# Patent

## Title
Methods for treating hematopoietic malignancies

## Abstract

The present invention relates to methods for treating neoplasias in a mammalian subject. In particular, the invention provides methods for treating lymphomas, including forms of non-Hodgkin lymphoma. In one embodiment, these methods involve reducing tumor necrosis factor signaling.

## Background
This application claims priority to co-pending U.S. provisional Application Ser. No. 61/062,725, filed Jan. 28, 2008, which is herein incorporated by reference in its entirety for all purposes.
A Sequence Listing has been submitted in ASCII text file named 16915_ST25.txt, created on Feb. 25, 2013, consisting of 34.3 KB, the entire content of which is herein incorporated by reference.


This invention was made with government support under contract numbers T32 CA09523, K01 CA122192, R01 HL088686, and R01CA135531 awarded by the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates to methods for treating neoplasias in a mammalian subject. In particular, the present invention provides methods for treating lymphomas, including forms of non-Hodgkin lymphoma. In one embodiment, these methods involve reducing tumor necrosis factor-alpha signaling.
BACKGROUND OF THE INVENTION
Despite years of research into the development of new methods of treatment, cancers of the lymphatic system, or lymphomas, remain quite common. For example, more than 60,000 people in the United States are diagnosed with lymphoma each year, including more than 55,000 cases of non-Hodgkin lymphoma (NHL), and these numbers are constantly increasing. In addition, the prognosis for those affected by these diseases is often poor, as the survival rates for lymphoma patients remain low.
While traditional treatments for lymphoma typically depend on the type of lymphoma as well as the medical history of the patient, first-line treatments typically include chemotherapy. Such chemotherapy generally entails the administration of a mixture of compounds (e.g., the formulation referred to as CHOP that includes cyclophosphamide, doxorubicin, vincristine and prednisone. Cancer treatments also frequently include other forms of therapy (e.g., radiation). In many cases, patients respond initially to such first-line treatments, but subsequently suffer a relapse (e.g., tumor reappears or resumes growing). Following one such relapse, patients are often treated with further chemotherapy, or with other procedures such as bone marrow transplantation. Again, in many cases, patients initially respond to such additional treatments, but subsequently suffer another relapse. In other cases, a patient fails to respond at all to a treatment, even initially, and is thus said to have a refractory cancer. In such cases little agreement exists in the art regarding optimal subsequent treatment.
Thus there remains a need in the art for methods suitable for treating relapsing and/or refractory lymphomas, as well as for treating other poor-prognosis hematopoietic malignancies.
SUMMARY OF THE INVENTION
The present invention relates to methods for treating neoplasias in a mammalian subject. In particular, the invention provides methods for treating lymphomas, including forms of non-Hodgkin lymphoma. These methods involve reducing tumor necrosis factor-alpha signaling. Specifically, the present invention provides methods of treating a hematopoietic neoplasm in a human subject, comprising administering to the human subject an effective amount of a tumor necrosis factor-alpha (TNF-alpha) inhibitor. In some embodiments the TNF-alpha inhibitor is selected from the group consisting of an anti-TNF-alpha antibody and a soluble TNF-alpha receptor. In one embodiment, the TNF-alpha inhibitor comprises Pentoxifylline (Trental®).
In some embodiments, the anti-TNF-alpha antibody is a chimeric monoclonal antibody comprising a murine variable region or a TNF-binding portion thereof, and a human constant region. Anti TNF-alpha antibodies are exemplified by those described in U.S. Pat. Nos. 5,795,967, 5,223,395, 5,672,347, 6,537,549, 5,654,407, 6,649,589, 6,982,089, 6,419,944, and 6,379,666, including adalimumab (Humira®), and infliximab (Remicade®). In some preferred embodiments, the chimeric monoclonal antibody comprises infliximab (Remicade®). In some embodiments, the anti-TNF-alpha antibody is a humanized monoclonal antibody. In some embodiments, the anti-TNF-alpha antibody is a fully human monoclonal antibody. In some preferred embodiments, the human monoclonal antibody is adalimumab. In some embodiments, the soluble TNF-alpha receptor is a chimeric protein comprising human immunoglobulin G (IgG). In particular embodiments, the TNF-alpha receptor comprises eternacept (Enbrel®), an antagonistic soluble TNF-alpha receptor. In some preferred embodiments, the IgG fusion protein is etanercept. In additional preferred embodiments, the methods further comprise administering to the human subject a pharmaceutical composition comprising cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP). In some embodiments, the methods further comprise administering to the human subject an anti-CD20 monoclonal antibody. In some preferred embodiments, the anti-CD20 monoclonal antibody is rituximab. In some embodiments, the hematopoietic neoplasm is a leukemia or a lymphoma. In some preferred embodiments, the lymphoma is a non-Hodgkin lymphoma. In some particularly preferred embodiments, the non-Hodgkin lymphoma is a diffuse large B-cell lymphoma (DLBCL) of an activated B-cell (ABC) subtype. In some embodiments, cells of the non-Hodgkin lymphoma express elevated levels of one or both of miR-21 and miR-155 in relation to germinal center B-cell (GCB) subtype lymphoma cells. In some embodiments, cells of the non-Hodgkin lymphoma express reduced levels of one or both of PTEN and SHIP-1 in relation to germinal center B-cell (GCB) subtype lymphoma cells.
The present invention also provides methods of treating a hematopoietic neoplasm in a human subject, comprising: a) detecting altered expression of one or more of miR-21, miR-155, PTEN, and SHIP-1 in cells of the hematopoietic neoplasm in relation to their expression in germinal center B-cell (GCB) subtype lymphoma cells; and b) administering to the human subject an effective amount of a tumor necrosis factor-alpha (TNF-alpha) inhibitor. In some embodiments, the hematopoietic neoplasm is a leukemia or a lymphoma. In some preferred embodiments, the lymphoma is a non-Hodgkin lymphoma. In some embodiments, the non-Hodgkin lymphoma is a diffuse large B-cell lymphoma (DLBCL). In some particularly preferred embodiments, the DLBCL is an activated B-cell (ABC) subtype DLBCL. Moreover, the present invention provides methods further comprising one or more of detecting increased expression of the miR-21, detecting increased expression of the miR-155, detecting reduced expression of the PTEN, and detecting reduced expression of the SHIP-1. In some embodiments the TNF-alpha inhibitor is selected from the group consisting of an anti-TNF-alpha antibody and a soluble TNF-alpha receptor. In some embodiments, the anti-TNF-alpha antibody is a chimeric monoclonal antibody comprising a murine variable region or a TNF-binding portion thereof, and a human constant region. In some preferred embodiments, the chimeric monoclonal antibody is infliximab. In some embodiments, the anti-TNF-alpha antibody is a humanized monoclonal antibody. In some embodiments, the anti-TNF-alpha antibody is a fully human monoclonal antibody. In some preferred embodiments, the human monoclonal antibody is adalimumab. In some embodiments, the soluble TNF-alpha receptor is a human immunoglobulin G (IgG) fusion protein. In some preferred embodiments, the IgG fusion protein is etanercept. In additional preferred embodiments, the methods further comprise administering to the human subject a pharmaceutical composition comprising CHOP. In some embodiments, the methods further comprise administering to the human subject an anti-CD20 monoclonal antibody. In some preferred embodiments, the anti-CD20 monoclonal antibody is rituximab.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B show that PTEN and SHIP expression are significantly down-regulated in diffuse large B cell lymphoma (DLBCL) as compared to chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL) or CLL alone. The ONCOMINE program was used to query published cDNA array data. The results of two independent expression studies are shown (Alizadeh et al., Nature, 403:503-511, 2000; Lawrie et al., Int J Cancer, 121:1156-1161, 2007; Rosenwald et al., J Exp Med, 198:851-862, 2000; and Shipp et al., Nat Med, 8:68-74, 2002, all herein incorporated by reference). The Y-axis units are normalized expression values (standard deviations above and below the median per array, with P values indicated).
FIG. 2 illustrates that low SHIP expression levels correlate with poor survival of DLBCL patients as determined through analysis of the published cDNA array data using the ONCOMINE program. The graph shown is a Kaplan-Meier plot of overall survival of DLBCL patients using defined cut-off values for SHIP.
FIG. 3 provides a sequence alignment of the SHIP 3′ UTR (SEQ ID NO:1) and miR-155 (SEQ ID NO:2). The sequence match in the crucial seed sequence is highlighted.
FIGS. 4A and 4B provide graphs showing that miR-155 regulates SHIP expression levels. CEM cells were transfected with 50 □M of a non-specific control miR, miR-1, miR-155, anti-control miR, or anti-miR155. SHIP mRNA levels were determined by QPCR after 12 hours.
FIGS. 5A and 5B provide graphs showing a tumor necrosis factor-alpha (TNF-alpha)-mediated auto-stimulatory loop in ABC-like DLBCL cells. Toledo cells were cultured in the presence or absence of 5 ng/ml of an anti-TNF-alpha monoclonal antibody (infliximab) for 8 hours, before isolation of total RNA and determination of miR-155 and SHIP mRNA expression levels by QPCR.
FIG. 6 provides a graph further illustrating a TNF-alpha-mediated auto-stimulatory loop in ABC-like DLBCL cells. Toledo cells were cultured in the presence or absence of 100 ng/ml of a soluble TNF-alpha receptor antagonist (eternacept) for 24 hours prior to analysis of SHIP mRNA expression levels. Transfection of cells with anti-miR-155 was included for comparison.
FIGS. 7A, 7B and 7C show that miR-21 and miR-155 expression levels are elevated, and that SHIP expression is reduced in specimens from patients with poor prognosis (ABC sub-type) diffuse large B cell lymphoma (DLBCL) as compared to the levels of specimens from patients with good prognosis (GC sub-type) DLBCL, respectively. Laser-capture-micro-dissection was performed on frozen lymph node sections from patients diagnosed with ABC DLBCL (n=6) or diagnosed with GC DLBCL (n=6). Quantitative-PCR (Q-PCR) analysis was done on 1000 cells/section using specific primers for miRs and SHIP. Q-PCR was normalized to U6B and GAPDH expression levels.
FIG. 8 shows that anti-TNF-alpha (infliximab) treatment of diffuse large B cell lymphoma (DLBCL) reduces expression of miR-155 and increases expression of SHIP1 in cells representing poor prognosis (ABC sub-type) DLBCL, but not in cells representing good prognosis (GC sub-type) DLBCL. Briefly, DLBCL cells were cultured in the presence or absence of 5 ng/ml anti-TNF alpha (infliximab) for eight hours. Afterwards the cells were harvested and washed in PBS before RNA was isolated using Trizol as directed by the manufacturer. Levels of miR-155 and SHIP1 mRNAs were determined by Q-PCR, normalized to U6B expression.
FIG. 9 shows that differential SHIP expression correlates with prognosis of DLBCL. (A) Two independent cDNA arrays (Alizadeh et al. (2000) Nature 403:503-511, Rosenwald et al. (2002) N Engl J Med 346:1937-1947), were analyzed in Oncomine on the world wide web oncomine.org for expression of SHIP in DLBCL, Chronic Lymphocytic Leukemia (CLL) and Follicular Lymphoma (FL). Y-axis represents normalized expression values (mean+/−standard deviation; p-values are derived from Student's t-test). (B) Kaplan-Meier plots (Oncomine; (7)) showing overall survival of 72 non-GC-DLBCL patients using defined cut-off values for SHIP expression (patient sub-groups defined as having the lowest 50% (50% low: 34 patients), lowest 25% (25% low: 18 patients) or lowest 10% (10% low: 7 patients) relative SHIP expression). (C) Sequence alignment of the SHIP 3′ UTR and miR-155 (TargetScan; seed sequence is highlighted in gray). (D) Frozen lymph node sections of ABC (non-GC) and GC-DLBCL patients were stained with H&E, and tumor cells (>1000 cells/specimen) were isolated by Laser-Capture Microdissection. RNA was analyzed for miR-155, SHIP, U6 and GAPDH expression. Data represent 5 (GC) and 6 (non-GC) specimens, respectively (Wilcoxon rank sum test; p-value miR-155:0.08225; p-value SHIP:0.08125).
FIG. 10 shows that miR-155 attenuates SHIP expression. Non-GC-DLBCL cell lines OCILY-3 and Toledo were transfected with 50 μM of either non-specific miR (miR-CTL), miR-1, miR-155, non-specific anti-miR (αmiR-CTL) or anti-miR-155 (αmiR-1500). (A) Cells were harvested 12 hours post transfection, SHIP mRNA levels were determined by qPCR and normalized to GAPDH (mean+/−STD; n=4). (B) Same as (A), except cells were lysed after 72 hours, and Western blot analysis was performed using antibodies against human SHIP and GAPDH.
FIG. 11 shows autocrine stimulation of non-GC DLBCL by TNF-alpha. Non-GC-DLBCL cell lines OCILY-3, OCILY-10, Toledo, and the GC-DLBCL cell line SUDHL-4 and the Burkitt lymphoma Daudi were treated with 100 ng/ml eternacept as indicated. (A) miR-155 expression levels were determined by qPCR and normalized to U6 RNA (mean+/−STD; n=3); (B) Cells were harvested after 72 hrs, and SHIP protein expression was determined by Western blot analysis; (C) Cell proliferation was determined in triplicate cultures 24, 48, and 72 hours after addition of eternacept (mean+/−SD of at least three independent experiments).
FIG. 12 shows anti-TNF-alpha regimen inhibits DLBCL growth in vivo. Sub-lethally irradiated (4 Gy) NOD/SCID mice were injected subcutaneously with 107 non-GC-DLBCL cells (Toledo). Upon establishment of palpable tumors, mice were injected every three days with 100 μg eternacept intravenously (i.v.), and tumor size was measured every second day with callipers.
FIGS. 13A-B shows the coding and protein sequences of exemplary human TNF-alpha (GENBANK Accession No. NM—000594) (SEQ ID NOS: 4, 5).
FIGS. 14A-F shows the coding and protein sequences of exemplary human PTEN (GENBANK Accession No. NM—000314) (SEQ ID NOS: 6, 7).
FIGS. 15A-E shows the coding and protein sequences of exemplary human SHIP-1 (GENBANK Accession No. BC113582) (SEQ ID NOS: 8, 9).
FIG. 16 shows the coding and protein sequences of exemplary human CD20 (GENBANK Accession No. DQ896499) (SEQ ID NOS: 10, 11).



DEFINITIONS
The terms “neoplasm” and “tumor” refer to a tissue growth that is characterized, in part, by increased angiogenesis and/or increased cell proliferation. This includes tissue that has aberrant growth of cells, tumors, malignant effusions, warts, polyps, nonsolid tumors, cysts and other growths. A site of neoplasia can contain a variety of cell types, including but not limited, to neoplastic cells, vascular endothelia, or immune system cells, such as macrophages and leukocytes, etc. Neoplasms may be benign or malignant. The terms “malignant neoplasm” and “malignant tumor” refer to a neoplasm that contains at least one cancer cell. A “cancer cell” refers to a cell undergoing early, intermediate or advanced stages of multi-step neoplastic progression as previously described (H. C. Pitot (1978) in “Fundamentals of Oncology,” Marcel Dekker (Ed.), New York pp 15-28).
The term “cancer” in a mammal refers to any of a number of conditions caused by the abnormal, uncontrolled growth of cells. Cells capable of causing cancer, called “cancer cells”, possess a number of characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain typical morphological features. Often, cancer cells will be in the form of a tumor, but such cells may also exist alone within a mammal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. A cancer can be detected in any of a number of ways, including, but not limited to, detecting the presence of a tumor or tumors (e.g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicative of cancer (e.g., CA125, PAP, PSA, CEA, AFP, HCG, CA 19-9, CA 15-3, CA 27-29, LDH, NSE, and others), and detecting a genotype indicative of a cancer (e.g., TP53, ATM, etc.). However, a negative result in one or more of the above detection methods does not necessarily indicate the absence of cancer, e.g., a patient who has exhibited a complete response to a cancer treatment may still have a cancer, as evidenced by a subsequent relapse.
The term “systemic delivery” as used herein, refers to delivery that leads to a broad bio-distribution of a compound within an organism. The term “systemic delivery,” means that a useful, preferably therapeutic, amount of a compound is exposed to most parts of the body. To obtain broad bio-distribution generally requires a route of introduction such that the compound is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching a disease site.
“Hematopoietic neoplasm” refers to a neoplasm that is located in the blood and/or blood-forming tissue (such as the bone marrow, lymphatic tissue, etc.). The commonest forms are the various types of leukemia, of lymphoma, and of the progressive, life-threatening forms of the myelodysplastic syndromes. Hematopoietic neoplasms may originate from a hematopoietic cell of any lineage, including the lymphoid, myeloid and erythroid lineages. Hematopoietic neoplasms are exemplified by leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome (MDS), myeloproliferative disease, and amyloid disease.
The term “leukemia” refers to a progressive, malignant disease of the blood or bone marrow, characterized by abnormal proliferation and development of blood cells, usually white blood cells (leukocytes). Leukemias are classified according to the degree of cell differentiation as acute or chronic, and according to predominant type of cell involved as myelogenous or lymphatic. Leukemias are exemplified by acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, etc.
The term “lymphoma” refers to a malignant growth of B or T cells in the lymphatic system, presenting as an enlargement of the node (a tumor). The term “lymphoma” includes numerous types of malignant growths, including Hodgkin's lymphoma and non-Hodgkin lymphoma (NHL).
“Hodgkin lymphomas” are exemplified by classical Hodgkin lymphomas and nodular lymphocyte-predominant Hodgkin lymphoma.
The term “non-Hodgkin lymphoma” refers to a malignant growth of B or T cells in the lymphatic system that is not otherwise classified as a Hodgkin's lymphoma (e.g., having Reed-Sternberg cells in the cancerous area). Non-Hodgkin lymphomas encompass over 29 types of lymphoma, the distinctions between which are based on the type of cancer cells. The particular classification depends on the particular system of classification used, such as the Working formulation, the Rappaport classification, and the REAL classification. In preferred embodiments, the REAL classification is used. For example, Non-Hodgkin lymphomas include mature B cell neoplasms and T cell and natural killer cell neoplasms. Mature B cell neoplasms are exemplified by diffuse large B-cell lymphoma (DLBCL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenström macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasms, extranodal marginal zone B cell lymphoma (also called MALT lymphoma), nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, and Burkitt lymphoma/leukemia. Mature T cell and natural killer (NK) cell neoplasms are exemplified by T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, and anaplastic large cell lymphoma.
The term “relapsed cancer” or “relapsed lymphoma” refers to a cancer or lymphoma that has recurred following prior complete or partial remission in response to a prior treatment. Recurrence can be defined in any way, including a reappearance or re-growth of a tumor as detected by clinical, radiological, or biochemical assays, or by an increased level of a cancer marker. Prior treatments can include, but are not limited to, chemotherapy, radiation therapy, and bone marrow transplantation.
The term “indolent” non-Hodgkin lymphoma is a classification that includes slow growing forms of lymphoma. They encompass what are called low grade and some categories of intermediate grade NHL. Indolent NHLs are sometimes not responsive to conventional cancer therapies such as chemotherapy and radiation therapy.
Patients with “refractory cancer” or “refractory lymphoma” are those who have failed to achieve complete remission on their first course of combination chemotherapy, or to patients who have failed to achieve complete or partial remission on subsequent chemotherapy. “Primary refractory” patients are those who have never achieved complete remission even at first treatment.
A “stable disease” is a state wherein a therapy causes cessation of growth or prevalence of a tumor or tumors as measured by the usual clinical, radiological and biochemical means, although there is no regression or decrease in the size or prevalence of the tumor or tumors, i.e., cancer that is not decreasing or increasing in extent or severity.
“Partial response” or “partial remission” refers to the amelioration of a cancerous state, as measured by tumor size and/or cancer marker levels, in response to a treatment. Typically, a “partial response” means that a tumor or tumor-indicating blood marker has decreased in size or level by about 50% in response to a treatment. The treatment can be any treatment directed against cancer, but typically includes chemotherapy, radiation therapy, hormone therapy, surgery, cell or bone marrow transplantation, immunotherapy, and others. The size of a tumor can be detected by clinical or by radiological means. Tumor-indicating markers can be detected by means well known to those of skill (e.g., ELISA or other antibody-based tests).
A “complete response” or “complete remission” means that a cancerous state, as measured by, for example, tumor size and/or cancer marker levels, has disappeared following a treatment such as chemotherapy, radiation therapy, hormone therapy, surgery, cell or bone marrow transplantation, or immunotherapy. The presence of a tumor can be detected by clinical or by radiological means. Tumor-indicating markers can be detected by means well known to those of skill (e.g., ELISA or other antibody-based tests). A “complete response” does not necessarily indicate that the cancer has been cured, however, as a complete response can be followed by a relapse.
The term “chemotherapy” refers to the administration of chemical agents that inhibit the growth, proliferation and/or survival of cancer cells. Such chemical agents are often directed to intracellular processes necessary for cell growth or division, and are thus particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, and thus inhibits cells from entering mitosis. In general, chemotherapy can include any chemical agent that inhibits, or is designed to inhibit, a cancerous cell or a cell likely to become cancerous. Such agents are often administered, and are often most effective, in combination (e.g., CHOP formulation).
The term “radiation therapy” refers to the administration of radioactivity to an animal with cancer. Radiation kills or inhibits the growth of dividing cells, such as cancer cells.
The term “immunotherapy” refers to methods of enhancing the ability of an animal's immune system to destroy cancer cells within the animal.
As used herein the terms “tumor necrosis factor-alpha,” “TNF-alpha” and “cachectin” refer to a multifunctional proinflammatory cytokine secreted predominantly by monocytes/macrophages that has effects on lipid metabolism, coagulation, insulin resistance, and endothelial function. The coding and protein sequences of exemplary human TNF-alpha are described in GENBANK Accession No. NM—000594 (SEQ ID NOS: 4, 5).
As used herein the terms “microRNAs” and “miRNAs” refer to endogenous noncoding RNAs of about 22 nucleotides that regulate mRNAs by targeting them for cleavage or translational repression. The sequence of “miR-155” is 3′ GGGGAUAGUGCUAAUCGUAAUU-5′ (SEQ ID NO:2). The sequence of “miR-21” is 5′- AGUUGUAGUC AGACUAUUCG AU -3′ (SEQ ID NO:3).
The terms “phosphatase and tensin homolog” deleted on chromosome ten, “PTEN,” “mutated in multiple advanced cancers 1” and “MMACI” refer to a tumor suppressor protein having a tyrosine phosphatase domain and a region of homology to tensin. The coding and protein sequences of exemplary human PTEN are described in GENBANK Accession No. NM—000314 (SEQ ID NOS: 6, 7).
The terms “SH2-containing inositol phosphatase,” “SHIP-1” “inositol polyphosphate-5-phosphatase” and “1NPP5D” refer to an enzymes that hydrolyzes inositol 1,4,5-triphosphate in a signal-terminating reaction. The coding and protein sequences of exemplary human SHIP-1 are described in GENBANK Accession No. BC113582 (SEQ ID NOS: 8, 9).
The terms “B-lymphocyte surface antigen B 1,” “B 1” and “CD20” refer to an antigen having four highly conserved transmembrane domains, flanked by N- and C-terminal cytoplasmic regions, which is widely expressed during B-cell development from early pre-B cell stages until differentiation into plasma cells. The coding and protein sequences of exemplary human CD20 are described in GENBANK Accession No. DQ896499 (SEQ ID NOS: 10, 11).
Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. A monoclonal antibody (mAb) contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. MAbs may be obtained by methods known to those skilled in the art (e.g., Kohler and Milstein, Nature 256:495-497, 1975; Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, NY, 1992; and Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, 1993; the contents of which are incorporated herein in their entirety). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. A hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production.
Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region, which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc Natl Acad Sci USA, 81:3273-3277, 1984; Morrison et al., Proc Natl Acad Sci USA, 81:6851-6855, 1984; Boulianne et al., Nature, 312:643-646, 1984; Neuberger et al., Nature, 314:268-270, 1985; Sahagan et al., J Immunol, 137:1066-1074, 1986; Liu et al., Proc Natl Acad Sci USA, 84:3439-3443, 1987; Sun et al., Proc Natl Acad Sci USA, 84:214-218, 1987; Better et al., Science, 240:1041-1043, 1988; and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
Anti-TNF antibodies of the present invention can include at least one of a heavy chain constant region (Hc), a heavy chain variable region (Hv), a light chain variable region (Lv) and a light chain constant region (Lc), wherein a polyclonal Ab, monoclonal Ab, fragment and/or regions thereof include at least one heavy chain variable region (Hv) or light chain variable region (Lv) which binds a portion of a TNF and inhibits and/or neutralizes at least one TNF biological activity.
As used herein, the term “chimeric antibody” includes monovalent, divalent or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain. A divalent chimeric antibody is tetramer (H2L2) formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric antibody can also be produced, for example, by employing a Hc region that aggregates (e.g., IgM H chain).
Murine and chimeric antibodies, fragments and regions of the present invention comprise individual heavy (H) and/or light (L) immunoglobulin chains. A chimeric H chain comprises an antigen-binding region derived from the H chain of a non-human antibody specific for TNF, which is linked to at least a portion of a human Hc region, such as CH1 or CH2. A chimeric L chain according to the present invention comprises an antigen-binding region derived from the L chain of a non-human antibody specific for TNF, linked to at least a portion of a human Lc region.
The terms “treating,” “treatment” and grammatical equivalents when in reference to a disease (e.g., cancer) encompasses delaying and/or reducing one or more objective symptoms (such as tumor size, blood or urine levels of metabolic compounds such as glucose, body weight, etc.) and/or subjective symptoms (e.g., pain, fatigue, difficulty in breathing, clarity of vision, nausea, etc.) of the disease.
When in reference to the level of molecules and/or phenomena, the terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” and grammatical equivalents (including “lower,” “smaller,” etc.) when in reference to the level of any molecule (such as SHIP, miR-21, miR155, TNF-alpha, anti-TNF-alpha antibody, TNF-alpha receptor, etc., and nucleic acid sequences encoding any of the polypeptides described herein), and/or phenomenon (e.g., cell number, cell death, cell apoptosis, cell viability, cell survival, binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, etc.) in a first sample relative to a second sample, mean that the quantity of molecule and/or phenomenon in the first sample is lower than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis. In one embodiment, the reduction may be determined subjectively, for example when a patient refers to their subjective perception of disease symptoms, such as pain, fatigue, difficulty in breathing, clarity of vision, nausea, etc. In another embodiment, the quantity of molecule and/or phenomenon in the first sample is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and/or at least 90% lower than the quantity of the same molecule and/or phenomenon in a second sample.
When in reference to the level of molecules and/or phenomena, the terms “increase,” “elevate,” “raise,” and grammatical equivalents (including “higher,” “greater,” etc.) when in reference to the level of any molecule (such as SHIP, miR-21, miR155, TNF-alpha, anti-TNF-alpha antibody, TNF-alpha receptor, etc., and nucleic acid sequences encoding any of the polypeptides described herein), and/or phenomenon (e.g., cell number, cell death, cell apoptosis, cell viability, cell survival, binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, etc.) in a first sample relative to a second sample, mean that the quantity of the molecule and/or phenomenon in the first sample is higher than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis. In one embodiment, the increase may be determined subjectively, for example when a patient refers to their subjective perception of disease symptoms, such as pain, fatigue, difficulty in breathing, clarity of vision, nausea, etc. In another embodiment, the quantity of the molecule and/or phenomenon in the first sample is at least 10% greater than, at least 25% greater than, at least 50% greater than, at least 75% greater than, and/or at least 90% greater than the quantity of the same molecule and/or phenomenon in a second sample.
“Subject” and “animal” include mammals, such as humans and non-human mammals that include primates, murines, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, guinea pig, hamster, ferret, chinchilla, mouse and cotton rat.
“Tumor necrosis factor-alpha (TNF-alpha) inhibitor” refers to a molecule that reduces the level of binding of TNF-alpha to its receptor by any statistically significant amount, including a reduction in binding to a quantity that is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and/or at least 90% lower than the quantity of binding when compared to the quantity of binding in the absence of the TNF-alpha inhibitor. TNF-alpha inhibitors are exemplified by anti-TNF-alpha antibody, TNF-alpha receptor, and other compounds, such as Pentoxifylline, which is the International Nonproprietary Name (INN) of a is a xanthine derivative drug sold by Aventis under the name Trental®. Its chemical name is 1-(5-oxohexyl)-3,7-dimethylxanthine.
The terms “chimeric” and “fusion” when in reference to a protein refer to a protein that contains at least two amino acid sequences that are operably linked together. The amino acid sequences may be derived from different sources (e.g., different organisms, different tissues, different cells, etc.) or may be different sequences from the same source. In one embodiment, the chimeric protein is a recombinant protein that is produced by expressing operably linked nucleotide sequences that encode the amino acid sequences. The term “operably linked” when in reference to the relationship between nucleic acid sequences and/or amino acid sequences refers to linking the sequences such that they perform their intended function. For example, operably linking a promoter sequence to a nucleotide sequence of interest refers to linking the promoter sequence and the nucleotide sequence of interest in a manner such that the promoter sequence is capable of directing the transcription of the nucleotide sequence of interest and/or the synthesis of a polypeptide encoded by the nucleotide sequence of interest. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
“Pentoxifylline” is the generic name of the brand name drug “Trental®.” It is a tri-substituted xanthine derivative designated chemically as 1-(5-oxohexyl)-3,7-dimethylxanthine (CAS Registry Number 6493-05-6).
“Adalimumab” and “Humira®” refer to a recombinant human IgG1 monoclonal antibody specific for human tumor necrosis factor (TNF). Humira® was created using phage display technology resulting in an antibody with human derived heavy and light chain variable regions and human IgG1:K constant regions. Adalimumab is produced by recombinant DNA technology in a mammalian cell expression system and is purified by a process that includes specific viral inactivation and removal steps. It consists of 1330 amino acids and has a molecular weight of approximately 148 kilodaltons.
“Infliximab” and “Remicade®” refer to a chimeric IgG1κ monoclonal antibody with an approximate molecular weight of 149,100 daltons. It is composed of human constant and murine variable regions. Infliximab binds specifically to human tumor necrosis factor alpha (TNFα) with an association constant of 1010 M−1. Infliximab is produced by a recombinant cell line cultured by continuous perfusion and is purified by a series of steps that includes measures to inactivate and remove viruses.
“Etanercept,” “eternacept” and “Enbrel®” interchangeably refer to a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kd (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. The Fc component of etanercept contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgG1. Etanercept is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 KD.
DETAILED DESCRIPTION OF THE INVENTION
Data herein (Examples 1-6) identifies the inositol phosphatase SHIP, a previously unidentified tumour suppressor, as a bona-fide target of the oncogenic miR-155, an onco-miR whose expression in B cells alone is sufficient to trigger malignant transformation. In particular, data herein demonstrate that in diffuse large B cell lymphoma (DLBCL), elevated levels of miR-155, and consequent diminished SHIP expression are the result of autocrine stimulation by the pro-inflammatory cytokine TNF-alpha. Anti-TNF-alpha regimen such as eternacept or infliximab were sufficient to reduce miR-155 levels and restored SHIP expression in DLBCL cells with an accompanying reduction in cell proliferation. Furthermore, data herein demonstrate a substantial decrease in tumor burden in DLBCL xenografts in response to eternacept. This data strongly support the concept that cytokine-regulated inflammatory miRs can function as a link between inflammation and cancer, can contribute to the development and/or progression of cancer, and illustrate the feasibility of anti-TNF-alpha therapy as a novel and immediately accessible treatment for hematopoietic cancers, such as DLBCL, alone or in combination with other treatments for DLBCL.
While an understanding of the mechanism of action of the invention is not necessary, and without limiting the invention to any particular mechanism, it is the inventors' view that aberrant miR-155 expression in non-GC-DLBCL appears to be the result of an autocrine stimulation by TNF-alpha, as miR-155 and SHIP levels can be reduced or increased, respectively, through the administration of neutralizing anti-TNF-alpha antibodies or soluble TNF-alpha receptor.
B cell survival and fate determination are strongly dependent upon phosphatidylinositol 3-kinase (PI3K) signalling (Fruman et al. (2004) Curr Opin Immunol 16:314-320, Alizadeh et al. (2000) Nature 403:503-511). PI3K catalyzes the conversion of membrane phosphatidylinositol-(4,5)-bisphosphate to phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), which acts as a second messenger to recruit pleckstrin-homology domain containing adapters and kinases such as PDK, AKT, PLCγ2, BTK, DOK, and others. Subsequent activation/inactivation of additional effectors including SGK, TOR, PP2A, FOXO, and Cyclin D/E mediates diverse biological responses such as survival, proliferation, migration, adhesion, and differentiation.
PI3K signals are antagonized by two lipid phosphatases: the 3′-inositol phosphatase PTEN and the 5′-inositol phosphatase SHIP. Mills et al. recently discovered that in contrast to T cells, B cells do not undergo malignant transformation upon PTEN deletion; similarly, deletion of SHIP in B cells is insufficient to generate B cell lymphoma. However, concomitant ablation of both SHIP and PTEN in murine B cells induces lethal lymphoma resembling DLBCL with 100% penetrance, revealing a novel role for SHIP as a tumor-suppressor (Mills et al., manuscript in preparation).
DLBCL is clinically, morphologically and genetically a heterogeneous group of malignant proliferation of large lymphoid B cells that accounts for approximately 40% (25,000 cases/yr) of adult non-Hodgkin lymphomas (3). Standard chemotherapy has been recently expanded from CHOP (Cyclophosphamide/Doxorubicin/Vincristine/Prednisolone) to R-CHOP with the inclusion of Rituximab, an anti-CD20 monoclonal antibody, which improved treatment success to an overall 3-year relapse free survival of DLBCL patients at 53-63.1% (Habermann et al. (2006) J Clin Oncol 24:3121-3127, Pfreundschuh et al. (2008) Lancet Oncol 9:435-4444). Two prognostically different subgroups of DLBCL have been identified with distinct gene expression profiles either characteristic of normal germinal center B cells or of activated memory B cells. The germinal center B-cell-like (GC) subgroup was correlated with a significantly better prognosis (5-year survival: 76%) in comparison to the activated B-cell-like (ABC or non-GC) subgroup (5-year survival: 16%) (Alizadeh et al. (2000) Nature 403:503-511, Shipp et al (2002) Nat Med 8:68-74).
Recently, SHIP-1 has been identified as a tumor suppressor. Now as described herein, poor-prognosis large B-cell lymphomas were found express lower levels of SHIP-1, which is a target for the micro-RNA miR-155. Ectopic overexpression of miR-155 has been shown to cause lymphomas in mice (Costinean et al., Proc Natl Acad Sci USA, 103:7024-7029, 2006), and miR-155 RNA has been shown to accumulate in various types of human B cell lymphoma (Eis et al., Proc Natl Acad Sci USA, 102:3627-3632, 2005). In addition, miR-155 expression has been shown to be can be induced by TNF-alpha (O'Connell et al., Proc Natl Acad Sci USA, 104:1604-1609, 2007). Now as determined during development of the present invention, reducing TNF-alpha signaling in poor-prognosis large B-cell lymphoma cells leads to the down-regulation of miR-155 and consequent up-regulation of SHIP-1. Thus reducing TNF-alpha signaling provides a new and counter-intuitive therapy for poor-prognosis large B-cell lymphomas and other hematopoietic malignancies.
I. Lymphoma
Disease in B cell non-Hodgkin lymphoma (NHL) patients is graded according to the International Prognostic Index (IPI), which assigns low scores for the involvement of single lymph nodes, intermediate scores for multiple lymph node involvement, and high scores for systemic nodal and non-lymphoid metastases (See, e.g., Blood, 89:3909-3918, 1997). Diffuse large B-cell lymphoma (DLBCL) accounts for approximately 40% of adult non-Hodgkin lymphomas. DLBCL is clinically, morphologically and genetically a heterogeneous group of tumors composed of large B cells. Several microarray studies performed on untreated, de novo DLBCL, identified two main, prognostically different subgroups (Alizadeh et al., Nature, 403-503-511, 2000). Both were characterized by a distinct gene expression profile either characteristic of normal germinal center B-cells or of activated blood memory B-cells. The germinal center B-cell-like (GCB-like) subgroup was correlated with a significantly better prognosis in comparison to the activated B-cell-like (ABC-like) subgroup (Rosenwald et al., N Engl J Med, 346:1937-1947, 2002; and Rosenwald et al., J Exp Med, 198:851-862, 2003). In particular the five-year overall survival rate for the GCB-like is 70%, while the rate for the ABC-like group is 12%.
Standard chemotherapy has been recently expanded from cyclophosphamide/doxorubicin/vincristine/prednisolone (CHOP) formulation to R-CHOP with the further inclusion of rituximab (See, e.g., U.S. Pat. No. 7,244,450 to Sarris et al., herein incorporated by reference). Rituximab is an anti-CD20 monoclonal antibody (See, e.g., U.S. Pat. No. 5,736,137 to Anderson et al., herein incorporated by reference). Rituximab, which is marketed as RITUXIN by Genentech, South San Francisco, Calif. and Biogen Idec, Cambridge, Mass., has improved treatment success of specific DLBCL subgroups. Although this B cell-depleting/inactivating monoclonal antibody has greatly advanced DLBCL therapy and significantly slowed disease progression in certain patient subgroups with low IPI scores, the lifespan of high-risk patients with disseminated lymphoma is not significantly extended by this treatment. Significant effort toward understanding disease progression in human patients has suggested that B cell NHL, in addition to multiple cancer-promoting genetic hits is fueled by non-specific stimuli, and B cell antigen receptor-mediated recognition of undefined self-antigens. In support of this latter idea, B-cell NHL tumors often display several hallmarks of antigen-mediated clonal selection. Additionally, B-cell NHL patients often have high serum autoantibody titers, suggesting autoantigen-mediated terminal differentiation.
II. microRNAs (miRNAs) and Cancer
Soon after the discovery of the first mammalian miRNA some 12 years ago, it became evident that this class of molecules plays a critical role in global gene regulation, and likely impacts cellular survival and death pathways. More recently high throughput analyses have demonstrated that miRNA expression is commonly dysregulated in a multitude of human cancers (Calin, Nature Rev, 6:957-866, 2996; Lu, Nature, 435:834-838, 2005; and Roldo, J Clin Invest, 24:4677-4684, 2006), and miRNA expression profiling has shown promise in defining malignant status in retrospective studies.
Interestingly, more than 50% of annotated human miR genes are located in fragile chromosomal regions that are susceptible to amplification, deletion, or translocation during the course of tumor development (Calin, Proc Nat Acad Sci USA, 101:2999-3004, 2004). Moreover, recent evidence indicates that some miRs functions either as oncogenes or tumor suppressors (Eswuela-Kerscher, Nature Rev Cancer, 6:259-269, 2006; He, Nature, 435:828-833, 2005; and Johnson, Cell, 120:635-647, 2005). The first study documenting abnormalities in miRNA expression in tumor samples focused on chronic lymphocytic leukemia (CLL). Deletion of chromosome 13q14 is the most frequent chromosomal abnormality in this disorder. Croce and co-workers demonstrated that tumor suppressor activity is likely provided by two miRNAs, miR-15a and miR-16-1 (Calin et al., Proc Nat Acad Sci USA, 99:15524-15529, 2002). In addition, a conserved site for miR-15a and miR-16-1 was identified in the 3′ UTR of the bcl-2 mRNA, which encodes the anti-apoptotic protein Bcl-2. Loss of miR-15a and miR-16-1 are thought to contribute to elevated Bcl-2 expression and pathological cell survival in CLL (Cimmino et al., Proc Nat Acad Sci USA, 102:13944-13949, 2005). In contrast, the miR-17-92 polycistron is located in a region of DNA that is amplified in human B cell lymphomas (Ota et al., Cancer Res, 64:3087-3095, 2004), and enforced expression of the miR-17-92 cluster acts to accelerate tumor development in mouse B cell lymphoma (He et al., Nature, 435:828-833, 2005). Similarly, miR-17-5p and miR-20a have been demonstrated to control the balance of cell death and proliferation through c-myc (O'Donnell, Nature, 435:839-843, 2005) and the miR-34 family of microRNAs has been found to target the tumor suppressor gene p53 thereby encouraging inappropriate cell proliferation. In addition, miR-10b is highly expressed in breast cancer and has recently been found to inhibit the translation of the homeobox gene D10 resulting in increased expression of the pro-metastatic gene RHOC (Ma, Nature, 499:682-688, 2007).
Several studies have illustrated the importance of miRs in B cell lymphomas. For instance, miR-155 was found to be highly expressed in Hodgkin lymphoma and DLBCL (Lawrie et al., Expert Opin Biol Ther, 7:1363-1374, 2007; Lawrie et al., Br J Haematol, 137:503-512, 2007; and Eis et al., Proc Natl Acad Sci USA, 102:3627-3632, 2005). Using a prototypic cell line model of the GCB and ABC subtypes of DLBCL, microRNAs miR-221 and miR-21 as well as miR-155 were found to be over-expressed in ABC-type but not GCB-type lymphoma cells. In addition, these microRNAs were found to be over-expressed in clinical cases of DLBCL and FCL, and in DLBCL cases that had undergone high-grade transformation from previously diagnosed FCL. Consistent with the cell line model, these microRNAs were more highly expressed in de novo DLBCL cases that were immunophenotypically classified as ABC-type than those that were classified as GCB-type. While many of these studies are of a correlative nature, conclusive proof for the oncogenic potential of miRs has been demonstrated by transgenic expression of miR-155, resulting in a preleukemic pre-B cell proliferation evident in spleen and bone marrow, followed by frank B cell malignancy (Costinean et al., Proc Nat Acad Sci USA, 103:7024-7029, 2006). This direct evidence that overexpression of a miRNA can result in the development of a neoplastic disease, highlighting the potential role of miRNA in human malignancies.
III. Regulation of Lipid Phosphatases and Cancer
Survival of all cell types is mediated by phosphatidylinositol-3-kinase (PI3K) signaling, and is important in B cell fate determination. PI3K signals are antagonized by two lipid phosphatases PTEN and SHIP. To determine if attenuated SHIP expression occurs in human B cell malignancies, during development of the present invention, the ONCOMINE program was used to query published cDNA array data (Rhodes et al., Neoplasia, 6:1-6, 2004; Rhodes et al., Proc Natl Acad Sci USA, 101:9309-9314, 2004; Rhodes et al., Nat Genet, 37:579-583, 2005; and Rhodes et al., Neoplasia, 9:166-180, 2007). In particular the ONCOMINE program was utilized to look for a correlation between expression profiles of PTEN and/or SHIP, and prognosis of patients with B cell tumors. Through analysis of the data sets provided by two groups (Alizadeh Nature, 403:503-511, 2000; and Rosenwald et al., J Exp Med, 198:851-862, 2003) expression of both SHIP and PTEN was found to be significantly reduced in different human lymphoma patient specimens, such as Diffuse Large B-Cell Lymphoma (DLBCL) or Follicular Lymphoma (FL), as compared to normal tissue. In addition, the levels of both tumor suppressor genes were found to be reduced in a pattern that correlated with the aggressiveness of the hematological malignancy. Specifically as described herein, the more indolent diseases such as CLL (Chronic Lymphocytic Leukemia), and (FL) (Follicular Lymphoma) express significantly higher levels of PTEN and SHIP, as compared to the more aggressive B cell malignancy DLBCL (Diffuse Large B cell Lymphoma) (FIG. 1). Furthermore, the expression levels of PTEN or SHIP were found to be lowest in patients of the ABC subgroup of DLBCL patients, as compared to the GCB subgroup of patients. Further analysis of the published cDNA array data revealed that SHIP expression levels in DLBCL specimens correlate with overall patient survival (FIG. 2).
To explore whether the reduced SHIP expression in DLBCL, particularly of the ABC subtype were a consequence of misexpression of cellular microRNAs (miRNAs), algorithms provided by the miRBase were used to identify miRNAs likely to target SHIP. As shown in FIG. 3, the 3′ UTR of the SHIP mRNA was found to contain a very promising target site for onco-miR 155. Indeed transfection of a synthetic mimic of miR-155, but not a non-specific control miRNA or an irrelevant miRNA (e.g., miR-1) was found to substantially attenuate SHIP expression. Conversely introduction of a neutralizing anti-miR against miR-155 caused a significant increase in SHIP mRNA levels.
miR-155 is one of only a few microRNAs whose expression had been shown to be regulated by extracellular ligands (O'Connell et al., Proc Nat Acad Sci USA, 104:1604-1609, 2007). In particular, miR-155 expression can be induced in macrophages by stimulation with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNFα). Analysis of the activation pathway indicated that miR-155 is an NFκB-responsive transcript. Strikingly, the cDNA array studies that characterized DLBCL based on distinct gene expression profiles found that poor-prognosis ABC subtype cells displayed a gene expression profile consistent with elevated NFκB activation. Accordingly, the premise that TNFα induces miR-155 in B cells was tested during development of the present invention by exposing splenic B cell from wild type mice with TNFα in vitro. Indeed TNFα exposure induced expression of miR-155 in primary murine B cells.
Toledo cells are a widely used cell line representative of ABC subtype DLBCL. Toledo cells were found to express higher miR-155, and consequently lower SHIP levels, when compared to Forage or Dohh2 cell lines representative of GC subtype DLBCL. Since B cells are known to produce TNFα, the possibility exists that DLBCL cells create an auto-stimulatory loop leading to elevated miR-155 levels by TNFα production. As described herein, Toledo cells were cultured in the presence of infliximab, a neutralizing humanized monoclonal antibody against TNFα (marketed as REMICADE by Centocor, Malvern, Pa.). Infliximab (U.S. Pat. No. 7,252,823 to Le et al., herein incorporated by reference) finds widespread clinical use in the treatment of inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Strikingly anti-TNFα treatment of Toledo cells lead to a substantial reduction in miR-155 expression, with a concomitant increase in SHIP mRNA levels (FIG. 5). Toledo cells were also cultured in the presence of Eternacept, an antagonistic soluble TNFα receptor (co-marketed as ENBEL by Wyeth and Amgen). As was observed with infliximab treatment, eternacept treatment of Toledo cells lead to a remarkable increase in SHIP mRNA levels (FIG. 6). In addition, induction of SHIP expression in Toledo cells was accomplished by transfection with anti-miR-155.
Thus as determined during development of the present invention, miR-155 inhibits SHIP mRNA and protein expression. In addition, enhanced miR-155 expression in ABC-like DLBCL cells was found not to be a product of endogenous mutations, but rather the result of a TNFα-mediated autocrine feedback loop. Consequently, anti-TNFα regimens were found to be sufficient to reduce miR-155 levels, and restore expression of the tumor suppressor SHIP. By extension, downregulation of TNF alpha signaling in hematopoietic malignancies is contemplated to be an effective treatment when used alone or in conjunction with other treatment regimens.
IV. Therapeutic Formulations
Suitable TNFα inhibitors for use in the methods of the present invention to treat hematopoietic malignancies such as non-Hodgkin lymphoma include but are not limited to anti-TNFαantibodies and soluble TNFα receptors. Exemplary anti-TNFα antibodies include but are not limited to infliximab (marketed as REMICADE by Centocor) and adalimumab (marketed as HUMIRA by Abbott Laboratories). An exemplary Soluble TNFα receptor is the human immunoglobulin (IgG) fusion protein etanercept (co-marketed as ENBEL by Wyeth and Amgen). The treatment regimens of the present invention employing TNFα inhibitors may be used alone or in conjunction with other treatment regimens such as chemotherapy with CHOP, CHOP with rituximab, radiation, and/or surgery.
The antibodies and antibody fragments of some embodiments of the present invention may be administered by any suitable means, including parenteral, non-parenteral, subcutaneous, topical, intraperitoneal, intrapulmonary, intranasal, and intraregional administration (e.g., for local immunosuppressive treatment). Parenteral infusions include, but are not limited to, intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In addition, antibodies are suitably administered by pulse infusion, particularly with declining doses. Preferably, the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The dosages of the antibodies of the present invention are generally dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody fragment is 0.1-20 mg/kg, more preferably 1-10 mg/kg. In some embodiments, the dosage is from 50-600 mg/m2 (e.g. 375 mg/m2). It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the present invention.
The dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, its mode and route of administration, the age, health, and weight of the recipient, the nature and extent of symptoms, the kind of concurrent treatment, the frequency of treatment, and the effect desired. For example, a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1 to 5, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form, may be effective to obtain desired results.
As a non-limiting example, treatment can be provided as a daily dosage of anti-TNF-alpha peptides, monoclonal chimeric and/or murine antibodies of the present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
In another example, treatment with infliximab (Remicade®) may be by intravenous administration at dosages less than 3 mg/kg (e.g., from 0.01 to 2.9 mg/kg), more than 5.7 mg/kg (e.g., from 5.8 to 500 mg/kg), on a daily basis for at least 2 days, and/or weekly basis for at least 2 weeks, monthly basis for at least two months, or any combination thereof.
In a further embodiment, treatment with etanercept (eternacept, Enbrel®) may be at a dosage less than 25 mg, more than 50 mg, on a daily basis for at least 2 days, and/or weekly basis for at least 2 weeks, monthly basis for at least two months, or any combination thereof.
In yet another embodiment, treatment with adalimumab (Humira®) may be by subcutaneous injection at dosages between 40 and 80 mg, less than 40 mg (e.g., from 0.1 to 39 mg) more than 80 mg (e.g., from 81 to 1000 mg) on a daily basis for at least 2 days, and/or weekly basis for at least 2 weeks, monthly basis for at least two months, or any combination thereof.
The antibody and antibody fragments of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. For example, the pharmaceutical composition may comprise an antibody or antibody fragment and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibodies of the present invention.
The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies.
Therapeutic compositions typically are sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody fragment) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption (e.g., monostearate salts and gelatin).
In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art (see, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson. ed., Marcel Dekker, Inc., New York, 1978).
In certain embodiments, the binding molecules of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
The pharmaceutical compositions of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antibody fragment of the invention.
A “therapeutically effective amount,” “effective amount,” “prophylactically effective amount,” and “protective amount” of a composition with respect to a hematopoietic neoplasm interchangeably refer to, in one embodiment, an amount of the composition that delays, reduces, palliates, ameliorates, stabilizes, prevents and/or reverses one or more symptoms (e.g., clinical symptom, biochemical symptom, etc.) that are associated with the hematopoietic neoplasm compared to in the absence of the composition. This includes using dosages and periods of time necessary, to achieve the desired therapeutic result. The term “delaying” symptoms refers to increasing the time period between exposure to the immunogen or virus and the onset of one or more symptoms of the exposure. The term “eliminating” symptoms refers to 100% reduction of one or more symptoms of exposure to the immunogen or virus. A therapeutically effective amount also includes one in which any toxic or detrimental effects of the composition (e.g., antibody or antibody fragment) are outweighed by therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. In some embodiments, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. Specific dosages can be readily determined by clinical trials and depend, for example, on the route of administration, disease state, age, sex, and weight of the individual (e.g. milligrams of drug per kg body weight). Dosages are further discussed below.
EXPERIMENTAL
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: N (normal); M (molar); mM (millimolar); μM (micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg (micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); C (degrees Centigrade); CHOP (cyclophosphamide/doxorubicin/vincristine/prednisone); IFN (interferon); TNF (tumor necrosis factor); DLBCL (diffuse large B-cell lymphoma); NHL (non-Hodgkin lymphoma); miR or miRNA (microRNA); PCR (polymerase chain reaction), QPCR (quantitative PCR).
Example 1
Treatment of Non-Hodgkin's Lymphoma
Patients with non-Hodgkin's lymphoma receive either a placebo and CHOP or one of several doses/schedules of a TNF-alpha inhibitor such as an anti-TNF-alpha antibody (e.g., infliximab marketed as REMICADE by Centocor) and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy. The CHOP chemotherapy regimen is as described in U.S. Pat. No. 5,736,137. Exemplary anti-TNF-alpha antibody doses/schedules are as follows: 3 mg/kg or 10 mg/kg by IV infusion at weeks 0, 2 and 6, followed by additional infusions every 4 or 8 weeks. After treatment, the patient is monitored to evaluate the effect on lymphoma status (e.g., number and size of tumors).
Example 2
Material and Methods
The following are exemplary materials and methods that were used to obtain data herein, including those described in Examples 3-5.
1. Cell Culture.
OCILY-3, OLILY-10 and Toledo cells were maintained in complete Iscove's DMEM+20% human serum+100 mg/ml penicillin/streptomycin+2 mM L-glutamine (Invitrogen). SUDHL-4 and Daudi cells were maintained in complete RPMI+10% FCS+100 mg/ml penicillin/streptomycin+2 mM L-glutamine. Cell proliferation was determined using a Coulter Counter.
2. Patient Samples
Frozen lymph node biopsies were stained with H&E, and lymphoma cells were isolated by laser-capture microdissection. All human material was handled in full compliance with NIH guidelines and IRB approval.
3. Transfection of miRs
OptiMem (Invitrogen) and Mirus transfection reagent (Mirus Bio/Fisher Scientific) were combined according to the manufacturer instructions, and miR mimics or anti-miRs (mimics from Dharmacon, Lafayette, Colo.; anti-miRs from Applied Biosystems, Foster City, Calif.) were introduced prior to addition of the transfection mix to the cells.
4. RNA Isolation and qPCR
RNA was extracted by TriZol, and qPCR was performed using primers targeting human SHIP (SuperArrays/SABiosciences, Frederick, Md.) and GAPDH (Dharmacon) for analysis of SHIP and GAPDH mRNA, miR-155, or U6 RNA by qPCR. Alternatively, miR kits (RT and TaqMan q-PCR primers, Applied Biosystems, Foster City, Calif.) were used for qPCR analysis of miR-155, U6 and U43 according to the manufactures instructions.
5. Western Blot Analysis
Cell lysates were subject to Western blot analysis using rabbit monoclonal antibodies against SHIP or GAPDH (Cell Signaling Tech, Danbars, Mass.), and blots were developed using ECL (GE Healthcare).
Example 3
Differential SHIP Expression Correlates with Prognosis of DLBCL
To determine whether a correlation exists between survival of patients with B cell malignancies and expression levels of PTEN and SHIP, we utilized ONCOMINE to query published cDNA array results. Analysis of data originating from gene expression studies by Alizadeh and Rosenwald (Alizadeh et al. (2000) Nature 403:503-511, Rosenwald et al. (2002) N Engl J Med 346:1937-1947), revealed that SHIP levels are significantly decreased in DLBCL compared to more indolent Chronic Lymphocytic Leukemia (CLL) or Follicular Lymphoma (FL) (FIG. 9A), moreover, SHIP levels were lower in non-GC (ABC)—compared to GC-DLBCL (not shown). Strikingly, SHIP levels within the non-GC subset displayed strong correlation with overall survival (FIG. 9B). These results suggest that SHIP expression levels are useful prognostic indicators of survival among DLBCL patients.
In order to determine the molecular mechanisms that mediate the observed decrease in SHIP expression in DLBCL, we determined the methylation status of the SHIP promoter in 44 non-GC and GC-DLBCL specimens, as well as screened for mutations in the coding regions and splice sequences, but did not detect any differences among the samples (not shown). We next considered the possibility that post-transcriptional regulatory events mediated by small non-coding RNAs might alter the expression of SHIP in these hematopoietic malignancies. Indeed, in addition to differential expression of coding genes, non-GC-type lymphoma cells express elevated levels of several microRNAs (miRs). Scanning of the SHIP 3′ UTR revealed perfect sequence complementarity with the seed sequence of miR-155 (FIG. 9C), an onco-miR whose ectopic expression gives rise to B cell malignancies (Costinean et al. (2006) Proc Natl Acad Sci USA 103:7024-70298), but whose cellular targets have remained elusive.
Using a prototypic cell line model of the GC- and a non-GC-subtypes of DLBCL, Lawrie et al. (Lawrie et al. (2007) Int J Cancer 121:1156-1161) showed that in addition to miR-155, microRNAs miR-221 and miR-21 were also over-expressed in non-GC-type but not GC-type lymphoma cells. To determine miR-155 and SHIP-mRNA levels in primary DLBCL patient samples without concern of potential contamination by non-malignant tissue, we isolated tumor cells by Laser-Capture-Micro-Dissection from frozen lymph node biopsies. As shown in FIG. 9D, miR-155 expression was significantly higher in non-GC-DLBCL compared to GC-DLBCL, whereas SHIP mRNA levels were lower in non-GC-DLBCL, consistent with the notion of attenuation of SHIP expression by miR-155.
Example 4
Autocrine Stimulation of Non-GC DLBCL by TNFα
To establish a link between miR-155 and SHIP expression beyond mere correlation, the non-GC-DLBCL cell lines OCILY-3 was transfected either with a non-specific control-miR, miR-1, or miR-155. Neither miR-1 nor the control miR affected SHIP mRNA levels, whereas introduction of miR-155 resulted in a clear decrease in SHIP mRNA (FIG. 10A). More importantly, neutralization of endogenous miR-155 by means of a synthetic anti-miR resulted in a dramatic increase in SHIP mRNA compared to a transfection of a non-specific anti-miR control (FIG. 10A). As anticipated, the modulation of SHIP mRNA levels by miR-155 or anti-miR-155 is reflected by accompanying changes in SHIP protein expression (FIG. 10B, left panel), and is not unique to OCILY-3 cells, but is also observed in the Toledo cells, a widely used cell line model representative of non-GC-DLBCL (FIG. 10B, right panel).
The biological significance of these observations is evidenced by the finding that eternacept imposes significant antiproliferative effects upon the three non-GC-DLBCL cell lines, but did not produce any growth modulation in the GC-DLBCL cells, or in Daudi Burkitt lymphoma (FIG. 11C).
Example 5
Anti-TNFα Regimen Inhibits DLBCL Growth in vivo
The above data demonstrated that in non-GC-DLBCL, elevated levels of miR-155, and consequent abrogation of SHIP expression, are mediated through autocrine stimulation of cells by TNFα, a proinflammatory cytokine whose serum levels are known to be elevated in DLBCL patients (Pedersen et al. (2005) Br J Haematol 128:813-81913). To explore the potential efficacy of anti-TNFα regimen as a treatment for non-GC-DLBCL patients, we employed xenograft models in which Nod/SCID mice were subcutaneously inoculated with non-GC-DLBCL Toledo cells. Upon establishment of palpable tumors, the animals received either 100 μg eternacept or solvent intravenously every three days, and tumor size was measured after 2, 4 or 6 days. As shown in FIG. 12, eternacept treatment resulted in a slight, but detectable inhibition in tumor growth at day 4, and produced a substantial reduction in tumor burden after 6 days.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in the relevant fields, are intended to be within the scope of the following claims.

## Claims

1. A method of treating non-Hodgkin diffuse large B-cell lymphoma (DLBCL) of an activated B-cell (ABC) subtype in a mammalian subject, comprising administering to the mammalian subject having an ABC-subtype DLBCL an effective amount of adalimumab that reduces expression of inositol polyphosphate-5-phosphatase (SHIP-1) and increases expression of miR-155 in said non-Hodgkin diffuse large B-cell lymphoma (DLBCL) of an activated B-cell (ABC) subtype.

2. A method of treating non-Hodgkin diffuse large B-cell lymphoma (DLBCL) in a human subject, comprising
a) detecting increased expression of miR-155 and decreased expression of SHIP-1 in cells of said non-Hodgkin DLBCL in relation to their expression in germinal center B-cell (GCB) subtype DLBCL cells, and
b) administering to the human subject an effective amount of adalimumab.

3. The method of claim 2, wherein said DLBCL is an activated B-cell (ABC) subtype DLBCL.

4. A method of treating an activated B-cell (ABC) subtype diffuse large B-cell lymphoma (DLBLC) in a mammalian subject, comprising administering adalimumab to a mammalian subject having ABC subtype DLBLC in an amount effective to reduce one or more symptoms of said ABC subtype DLBLC.

5. A method of treating an activated B-cell (ABC) subtype diffuse large B-cell lymphoma (DLBLC) in a mammalian subject, comprising
a) obtaining ABC subtype DLBLC tumor cells from the human subject,
b) detecting increased expression of miR-155 and decreased expression of SHIP-1 in said tumor cells obtained from the human subject in relation to the expression of miR-155 and SHIP-1 in germinal center B-cell (GBC) subtype DLBCL cells, and
c) administering to the human subject having ABC subtype DLBLC tumor cells with increased expression of miR-155 and decreased expression of SHIP-1 adalimumab in an amount effective to reduce one or more symptoms of said ABC subtype DLBLC tumor cells.