIN ligands, Genentech/Amgen); and
        drugs targeting poly(ADP-ribose) polymerase (PARP), such as AG014699 (small molecule, Cancer Res. Tech.);
        drugs targeting the proteosome, such as bortezomib (Velcade) (26S proteosome inhibitor, Millennium Pharma.); and
        kinase inhibitors, such as herceptin (mAb targeting HER2, Roche), centuximab (mAb targeting HER1, Imclone/BMS), gefitinib (Iressa) and erlotinib (Tarceva) (small molecule inhibitors of HER1, AstraZeneca and Genentech/OSI respectively, CCI-779 (small molecule acting on mTOR, Novartis, Bay 43-9006 (small molecule inhibitor of kinases including Raf and VEGFR, and imatinib mesylate (Gleevec, STI-571) (small molecule inhibitor of cKit, PDGFR, Bcr-Abl, Novartis).
    
    


The TLR3 agonist compositions described above may also include other therapeutic agents such as immunomodulatory agents such as tumor necrosis factor, interferon alpha, beta, and gamma, IL-2, IL-12, IL-15, IL-21, CpG-containing single-stranded DNA, agonists of other TLRs, other cytokines and immunosuppression agents; F42K and other cytokine analogs; or MIP-1, MIP-I beta, MCP-1, RANTES, and other chemokines; agents that affect the upregulation of cell surface receptors and GAP junctions; cytostatic and differentiation agents; or inhibitors of cell adhesion.
It will be recognized by those of skill in the art that certain therapeutic agents set forth above fall into two or more of the categories disclosed above. For the purpose of this invention, such therapeutic agents are to be considered members of each of those categories of therapeutics and the characterization of any therapeutic agent as being in a certain specified category does not preclude it from also being considered to be within another specified category.
In yet another embodiment, the invention provides a composition of matter comprising a TLR3 agonist and another therapeutic agent useful in the treatment of cancer in separate dosage forms, but associated with one another. The term “associated with one another” as used herein means that the separate dosage follus are packaged together or otherwise attached to one another such that it is readily apparent that the separate dosage forms are intended to be sold and administered as part of the same regimen. The agent and the TLR3 agonist are preferably packaged together in a blister pack or other multi-chamber package, or as connected, separately sealed containers (such as foil pouches or the like) that can be separated by the user (e.g., by tearing on score lines between the two containers).
In another embodiment, a dsRNA TLR3 agonist may be formulated together in a composition with an endocytosis inhibitor. Dendritic cells express TLR3 on their endosomal surface and are believed to take up dsRNA through endocytosis. Agonism of TLR3 in dendritic cells causes maturation of dendritic cells, production of type I interferons and the secretion of various cytokines. In a tumor environment, certain of those cytokines may cause tumor growth. Thus, in certain embodiments it is desirable to prevent a dsRNA TLR3 agonist from entering into dendritic cells. Compositions comprising a dsRNA TLR3 agonist and an agent that blocks endocytosis can be used to treat cancer characterized by a TLR3-expressing cancer cell without stimulating TLR3 in dendritic cells. Examples of an agent that inhibits endocytosis include, but are not limited to, an actin stabilizing agent, such as latrunculin B, and an endosomal pH neutralizing agent, such as choloroquine or hydroxychloroquine.
In a related embodiment, the endocytosis inhibitor may be conjugated to the dsRNA. TLR3 agonist. The two agents may be either covalently bonded or conjugated directly to one other or attached via a linker or tether moiety.
In yet another related embodiment, a dsRNA TLR3 agonist and an endocytosis inhibitor may be present in separate dosage forms, but associated with one another.
Cancer and Therapeutic Methods
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 an TLR3 agonist for the manufacture of a medicament for the prevention, management, treatment or amelioration of cancer or one or more symptoms thereof.
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.
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. The invention also concerns the use of an TLR3 agonist for the manufacture of a medicament 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. Preferably, the TLR3 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.
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. The invention also concerns the use of an TLR3 agonist for the manufacture of a medicament for preventing, managing, treating or ameliorating cancer, wherein said TLR3 agonist is used in combination with one or more 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.
The invention provides methods for preventing, managing, treating or ameliorating cancer that has been 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. The invention also concerns the use of an TLR3 agonist for the manufacture of a medicament for preventing, managing, treating or ameliorating cancer that has been refractory to one or more therapeutic agents or therapies. 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.
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. Other therapies useful in combination with the administration of TLR3 agonist also include treatment with any of the agents described above that are useful in TLR3 agonist combination compositions.
In all of the treatment regimens it is preferred that the TLR3 agonist is a dsRNA compound. Treatments involving the administration of a TLR3 agonist in combination with one or more therapies include simultaneous administration and treatment either as separate therapies administered at the same time (such as multiple dosage forms or a physical treatment such as exposure to radiation, while administering the TLR3 agonist), or as a single combination therapy (such as the combination single dosage forms described above containing a TLR3 agonist and another therapeutic agent. A treatment involving the administration of a TLR3 agonist in combination with one or more therapies also includes consecutive or staggered treatments where the TLR3 agonist is administered at one point in time and the other therapy is administered from 1 minute to 1 month before or after that time.
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 μg/ml, 15 μg/ml, 20 μg/ml, 25 1 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45 μg/ml or 50 μg/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.
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.
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%, ate 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.
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.
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
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.
Leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, 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 myelomas 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, chordoma, 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, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, 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 pheochromocytoma 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 cystic 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, 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 (epidennoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), sperm atocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and venacous; 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, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/ or ureter); Wilkins' 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, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, 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).
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 promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosafcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xenoderma pigmentosum, keratoacanthoma, 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.
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
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.
The telin “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.
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:
Inflammatory (breast tissue is warm and appears red; tends to spread quickly)
Medullary carcinoma (originates in central breast tissue)
Mucinous carcinoma (invasive; usually occurs in postmenopausal women)
Paget's disease of the nipple (originates in the milk ducts and spreads to the skin of the nipples or areola)
Phyllodes tumor (tumor with a leaf-like appearance that extends into the ducts; rarely metastasizes)
Tubular carcinoma (small tumor that is often undetectable by palpation)
Rarely, sarcomas (cancer of the connective tissue) and lymphomas (cancer of the lymph tissue) develop in the breasts.
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.
Staging involves using the letters T, N, and M to assess tumors:
size of the primary tumor (T);
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
absence or presence of distant metastases (M)-cancer that has spread from the original (primary) tumor to distant organs or distant lymph nodes.
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.
Once the T, N, and M components are determined, a “stage” of I, II, III or IV is assigned:
Stage I cancers are small, localized and usually curable.
Stage II and III cancers typically are locally advanced and/or have spread to local lymph nodes.
Stage IV cancers usually are metastatic (have spread to distant parts of the body) and generally are considered inoperable.
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).
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.
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 metastases 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 I, 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.
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 as 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.
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.
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), HerceptinTM), 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
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.
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.
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
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 flutainide, 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 docetaxel, paclitaxel, estramustine/docetaxel, estramustine/etoposide, estramustine/vinblastine, and estramustine/paclitaxel.
Treatment of Melanoma
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-A), and interleukin-2 (IL-2).
Treatment of Ovarian Cancer
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
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.
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
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 (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitablen, navelbine, famesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.
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.
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 microtabule 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).
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).
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).
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).
Doxorubicin. Doxorubicin hydrochloride, 5,12-Naphthacenedione, (8s-ds)-10-[(3-amino-2,3,6trideoxy-a-L-Iyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,1 1-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.
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, myelonia, diffuse histiocytic lymphoina, 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.
Since doxorubicin is poorly absorbed it is administered intravenously. The phannacokinetics 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.
Appropriate doses are, for an adult, administered intravenously, are 60 to 75 mg/m2 at 21-day intervals, or 25 to 30 mg/m2 on each of 2 or 3 successive days repeated at 3- or 4-wk intervals, or 20 mg/m2 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 bilirabin 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/m2 in patients with normal heart function and 400 mg/m2 in persons having received mediastinal irradiation. Alternatively, 30 mg/m2 on each of 3 consecutive days, repeated every 4 wk may be administered. Exemplary doses may be 10 mg/m2, 20 mg/m2, 30 mg/m2, 50 mg/m2, 100 mg/m2, 150 mg/m2, 175 mg/m2, 200mg/m2, 225 mg/m2, 250 mg/m2, 275 nig/m2, 300 mg/m2, 350 mg/m2, 400 mg/m2, 425 mg/m2, 450 mg/m2, 475 mg/m2, 500 mg/m2. 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
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.
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.
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.
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
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 miscopically 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.
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
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
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-I beta, MCP-1, RANTES, and other chemokines.
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.
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
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 axillary lymph nodes (n=200).
Tumor biopsies from these patients were stained with TLR3-specific mAb and correlation between TLR3 expression and polyAU efficacy was determined.
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 TLR3 RNA interference.
Results: 182 tumor samples (91%) were available from the 200 pT×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:
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 have 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 axillary lymph node involvement, only the 200 patients with axillary node involvement were included in the present study.
Immunostainings:
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 Pobolsky, Massachusetts General Hospital, Boston) or rabbit preimmun 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:
Survival curves were determined according to Kaplan-Meier method. Survival curves were compared using Khi2 test.
Results:
Patients characteristics
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 1







Patients characteristics











TLR3− tumors
TLR3+ tumors




(n = 157)
(n = 18)















Poly

Poly
Total



Observation
AU
Observation
AU
(n =


Characteristics
(n = 77)
(n = 80)
(n = 10)
(n = 8)
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

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
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:
TLR3 is overexpressed by tumor cells in around 10% of cancer cases.
TLR3 expression correlates with the benefit of adjuvant therapy with polyAU in patients with lymph node positive breast cancer.

## Claims

1. A method for treating a subject having a melanoma comprising:
a) determining whether melanoma cells in said subject express a TLR3 receptor comprising SEQ ID NO: 2, the expression of a TLR3 receptor comprising SEQ ID NO: 2 being indicative of a subject responding to a TLR3 agonist selected from poly I: poly C (poly IC), poly A: poly U (polyAU), and
b) administering to said subject whose melanoma cells are determined to express a TLR3 receptor comprising SEQ ID NO: 2 a pharmaceutical composition comprising an effective amount of a TLR3 agonist selected from poly I: poly C (poly IC) or poly A: poly U (poly AU), and a pharmaceutically acceptable carrier.

2. The method according to claim 1, wherein said melanoma in said subject comprises at least 10% of cancer cells expressing a TLR3 receptor comprising SEQ ID NO: 2.

3. The method according to claim 1, wherein said determining whether cells of said melanoma in said subject express a TLR3 receptor comprising SEQ ID NO: 2 is performed ex vivo on a biopsied sample of said melanoma using a TLR3-specific antibody or an antigen binding fragment thereof.

4. The method according to claim 3, wherein said TLR3-specific ligand is capable of detecting a TLR3 receptor comprising SEQ ID NO: 2 expressed on the surface and in the cytoplasm of cancer cells.

5. A method for assessing the response of a subject having melanoma to a treatment using a TLR3 agonist selected from poly I: poly C (poly IC) or poly A:
poly U (polyAU), the method comprising determining whether cancer cells in said subject express a TLR3 receptor comprising SEQ ID NO: 2, the expression of a TLR3 receptor comprising SEQ ID NO: 2 being indicative of a responder subject, administering a therapeutically effective amount of said -TLR3 agonist to said subject and determining if there has been a reduction in size or spread of said cancer in said subject.

6. A method for selecting subjects having a melanoma that respond to a treatment using a TLR3 agonist selected from poly I: poly C poly IC) or poly A: poly U (polyAU), the method comprising determining whether melanoma cells in said subject express a TLR3 receptor comprising SEQ ID NO: 2, the expression of a TLR3 receptor comprising SEQ ID NO: 2 being indicative of a responder subject, administering a therapeutically effective amount of said TLR3 agonist to said subject if said subject has melanoma cells expressing a TLR3 receptor comprising SEQ ID NO: 2 and determining if there has been a reduction in size or spread of said melanoma in said subject.

7. A method for treating a subject having a melanoma, the method comprising determining whether melanoma cells in said subject express a TLR3 receptor comprising SEQ ID NO: 2, the expression of a TLR3 receptor comprising SEQ ID NO: 2 being indicative of a subject responding to a TLR3 agonist, and treating said subject whose melanoma cells express a TLR3 receptor comprising SEQ ID NO: 2 with a TLR3 agonist selected from poly I: poly C (poly IC) or poly A: poly U (polyAU).

8. The method according to claim 5, wherein the TLR3 agonist is poly AU.

9. The method according to claim 5, wherein the TLR3 agonist is poly IC.

10. The method according to claim 6, wherein the TLR3 agonist is poly AU.

11. The method according to claim 6, wherein the TLR3 agonist is poly IC.

12. The method according to claim 7, wherein the TLR3 agonist is poly AU.

13. The method according to claim 7, wherein the TLR3 agonist is poly IC.