Patent Application
20070202106
The present invention relates in general to the field of cancer treatment, and more particularly, to the characterization and development of novel diagnostics and treatments against cancer.
Without limiting the scope of the invention, its background is described in connection with the host response to cancers. Cancer growth and development depends on the interaction between cancer cells and surrounding nonmalignant stroma composed of non-hematopoietic cells (fibroblasts, endothelial cells) and immune cells from both the innate and the adaptive immune system (Coussens and Werb, 2002; Joyce, 2005). Innate immune cells consist of neutrophils, macrophages (MΦ), dendritic cells (DCs), mast cells, and NK cells (Janeway and Medzhitov, 2002). The adaptive immune cells are T and B lymphocytes capable of immune memory and rapid response upon antigen re-encounter. However, lymphocytes need to be educated as to the nature of the antigen. This task falls upon DCs that bridge the two arms of the immune system (Banchereau et al., 2000; Banchereau and Steinman, 1998; Shortman and Liu, 2002; Steinman, 1991). Indeed, DCs induce and maintain immune response and as opposed to MΦ, are able to prime naïve lymphocytes. Furthermore, vaccination with antigen loaded-DCs in both mouse and humans has shown that DCs can break tolerance to cancer and educate T cells (Banchereau and Palucka, 2005). Therefore, DCs represent an early target for manipulation by tumor.
Many studies have focused on the role of tumor associated macrophages (TAMs) and their subsets (Balkwill et al., 2005; Condeelis and Pollard, 2006). However, less attention has been given to DCs. Many studies in humans observed infiltration of tumors with DC (Gabrilovich, 2004). Yet, the immunological consequences of DC infiltration are less well understood. Tumors are thought to escape immune effectors via subverting DC function (Gabrilovich, 2004). For example, activation of STAT-3 in myeloid cells results in the increased production of vascular endothelial growth factor (VEGF) (Wang et al., 2004) that interferes with DC maturation (Gabrilovich et al., 1996). IL-6 secreted by breast cancer cells skews monocyte differentiation into TAM at the expense of DC (Chomarat et al., 2000) thereby skewing antigen presentation towards antigen degradation (Delamarre et al., 2005). Finally, tumors promote differentiation of IL-10 and/or TGF-β secreting subset of DCs that in turn expands CD4+CD25+ regulatory T cells (Enk et al., 1997; Ghiringhelli et al., 2005; Levings et al., 2005). However, there is still a need for compositions and methods for improving the type of immune response that is mounted by the host against cancer cells.
It has been found that blocking of IL-13 (interleukin-13) can be used to treat tumors of epithelial origin. The present invention includes compositions and methods of modulating a T cell response to cancer by identifying a patient in need of cancer treatment in which the predominate immune response includes the secretion of Type II cytokines; and treating the affected tissue with one or more Type II cytokine antagonists, wherein the Type II cytokine antagonists block CD4+ T cells that secrete Type II cytokines and increase the percentage of Th1 T cells in the affected tissue.
It has also been found that the present invention may be used for the treatment of cancers of epithelial origin, e.g., prostate and breast. The Type II cytokine antagonists include, e.g., IL-13, but may also include anti-IL-4, IL-5, IL-9, IL-13 or IL-25 antibody, a humanized anti-IL-4, IL-5, IL-9, IL-13 or IL-25 antibody, and combinations thereof. In another embodiment, Type II cytokine antagonists may be inactivated IL-4, IL-5, IL-9, IL-13 or IL-25 and combinations thereof. These antagonists may be provided in a single or multiple doses. Type II cytokines may be prevented using a variety and combinations of active agents, e.g., anti-cytokine receptors, neutralizing antibodies, receptor antagonists, soluble receptors, molecules interfering in the receptor-ligand binding, and inhibitors of downstream events of type II signaling pathways.
More particularly, the present invention includes compositions and methods for diagnosing and treating a patient suspected of having cancer by determining the level of IL-13 present in the tissue microenvironment of a biological sample, taken from the patient, suspected of having cancer; comparing the IL-13 level in the tissue microenvironment to the level of IL-13 known to be present in normal, non-cancerous tissue of the same type as the biological sample; and evaluating the prognosis of the patient, wherein an elevated level of IL-13 indicates cancer. In one embodiment, the method of determining the level of IL-13 is selected from the group consisting of Western blot analysis, immunoprecipitation, ELISA analysis, in situ fluorescence, in situ fluorescence resonance energy transfer, in situ immunoprecipitation and in situ immunohistochemistry. The method also includes measuring the effect of IL-13 on the tissue microenvironment as determined by measuring the level of phosphorylation of STAT6.
Samples for analysis using the method of the present invention include, e.g., tissue section, cryogenically frozen sections and the like. The level of IL-13 expression may be tracked over time and include the detection of IL-13 levels by hybridization to the IL-13 mRNA, quantitative RT-PCR and the like. Generally, the biological sample will be of epithelial origin and may be obtained from biological sample selected breast, prostate, lung, colon, ovary, endometrium, kidney, bladder, stomach, pancreas and secretory pituitary gland.
The present invention also includes a method of treating a cancer of epithelial origin by identifying a patient in need of treatment for the cancer; determining the level of IL-13 present in the tissue microenvironment of a biological sample, taken from the patient, suspected of having cancer tissue; comparing the IL-13 level in the tissue microenvironment to the level of IL-13 known to be present in normal, non-cancerous tissue of the same type as present in the biological sample; and treating the patient with one or more IL-13 antagonists in an amount sufficient to block cell-to-cell IL-13 communication in the tissue microenvironment. Examples of IL-13 antagonist for use with the present invention include, e.g., at least one of an anti-IL-13 antibody, a human anti-IL-13 antibody, a humanized anti-IL-13 antibody, a bivalent anti-IL-13 antibody, a bivalent humanized anti-IL-13 antibody, an anti-IL-13 monoclonal antibody, an anti-IL-13 antigen binding fragment selected from the group consisting of a Fab, Fab′, F(ab′)2, Fv, disulfide stabilized Fv and single-chain Fv fragments and combinations thereof, an anti-IL-13 binding fusion protein, a multivalent anti-IL-13 antibody and an anti-IL-13 chimeric antibody, a polyclonal anti-IL-13 antibody that targets IL-13 in the microenvironment of breast cancer cells. The method of treatment may further include the step of treating the patient with tumor-specific immune cells, e.g., an intratumoral vaccination comprising dendritic cells. Another embodiment of the present invention includes the step of treating the patient with vaccine that includes antigen-loaded dendritic cells. Other examples of IL-13 antagonist include at least one of a blocking anti-IL-13 cytokine receptor, an anti-IL-13 receptor neutralizing antibody, an anti-IL-13 receptor antagonist, a soluble IL-13 receptor, one or more molecules that interfere with the anti-IL-13 receptor-IL-13 binding, one or more inhibitors of downstream events of IL-13, an inactivated IL-13 and combinations thereof.
Yet another embodiment of the present invention includes a method for diagnosis of a patient suspected of exhibiting breast cancer by determining the level of IL-13 present in the tissue microenvironment of a biological sample, taken from the patient, suspected of having breast cancer; comparing the IL-13 level in the in the tissue microenvironment to the level of IL-13 known to be present in normal, non-oncogenic breast tissue, wherein an elevated level of IL-13 indicates breast cancer. Examples of methods for determining the level of IL-13 include, e.g., Western blot analysis, immunoprecipitation, ELISA analysis, in situ fluorescence, in situ fluorescence resonance energy transfer, in situ immunoprecipitation and in situ immunohistochemistry. The level of IL-13 may be determined by measuring the level of phosphorylation of STAT6, e.g., of a cryogenically frozen sample.
The present invention also includes a test kit for the detection of IL-13 in a breast sample, the kit including in packaged combination, a quantity of an IL-13-specific probe capable of detecting the level of expression of IL-13 in a breast tissue microenvironment; and instructions for use of the IL-13 probe, wherein the probe is detectable directly or indirectly.
Yet another embodiment of the present invention includes a method of treating a breast cancer by identifying a patient in need of treatment for the breast cancer; determining the level of IL-13 present in the tissue microenvironment of a breast tissue sample taken from the patient suspected of having breast cancer; comparing the IL-13 level in the sample to the level of IL-13 known to be present in normal breast tissue; and treating the breast cancer with one or more IL-13 antagonists. The present invention also includes a composition adapted for treatment of breast cancer by treating a patient with an effective amount of one or more IL-13 antagonists sufficient to treat the breast cancer.
Another method includes the steps of treating prostate cancer by identifying a patient in need of treatment for the cancer; determining the level of IL-13 present in the tissue microenvironment of a prostate tissue sample taken from the patient suspected of having prostate cancer; comparing the IL-13 level in the biological sample to the level of IL-13 known to be present in normal prostate tissue; and treating the prostate cancer with one or more IL-13 antagonists. The present invention also includes a composition adapted for treatment of prostate by treating a patient with an effective amount of one or more IL-13 antagonists sufficient to treat the prostate cancer.
Yet another method of the present invention includes a method of treating non-small cell lung cancer that includes identifying a patient in need of treatment for the cancer; determining the level of IL-13 present in the tissue microenvironment of a lung sample taken from the patient suspected of having lung cancer; comparing the IL-13 level in the biological sample to the level of IL-13 known to be present in normal lung tissue; and treating the lung cancer with one or more IL-13 antagonists. The present invention also includes a composition adapted for treatment of a non-small cell lung cancer by treating a patient with an effective amount of one or more IL-13 antagonists sufficient to treat a non-small cell lung cancer.
A method of the present invention also includes a method of inhibiting angiogenesis by treating a patient with an effective amount of one or more IL-13 antagonists sufficient to reduce angiogenesis.
Alternatively or in combination, a Type II cytokine antagonists may be used that is a soluble IL-4, IL-5, IL-9, IL-13 or IL-25 receptors and combinations thereof. In fact, the present invention may use combinations of one or more anti-cytokine antibodies (e.g., humanized antibodies), receptor antagonists and inactivated cytokine. One specific example may be a blocking IL-13 receptor binding antibody, a soluble IL-13R and combinations thereof. Other embodiments include the use of an IL-13 antagonist to decrease CD4+ T cells that secrete Type II cytokines (and increase CD4+ T cells that secrete Type I cytokines).
In certain embodiments it may also be useful to include anti-IFN-γ antibody, a humanized anti-IFN-γ antibody, a soluble IFN-γ receptor and combinations thereof to antagonize the effects of IFN-γ. In yet other embodiment, the antagonists may also include an anti-TNF antibody, a humanized anti-TNF antibody, a soluble TNF receptor and combinations thereof.
The present invention also includes compositions and methods for improving T cell responses to breast cancer by identifying a patient in need of treatment for a breast cancer and treating the affected tissue with one or more IL-13 antagonists, wherein the IL-13 antagonists block CD4+ T cells that secrete Type II cytokines. Examples of IL-13 antagonists include a blocking IL-13 receptor binding antibody, an inactivated IL-13, a soluble IL-13R and combinations thereof.
The composition of the present invention may be used in conjunction with a method to improve immunity against breast cancer by administering to a patient in need thereof a therapeutically effective amount of one or more Type II cytokine antagonists. Examples of Type II cytokine antagonists include, e.g., anti-IL-4, IL-5, IL-9, IL-13 or IL-antibody, a humanized anti-IL-4, IL-5, IL-9, IL-13 or IL-25 antibody; inactivated IL-4, IL-5, IL-9, IL-13 or IL-25 and combinations thereof, soluble IL-4, IL-5, IL-9, IL-13 or IL-25 receptors and combinations thereof, and combinations of two or more of the listed antagonists. The composition may also include anti-IFN-γ antibody, a humanized anti-IFN-γ antibody, a soluble IFN-γ receptor and combinations thereof and/or an anti-TNF antibody, a humanized anti-TNF antibody, a soluble TNF receptor and combinations thereof.
In yet another embodiment, the compositions and methods of the present invention may also includes one or more Type I cytokines to stimulate Th1 responses against the cancer. The one or more Type I cytokines may be provided in a single or multiple dose that stimulate Th1 responses.
The present invention also includes a method of reducing Th2 polarization by human breast cancer by providing an effective amount of one or more Type II cytokine antagonists selected from anti-IL-4, IL-5, IL-9, IL-13 or IL-25; soluble receptors for IL-4, IL-5, IL-9, IL-13 or IL-25 and combinations thereof, an anti-IFN-γ antibody, a humanized anti-IFN-γ antibody, a soluble IFN-γ receptor and combinations thereof and/or an anti-TNF antibody, a humanized anti-TNF antibody, a soluble TNF receptor and combinations thereof.
The present invention also includes compositions and methods for inhibiting angiogenesis in tumors in which a subject or patient in need thereof is provided with an effective amount of one or more IL-13 antagonists selected from a blocking anti-IL-13 cytokine receptor, anti-IL-13 neutralizing antibodies, anti-IL-13 receptor antagonists, anti-IL-13 soluble receptors, molecules that interfere with the anti-IL-13 receptor-ligand binding, inhibitors of downstream events of anti-IL-13, an inactivated IL-13 and combinations thereof. The method for inhibiting angiogenesis may be used alone or in combination with any of the therapies taught and discussed herein.
Alternatively, the present invention also includes compositions and method for inhibiting the function of tumor associated macrophages by cancers of epithelial cell origin in which an effective amount of one or more IL-13 antagonists selected from a blocking anti-IL-13 cytokine receptor, anti-IL-13 neutralizing antibodies, anti-IL-13 receptor antagonists, anti-IL-13 soluble receptors, molecules that interfere with the anti-IL-13 receptor-ligand binding, inhibitors of downstream events of anti-IL-13, an inactivated IL-13 and combinations thereof is provided to a patient in need thereof.
The present invention also includes compositions and methods for prevention of metastasis and/or the formation of tumor stroma by providing an amount effective to prevent the metastasis and/or formation of tumor stroma of one or more IL-13 antagonists selected from a blocking anti-IL-13 cytokine receptor, anti-IL-13 neutralizing antibodies, anti-IL-13 receptor antagonists, anti-IL-13 soluble receptors, molecules that interfere with the anti-IL-13 receptor-ligand binding, inhibitors of downstream events of anti-IL-13, an inactivated IL-13 and combinations thereof.
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
a and 2a shows the presence of Type 2 cytokines in the microenvironment of breast cancer samples from patients. Cytokine concentration measured by Multiplex Cytokine Bead assay (pg/ml, ordinate) in tumor cell suspensions from breast tumor (black, T) and surrounding tissue (white, ST) (
a to 2d show IL-13 secreting CD4+T cells in breast cancer samples from patients. Flow cytometry analysis of single cell tumor suspensions. (
a to 4e show that human breast cancer tumors developed in humanized mice are infiltrated with DCs. 10×106 tumor cells are inoculated subcutaneously into the flank of humanized mice four weeks post-CD34+HPC-transplant.
a through 5e show that the reconstitution with CD4+T cells is associated with accelerated early development of breast cancer tumors. PBS (100 μl), autologous CD8+T cells (10×106 cells/100 μl PBS) alone or together with CD4+T cells (10×106 cells/100 μl PBS) are injected at day 3, 6 and 9 post-tumor implantation into (
a and 6b show that accelerated breast cancer development requires CD4+T cells and autologous DCs. In
a and 7b show IL-13 expressing CD4+T cells in breast cancer tumors in humanized mice. In
a and 8b show that the breast cancer microenvironment modulates mDCs to induce CD4+T cells secreting type 2 cytokines. In
a to 9c shows the accelerated breast cancer development can be inhibited with IL-13 antagonists. In
a and 13b show the staining of IL-13 in tissue from lung tumor and
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
As used herein, “IL-13 antagonists” refers to active agents that interfere, reduce or eliminate the activity of IL-13, e.g., small molecules, peptides and proteins, which are provided in an amount sufficient to block cell-to-cell IL-13 triggered communication in a tissue microenvironment generally through the IL-13 receptor. Non-limiting examples of IL-13 antagonist for use with the present invention include, e.g., at least one of an anti-IL-13 antibody, a human anti-IL-13 antibody, a humanized anti-IL-13 antibody, a bivalent anti-IL-13 antibody, a bivalent humanized anti-IL-13 antibody, an anti-IL-13 monoclonal antibody, an anti-IL-13 antigen binding fragment selected from the group consisting of a Fab, Fab′, F(ab′)2, Fv, disulfide stabilized Fv and single-chain Fv fragments and combinations thereof, an anti-IL-13 binding fusion protein, a multivalent anti-IL-13 antibody and an anti-IL-13 chimeric antibody, a polyclonal anti-IL-13 antibody that targets IL-13 in the microenvironment of breast cancer cells. The method of treatment may further include the step of treating the patient with tumor-specific immune cells, e.g., an intratumoral vaccination comprising dendritic cells. Other examples of IL-13 antagonist include at least one of a blocking anti-IL-13 cytokine receptor, an anti-IL-13 receptor neutralizing antibody, an anti-IL-13 receptor antagonist, a soluble IL-13 receptor, one or more molecules that interfere with the anti-IL-13 receptor-IL-13 binding, one or more inhibitors of downstream events of IL-13, an inactivated IL-13 and combinations thereof.
As used herein, the term “Type II cytokine antagonists” refers to active agents that interfere, reduce or eliminate the activity of cytokines that normally would aid in the activation of a Type 2 helper T cell, e.g., anti-IL-4, IL-5, IL-9, IL-13 or IL-25 antibody, a humanized anti-IL-4, IL-5, IL-9, IL-13 or IL-25 antibody; inactivated IL-4, IL-5, IL-9, IL-13 or IL-25 and combinations thereof, soluble IL-4, IL-5, IL-9, IL-13 or IL-25 receptors and combinations thereof, as well as, combinations of two or more of the above.
As used herein, the term “oncohumammal” is used to refer to non-human mammal that is immune deficient into which a human immune system has been grafted and to which a human cancer has been implanted. As will be apparent to the skilled artisan, a number of existing animals may be used as the immune deficient animal. Also, a number of methods for the non-lethal manufacturing of immune deficient animals is available, including non-lethal doses of radiation, chemical treatments, animals with one or more genetic mutations, the genetic manipulation of the mammal by the making of a transgenic, a knock-out, a conditional knock-out, a knock-in and the like. One example of an “oncohumammal” is an “oncohumouse,” in which a mouse is used as the platform for the introduction of at least a portion of a human immune system and a human tumor. The tumor may one or more primary tumors (e.g., autologous with the immune system implanted, i.e., from the same patient), one or more tumor cell clones and/or one or more tumor cell lines.
As used herein, the terms “markers,” “detectable markers” and “detectable labels” are used interchangeably to refer to compounds and/or elements that can be detected due to their specific functional properties and/or chemical characteristics, the use of which allows the agent to which they are attached to be detected, and/or further quantified if desired, such as, e.g., an enzyme, radioisotope, electron dense particles, magnetic particles or chromophore. There are many types of detectable labels, including fluorescent labels, which are easily handled, inexpensive and nontoxic.
As used herein, the term “tissue microenvironment” refers to the local environment in which cell reside within a native tissue, e.g., in vivo. In the tissue microenvironment, the two or more tissue-specific cell types may interact with and/or influence one another, e.g., via contact (e.g., contact inhibition), cytokines, chemokines, etc. Most often, cells in close proximity within an organ will lead to almost concurrent exposure of substances in the immediate extracellular milieu with the two or more cells in the same tissue. In addition, two or more cell types exposed to the same microenvironment may react similarly upon exposure to a common agent introduced to the microenvironment. In vitro, a tissue microenvironment mimics the environment that the cells experience in vivo. In the context of this invention, a tissue microenvironment will often refer to an in vivo microenvironment within a tissue.
Direct Measurement of Cytokine Production. Direct measurements of IL-13 levels in assays include the direct measurement of nucleic acid or protein expression using methods such as, e.g., in situ hybridization, ELISA, radioimmunoassay, gel electrophoresis, western blotting, immunohistochemistry, and bioassays.
ELISA techniques for evaluating the skin immune response are well-known in the art. The ELISA protocol involves coating the wells of microtiter (ELISA) plates with a monoclonal or polyclonal antibody directed to the cytokine of interest, e.g., an anti-IL-13 antibody. Briefly, the antibody solution is allowed to incubate in the wells; the unbound antibody is washed away, and the open sites are blocked with an inert protein, e.g., bovine serum albumin (BSA) in PBS, which is allowed to incubate in the wells for 1-3 hours. The blocking solution is discarded and the sample suspected of having IL-13, e.g., a sample of breast cells that are suspected of having breast cancer that have be treated to remove the proteins while minimizing protease activity, is added to the ELISA wells. For quantitation the sample solution is serially diluted in PBS buffer and after an incubation period, the plates are again washed and the level of IL-13 detected directly or indirectly using, e.g., a second antibody directed to the cytokine, e.g., a polyclonal rabbit anti-IL-13 antibody. After an incubation period the plates are again washed and, e.g., a biotin-labeled second or third antibody, directed against the second antibody, is added (e.g., goat anti-rabbit) and incubated. After washing the unbound material, the level of IL-13 expression is detected using, e.g., horseradish peroxidase-labeled streptavidin is added and incubated for 0.5-1 hour at 37° C. Because the cytokine is situated between two layers of antibodies, this type of assay is often called a sandwich ELISA. Depending on the enzyme used for the detection, the detectable substrate is added to the well and presence/absence and/or the level of IL-13 is determined. The intensity of the signal (e.g., color, luminescence, etc.) is determined that equates (e.g., as compared to a known standard) to the amount of IL-13 specifically bound.
Radioimmunoassay. Radioimmunoassay may also be used to quantitate the production of IL-13 in a manner similar to that just described for quantitation by ELISA. Generally, an IL-13-specific antibody is attached to a substrate using standard techniques, such as those described in Harlow or Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor 1989), relevant portions incorporated herein by reference. The bound antibody is then exposed to the supernatant taken from the breast sample, biopsy or cells obtained from the sample and grown in culture. A second antibody, carrying a radioactive label such as 125I-modified tyrosine is added to the bound cytokine and allowed to incubate as described above at a specific activity that will provide a detectable signal to noise ratio. The unbound labeled material is washed away and the remaining label quantitated, using standard techniques, e.g., a scintillation counter.
Gel Electrophoresis and Western Blotting. As an alternative, the presence of IL-13 can be assessed by Western blot analysis (see, e.g., Didieijean, et al., J. Invest. Dermatol. 92:809 (1989), relevant portions incorporated herein by reference). Western blot analysis can be used to analyze samples that cannot be accurately tested in the ELISAs or functional assays (e.g., STAT6 phosphorylation) due to high protein concentration or the presence of various detergents or solubilizing agents (as is the case with skin homogenates and lamellar body preparations).
Cell supernatant samples are run on reducing or non-reducing SDS-polyacrylamide gels using standard electrophoresis techniques. The separated protein in the gel is electrophoretically blotted onto a substrate, e.g., nitrocellulose membrane, nylon membrane or some other suitable substrate, again using common techniques. The membrane may be blocked with an inert protein, then incubated with a solution containing an antibody to the protein of interest, such as one of the antibodies described above, to bind to the immobilized protein. The antibody may be an IL-13-specific monoclonal antibody (e.g., anti-human IL-13) or a specific anti-IL-13 antibody raised against the denatured cytokine or may be a high-titer polyclonal antiserum. The bound protein-antibody complex may be detected directly or indirectly. For indirect measurements, the target is subsequently incubated with a second labeled antibody specific for the first antibody to form a protein-antibody-antibody complex. For example, the second labeled antibody may be labeled with a calorimetric label, an enzymatic label, or may be radiolabeled. The bound antibody-cytokine complex is then assayed using the method appropriate for the label as described above.
Immunohistochemistry. Immunohistochemistry provides information of a qualitative and quantitative nature. In situ immunohistochemistry allows visualization and localization of the distribution of a specific cytokine among various cell types or within different regions of a tissue. Briefly, tissue samples may be flash frozen in an embedding compound, e.g., Tissue-Tek OCT (available commercially from Miles, Inc., Elkhart, Ind.), and stored at −70° C. until used. The tissue is cut into sections, typically 6 μm sections and placed on microscope slides. After fixing the tissue in acetone, the slides are incubated with a non-specific blocking agent, e.g., goat serum, to block non-specific binding sites. The samples are subsequently incubated with a specific antibody directed against the cytokine of interest. After thorough washing, a second, labeled antibody, e.g., a biotinylated antibody, is added and the amount of bound label is quantitated using standard techniques. When a biotinylated antibody is used, avidin conjugated with biotinylated horse radish peroxidase is added, followed by incubation with a chromogenic peroxidase substrate. The sample is then counterstained with hematoxylin. To ensure that the staining pattern is specific using a control antibody may be used for comparison with the anti-cytokine antibody. Untreated control tissue or cells are also compared to tissues or cells that have been subjected to an inflammatory stimulus.
Measurement of Cytokine Gene Expression. In addition to examining the regulation of cytokines at the level of protein production, direct assessment of IL-13 mRNA levels is a valuable indicator of changes in IL-13 expression levels. Although modulation of mRNA and protein levels often occur in tandem, this is not always the case.
Northern Blotting. Northern blotting has been used to determine the extent of IL-13 mRNA production of cytokines as an indirect measure of cytokine concentration. RNA from a beast cell sample is separated on an agarose gel and the separated RNA blotted onto a nylon or other suitable membrane. The membrane-bound RNA is probed with specific nucleic acid sequences which will bind to the mRNA encoding the amino acid sequences of the cytokines of interest. The probes are labeled, e.g., with 32P or chemiluminescence, to allow detection of probe binding to the appropriate mRNA.
In Situ Hybridization of mRNA. In situ hybridization is a qualitative procedure that allows the direct visualization of cellular mRNA levels in cultured cells or tissue sections. The relative expression of mRNA in different cell populations or in different regions of the tissue can be determined. Cell samples are affixed to microscope slides using standard methods and reagents. The fixed cells are incubated in ethanol, and the sample is hybridized with a DNA probe specific for the cytokine of interest by placing a small volume of the probe on the slide, covering the slide, and incubating the slide overnight in a humidified atmosphere. The probes are labeled as described hereinabove and/or as will be known to the skilled artisan. For DIG-labeled probes, a color development procedure is performed that is similar to that used for a Western blot. After counterstaining with hematoxylin, the distribution of the probe can be visualized at the level of the light microscope.
RT-PCR. Very sensitive and powerful techniques for assessing mRNA levels are based on reverse transcriptase-polymerase chain reaction. Total RNA is isolated from a sample and the mRNA copied to DNA (cDNA) using reverse transcriptase. The cDNA is then added to a PCR reaction with DNA primers that specifically target the mRNA species of interest. This PCR product is then detected using any of a variety of methods, e.g., electrophoresed on an agarose gel, stained with a fluorescent dye, and photographed. The intensity of the staining of the PCR product is proportional to the concentration of the product and can be quantitated using a densitometer. For use as a control, the housekeeping gene β-actin, whose expression is relatively constant under various treatment conditions, is analyzed in parallel with the cytokine genes. By normalizing the expression of various genes based on β-actin expression, semi-quantitative determination of mRNA concentrations can be achieved by RT-PCR.
Assay kits according to this invention include at least one probe against IL-13 or ots down stream effectors (e.g., phosphorylation of STAT6) and instructions for performing an assay. Kits may also include assay reagents, e.g., salts, buffers, nuclease inhibitors, restriction enzymes and denaturants. Kits may include a target or model target for a positive control test, and a target-less “sample” for a negative control test.
Amplification assay kits may include, in addition to some or all of the above, primers, nucleotides, polymerases and polymerase templates for the assay and for control assays. Vital stain kits may include, in addition to probe and instructions, permeabilizing agents, liposome precursors, buffers, salts, counterstains and optical filters. In situ kits may include, in addition to probe and instructions, fixatives, dehydrating agents, proteases, counterstains, detergents, optical filters and coated microscope slides.
Immune Deficient Animal Hosts. Any immunodeficient mammal may be used to generate the animal models described herein. As used herein, the term “immunodeficient” is used to describe an alteration that impairs the animal's ability to mount an effective immune response. As used herein, an “effective immune response” is used to describe a human immune response in the host animal that is capable or, e.g., destroying invading pathogens such as (but not limited to) viruses, bacteria, parasites, malignant cells, and/or a xenogeneic or allogeneic transplant. One example of an immunodeficient mammal is the immunodeficient mouse referred to as a severe combined immunodeficient (SCID) mouse, which generally lacks recombinase activity that is necessary for the generation of immunoglobulin and functional T cell antigen receptors, and thus does not produce functional B and T lymphocytes.
Immune deficient mice, rats or other animals may be used, including those that are deficient as a result of a genetic defect, which may be naturally occurring or induced. For example, heterologous or homologous: nude mice, immunodeficient nonobese diabetic/LtSz-scid/scid (NOD/SCID) mice with additional mutation in 132-microglobulin gene (NOD/SCID/β2m−/−), Rag 1−/−, Rag 2−/− mice and/or PEP−/− mice, mice that have been cross-bred with these mice and have an immunocompromised background may be used for implanting or engrafting a human immune system and/or cells as described herein. The deficiency may be, for example, as a result of a genetic defect in recombination, a genetically defective thymus or a defective T-cell receptor region, NK cell defects, Toll receptor defects, Fc receptor defects, immunoglobulin rearrangement defects, defects in metabolism, combinations thereof and the like. Induced immune deficiency may be as a result of administration of an immunosuppressant, e.g. cyclosporin, FK-506, removal of the thymus, radiation and the like.
Various transgenic immune deficient mice are currently available or can be mated or cross-bred and selected in accordance with conventional techniques. Generally, the immune deficient mouse will have a defect that inhibits maturation of lymphocytes, particularly lacking the ability to rearrange immunoglobulin and/or T-cell receptor regions, Toll receptors, and the like. Female, male, castrated or uncastrated mice may be used depending on the effect of the availability of, e.g., androgens, on the course of the tumor growth. In addition to mice, immune deficient rats or similar rodents may also be employed in the practice of the invention.
As used herein, the term “compounds,” “agent(s),” “active ingredient(s),” “pharmaceutical ingredient(s),” “active agents,” “bioactive agent” are used interchangeably and defined as drugs and/or pharmaceutically active ingredients. The present invention may use or release of, for example, any of the following drugs as the pharmaceutically active agent in a pool of test compounds to isolate one or more lead compounds. A number of test compounds may be tested, isolated and purified using the methods of the present invention.
Examples of test compounds include, antitumor agents, anti-miotics, steroids, sympathomimetics, local anesthetics, antimicrobial agents, antihypertensive agents, antihypertensive diuretics, cardiotonics, coronary vasodilators, vasoconstrictors, β-blockers, antiarrhythmic agents, calcium antagonists, anti-convulsants, agents for dizziness, tranquilizers, antipsychotics, muscle relaxants, respiratory agents, non-steroidal hormones, antihormones, vitamins, herb medicines, antimuscarinic, muscarinic cholinergic blocking agents, mydriatics, psychic energizers, humoral agents, antispasmodics, antidepressant drugs, anti-diabetics, anorectic drugs, anti-allergenics, decongestants, antipyretics, antimigrane, anti-malarials, anti-ulcerative, peptides, anti-estrogen, anti-hormone agents, antiulcer agents, anesthetic agent, drugs having an action on the central nervous system or combinations thereof. Additionally, one or more of the following bioactive agents may be combined with one or more carriers and the present invention (which may itself be the carrier).
Different lineages of immune-compromised mice may used in conjunction with the present invention. In one embodiment, the immune-compromised mouse may be made transgenic with one or more genes that are tumor suppressors, cytokines, enzymes, receptors, or even inducers of apoptosis. Alternatively, the second gene may be derived from an oncogene. Examples of oncogene include ras, myc, neu, raf erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl. Genes may also include a tumor suppressor, the tumor suppressor may be, e.g., p53, p16, p21, MMAC1, p73, zac1, BRCAI and Rb. Other genes may tumor cytokine, the cytokine is selected from the group consisting of IL-2, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, TNF, GMCSF, β-interferon and γ-interferon. In other embodiments the gene may be an enzyme, e.g., cytosine deaminase, adenosine deaminase, .beta.-glucuronidase, hypoxanthine guanine phosphoribosyl transferase, galactose-1-phosphate uridyltransferase, glucocerbrosidase, glucose-6-phosphatase, thymidine kinase and lysosomal glucosidase. In other embodiments, the gene may be a receptor, e.g., CFTR, EGFR, VEGFR, IL-2 receptor and the estrogen receptor. In other embodiment, the gene may be an inducer of apoptosis, e.g., Bax, Bak, Bcl-X.sub.s, Bik, Bid, Bad, Harakiri, Ad E1B and an ICE-CED3 protease. In certain embodiments, the cells that are made transgenic and/or transfected are human cells that are implanted in the mouse.
The present invention further provides a method of enhancing the effectiveness of ionizing radiotherapy by administering, to a tumor site in a mammal, an anti-angiogenic factor protein prior to radiation therapy; and ionizing radiation, wherein the combination of anti-angiogenic factor administration and radiation is more effective than ionizing radiation alone.
The present invention also includes pools and/or leads of therapeutic compounds in, e.g., a pharmaceutically acceptable carrier or diluent. With respect to in vivo applications, the compounds identified by screening methods may be administered to the oncohumouse in a variety of ways including, for example, parenterally, orally or intraperitoneally. Parenteral administration includes administration by the following routes: intravenous, intramuscular, interstitial, intraperitoneal, intradural, epidural, intraarterial, subcutaneous, intraocular, intrasynovial, transepithelial, including transdermal, pulmonary via inhalation, opthalmic, sublingual and buccal, topical, including ophthalmic, dermal, ocular, rectal, vaginal and nasal inhalation via insufflation or nebulization.
The Type II cytokine antagonists (and others) may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, they can be enclosed in hard or soft shell gelatin capsules, or they can be compressed into tablets. For oral therapeutic administration, the active compounds can be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, sachets, lozenges, elixirs, suspensions, syrups, wafers, and the like. The pharmaceutical composition may include active compounds in the form of a powder or granule, a solution or suspension in an aqueous liquid or non-aqueous liquid, or in an oil-in-water or water-in-oil emulsion.
The tablets, troches, pills, capsules and the like can also contain, for example, a binder, such as gum tragacanth, acacia, corn starch or gelatin. Excipients, such as dicalcium phosphate, a disintegrating agent, such as corn starch, potato starch, alginic acid and the like, a lubricant, such as magnesium stearate, and a sweetening agent, such as sucrose, lactose or saccharin, or a flavoring agent may also be included. When the dosage unit form is a capsule, it may include a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir may include the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring. Any material used in preparing any dosage unit will generally be pharmaceutically pure and substantially non-toxic. The active compound may be incorporated into sustained-release preparations and formulations.
The Type II cytokine antagonists (and others) may be administered parenterally or intraperitoneally. Solutions of the compound as a free base or a pharmaceutically acceptable salt may be prepared in water mixed with a suitable surfactant, e.g., hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative and/or antioxidants to prevent the growth of microbes and/or chemical degeneration.
The pharmaceutical forms of the Type II cytokine antagonists (and others) may be prepared for injectable use by including sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or other like use, the compounds are generally sterile and may be provided in liquid suspension and/or resuspended for delivery via syringe. It can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained by the use of a coating, e.g., lecithin, and incorporation into a particle of the required size (in the case of a dispersion) and by the use of surfactants as is well known to the skilled artisan. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars or sodium chloride may be used.
Sterile injectable solutions are prepared by incorporating the Type II cytokine antagonists (and others) in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that includes the basic dispersion medium and any of the other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation may include, e.g., vacuum drying, freeze-spraying, heat-vacuum and/or freeze drying techniques. Pharmaceutical compositions that are suitable for administration to the nose or buccal cavity include, e.g., powder, self-propelling and spray formulations, such as aerosols, atomizers and nebulizers.
The therapeutic Type II cytokine antagonists (and others) of this invention may be administered to a mammal alone or in combination with pharmaceutically acceptable carriers or as pharmaceutically acceptable salts, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice. The compositions may also include other therapeutically active compounds that are usually applied in the treatment of the diseases and disorders, e.g., cancer. Treatments using the present compounds and other therapeutically active compounds may be simultaneous or by intervals.
The in vivo analysis disclosed herein is based on immunodeficient mice reconstituted with CD34+HPCs and human tumors might prove useful in the analysis of the human immune system and human cancer. Immunodeficient mice implanted with human tumor xenografts, with or without adoptively transferred human peripheral blood leukocytes (PBL) (Mosier et al., 1988), have been used for many years as models to study human cancer (Mueller and Reisfeld, 1991; Reddy et al., 1987). They have permitted the identification of clinically relevant prognostic factors in hematological malignancies (Kamel-Reid et al., 1991; Kamel-Reid et al., 1989; Palucka et al., 1996; Uckun et al., 1995); and have played a significant role in pre-clinical development of cancer drugs and immune therapies (Bankert et al., 2001; Kelland, 2004). Such models have also helped in assessing the mechanisms of metastasis in solid tumors (Muller et al., 2001).
A major limitation of earlier models was the lack of human immune microenvironment. Indeed, approaches that were undertaken include: (i) tumor xenografts in the absence of human immune cells (Bankert et al., 2001; Mueller and Reisfeld, 1991; Reddy et al., 1987); (ii) xenografts of tumor surgical biopsies, which harbor immune cells that have been imprinted by the tumor (Anderson et al., 2003; Twanuma et al., 1997; Sakakibara et al., 1996); and (iii) xenografts with human peripheral blood lymphocytes (SCID-huPBL) (Mosier et al., 1988), which are nearly devoid of human DCs. The approach used herein, mice engrafted with human CD34+HPCs and reconstituted with human immune system from a healthy volunteers, permits the analysis for the first time of early events in the biology of cancer cells implanted in the environment of human immune cells, which have not been previously exposed to tumor.
Generation of humanized mice: CD34+HPCs were obtained from apheresis of adult healthy volunteers mobilized with G-CSF and purified as previously described (Palucka et al., 2003). CD34− fraction of apheresis was Ficoll-purified, obtained PBMCs were stored frozen and used as a source of autologous T cells. 2.5×106 CD34+HPCs were transplanted intravenously into sublethally irradiated (12 cGy/g body weight of 137Cs γ-irradiation) NOD-SCID β2m−/− mice (Jackson Laboratories). 10×106 breast cancer cells (Hs578T, MCF7, 1806) harvested from long term cultures, were injected subcutaneously into the flank of the mice or in the mammary glands area. For experiments with NOD-SCID β2m−/− mice, they were sublethally irradiated (12 cGy/g body weight of 137Cs γ-irradiation) the day prior to tumor implantation. Tumor size was monitored every 2-3 days. Tumor volume (ellipsoid) was calculated using the formula: (short diameter)2×long diameter/2.
Monocyte-derived dendritic cells and T cell purification: Monocyte-derived dendritic cells were generated from adherent fraction of PBMCs by culturing with GM-CSF (100 ng/ml) (Immunex, Seattle, Wash.) and IL-4 (25 ng/ml) (R&D systems, Minneapolis, Minn.). CD4+ and CD8+T cells were positively selected from thawed PBMCs using magnetic selection according to manufacturer instructions (Myltenyi Biotec, Auburn, Calif.). The purity was routinely >90%.
Immunofluorescence: Tissues were frozen in Tissue-Tek (OCT, Allegiance, McGaw, Ill.), cryosectioned on Superfrost Plus slides (Fisher scientific, Pittsburgh, Pa.) and fixed with cold acetone. Direct staining: HLA-DR FITC (BD Pharmingen, San Diego, Calif.); CRTH2-PE; IL-13-PE; CD3-FITC. Indirect staining: DC-LAMP (Immunotech, Marseille, France) following by anti-mouse IgG conjugated to Texas-Red (Jackson Immunoresearch, West Grove, Pa.). Confocal microscopy was performed using a Leica TCS-NT SP (Leica, Deerfield, Ill.). To assess tumor vascularization, OncoHumouse were injected with FITC-lectin (150 microliters at 2 mg/ml) (Vector Laboratories, Burlingame, Calif.) intravenous (iv) 10 min later mice were anesthetised and infused with PFA 4% iv. 10 μm tumor sections were fixed and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, Calif.) and analyzed with Olympus BX51 equipped with planapo objectives and Photometrics coolsnaps HQ and Metamorph software (UIC).
Flow cytometry: Cell suspensions were obtained from tumor, lymph nodes, and spleens by digestion with collagenase D (2 mg/ml) (Roche Diagnostics, Indianapolis, Ind.) 45 min at 37° C. Bone marrow cells were washed out of the harvested bones. Cell suspensions were washed twice and stained in PBS 2 mM EDTA 5% AB serum using the following antibodies: Lin, CD45, IgD, CD80-FITC, CD123, CD86, HLA-ABC, CD3-PE, HLA-DR-PerCP, CD11c, CD14, CD19-APC (BD Pharmingen, San Diego, Calif.) and CD40-PE (Immunotech, Marseille, France).
T cell cytokines: Naive CD4+T cells were obtained from buffy coats after magnetic depletion using CD8, CD14, CD19, CD16, CD56 and glycophorine A microbeads (Miltenyi Biotec, Auburn, Calif.) and sorted based on the CD4+CCR7+CD45RA+ phenotype. NKT cells were depleted by exclusion of Vα24+CD4+T cells from the sort gate. DCs were sorted based on HLA-DR+Lin-CD11c+ and HLA-DR+Lin-CD123+ phenotype. Naive CD4+T cells (5×104/well) were cultured with DC (5×103/well) in RPMI 1640 supplemented with 10% human AB serum (Gemini BioProducts, Woodland, Calif.). To assess cytokine secretion by Luminex, T cells were harvested at day 5, washed twice, resuspended at a concentration of 1×106/ml and restimulated for 16 h with PMA (50 ng/ml) and ionomycin (1 μg/ml) (Sigma, St Louis, Mo.). To assess cytokine expression by intracellular staining, T cells were harvested on day 6 of the culture, washed twice and restimulated 5 hours with PMA and ionomycin. Brefeldin A (10 mg/ml) (BD Pharmingen, San Diego, Calif.) was added for the last 2.5 hours. T cells were labeled with anti-CD3 and Abs to IL-4, IL-13, TNF, IFN-γ and IL-2 (BD Pharmingen, San Diego, Calif.).
In vivo IL-13 blocking: mice were injected intratumorally at day 4, 6 and 8 post-tumor implantation with anti-IL-13 mAbs and rhIL-13Rα2/Fc chimera or goat IgG isotype control (100 μg/ml each) (R&D systems, Minneapolis, Minn.).
Tumor samples from patients diagnosed with infiltrating or invasive breast carcinoma were obtained Baylor University Medical Center Tissue Bank (IRB#005-145). Samples were minced into small fragments and digested in a triple enzyme mix containing collagenase 2.5 mg/ml, hyaluronidase 1 mg/ml, DNase 20 U/ml 2-3 hours at 37° C. The suspension was filtered, washed, obtained cells were resuspended at a concentration of 1×106/ml and activated with PMA (50 ng/ml) and ionomycin (1 g/ml) (Sigma, St Louis, Mo.) for 16 h. Cytokine production was analyzed in the culture supernatant by Luminex. Alternatively, whole tumor fragments were placed in the overnight culture with PMA and Ionomycin. For intracellular cytokine staining, cells were stimulated 5 hours with PMA and ionomycin. Brefeldin A (10 mg/ml) (BD Pharmingen, San Diego, Calif.) was added for the last 2.5 hours. Cells were labeled with anti-CD3 and anti-CD4 mAb and intracellular cytokine staining was performed using Abs to IL 13 and IFNγ (BD Pharmingen, San Diego, Calif.). For inhibition of IL13 staining, anti-IL13 mAb was incubated with recombinant human IL13 (5 μg/ml) for 1 hour at room temperature prior use. Cells were fixed in PFA 1% and analyzed by flow cytometry. A piece of each tissue was frozen in for immunofluorescence analysis. Sections were labeled with CD3 Alexa 488 mAb (BD Pharmingen, San Diego, Calif.) and mounted with DAPI.
Statistics: Parametric t-test and non-parametric Mann-Whitney and Wilcoxon tests were used to assess the significance of observed differences. Parametric Pearson Correlation and non-parametric Spearman correlation were used as indicated.
It was found that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which often are engaged in tight clusters with CD4+T cells. It was found that, in the breast cancer tumor microenvironment, CD4+T cells secreting type 1 (IFN-γ) and type 2 (mostly IL-13) cytokines were found. Immunofluorescence staining on frozen tissue sections revealed IL-13 expression on breast cancer cells. To demonstrate the link between breast cancer, DCs and CD4+T cell polarization NOD/SCID β2m−/− mice engrafted with human CD34+ hematopoietic progenitor cells (HPCs) and T cells from healthy volunteers and implanted with human breast cancer cell lines were used. There, breast cancer cells attract human DCs and imprint them to prime naïve CD4+T cells to secrete IL-13. CD4+T cells promote tumor development, which can be inhibited with IL-13 antagonists. Thus, breast cancer promotes skewed DC maturation to elicit pro-cancer immunity.
It has been found that breast cancer is rich in certain subsets of human DCs (Bell et al., 1999). These include large quantities of immature myeloid DC (mDC) subsets such as Langerhans cells and interstitial DCs. The presence of these cells per se might not be surprising. Indeed, it is the function of immature DCs to monitor epithelial surfaces. Therefore, a tumor might use the mechanisms of physiological tissue homeostasis such as infiltration with immature DCs. Interestingly, peri-tumoral areas of breast cancer tissue display mature DC-LAMP+ DCs, which under normal physiological conditions can only be found in lymphoid tissues. Presence of mature DC outside lymphoid organs is linked with inflammation and can be observed in aseptic synovial inflammation in rheumatoid arthritis (Radstake et al., 2005; Thomas et al., 1999) Radstake et al., 2005; (Gordon and Taylor, 2005) or in the blood of patients with systemic autoimmunity (Banchereau et al., 2004; Blanco et al., 2001). However, the immunological consequences of the presence of mature DC in tumors remain unknown.
The characteristics of CD4+T cells infiltrating breast cancer tissue samples from patients were determined. It was found that IL-13 is found in the breast cancer microenvironment and IL-13 staining on breast cancer cells. These observations prompted us to engage into mechanistic studies. However, studies in humans are hampered by limited availability of samples making it difficult to establish causative links. Therefore, a model of humanized mice was made in which (Palucka et al., 2003) breast cancer cell lines were also grafted into the a humouse. These mice may be immunodeficient nonobese diabetic/LtSz-scid/scid (NOD/SCID) β2 microglobulin-deficient (NOD/SCID/β2m−/−) mice transplanted with human CD34+ hematopoietic progenitor cells (CD34+HPCs) (Humouse). These mice develop the components of human innate immune system such as macrophages, all subsets of human DCs and component of human adaptive immune system, B cells (Palucka et al., 2003). Grafting autologous T cells permits us to analyze modulation of human T cell subsets. It is shown herein that DCs that infiltrate breast cancer tumors polarize naive CD4+T cells towards secretion of IL-13.
Microenvironment of breast cancer tumors from patients is rich in type 2 cytokines. Breast cancer tumors are infiltrated, in peri-tumoral areas, with mature DCs that are engaged in tight clusters with T cells (Bell et al., 1999) suggesting an ongoing immune response. To investigate the consequences of this interaction for breast cancer tumor microenvironment, the pattern of T cell cytokines in tumor biopsies from patients with breast cancer was analyzed. Samples from 21 patients were analyzed, which included in situ and invasive duct and/or mucinous carcinoma of the breast as well as lobular carcinoma. Whenever possible, tumor sites as well as surrounding tissue (macroscopically uninvolved) obtained from the same patient were analyzed. Single cell suspensions (
Analysis of single cell suspensions in the initial cohort of patients (Pt#1-6 in Table 1) demonstrated high levels of IL-2, IFN-γ and TNF in tumor samples, but not surrounding tissues (
Breast cancer tumors from patients are infiltrated with CD4+T cells secreting type 1 and type 2 cytokines. To determine whether T cells actually contribute to the production of cytokines detected in breast cancer microenvironment, the T cell composition and the cytokine expression pattern in single cell suspensions was determined. Flow cytometry indicate the prevalence of CD4+T cells (≧75%;
High level of IL-13 expression by breast cancer cells. Next, whether IL-13-expressing CD4+T cells are engaged in clusters with mature DCs infiltrating breast cancer tumors was analyzed. As shown in
Breast cancer tumors in Humouse are infiltrated with human myeloid DCs. To demonstrate the link between breast cancer, DCs and CD4+ T cell polarization an in vivo model of the human immune system and human breast cancer was developed and used. Sub-lethally irradiated adult NOD/SCID/β2m− mice were transplanted with human G-CSF mobilized CD34+HPCs isolated from the healthy volunteer blood apheresis. At 4 weeks later when these mice develop the components of the human immune system including DCs and B cells (Palucka et al., 2003), 107 human tumor cells were implanted subcutaneously (s.c.) into the flank. Three different breast cancer cell lines (Hs587T, MCF-7 and 1806) representing primary (Hs587T and 1806) and metastatic (MCF-7) tumors with different histopathological and phenotypic characteristics were tested (Table 2). At day 4, a clearly delineated tumor was measurable with three tested cell lines (
Single cell suspensions were prepared from tumors harvested at days 4 and 30 post-implant. Flow cytometry analysis was performed as illustrated in
Lymph nodes draining breast cancer tumors also showed DC infiltration as illustrated in
CD4+T cells promote development of breast cancer tumors. To determine whether CD4+T cells will be polarized similarly to the findings in patients, humanized mice bearing human breast cancer tumors were reconstituted with T cells by intratumoral injection. The T cells were isolated from the blood mononuclear cells of the CD34+HPCs donor and were thus autologous to the Antigen Presenting Cells (APCs) that had developed after CD34+HPC transplant in vivo but allogeneic to the implanted tumor cells.
Reconstitution of Hs578T breast cancer tumors with T cells isolated from the donor PBMC (both CD4+ and CD8+) resulted in accelerated tumor development (
Next, whether previously primed CD4+T cells can confer the acceleration of tumor development was analyzed. Humanized mice were constructed, Hs578T breast cancer tumors implanted and then injected with CD4+T cells isolated from the donor PBMC. At day 15, at the peak of breast tumor development, tumors were harvested, pooled from several mice and CD4+T cells were sorted. These in vivo primed CD4+T cells were then injected into “T cell naïve” Hs578T breast cancer tumors of recipient humanized mice constructed with CD34+HPCs from the same donor. Control mice received PBS. A single transfer of 1.5×106 in vivo primed CD4+T cells per mouse led to acceleration of breast cancer tumor development in three out of four tested mice in two independent experiments (mean tumor volume±SEM=51±17 in control mice that received PBS vs 167±27 in experimental mice that received T cells, p=0.06;
Increased tumor volume was associated with angiogenesis. Macroscopic analysis of Hs578T breast cancer tumors injected with PBS demonstrated at day 15 a clearly visible tumor and a small draining lymph node (
CD4+T cells require DCs to promote breast cancer tumor development. To determine whether CD4+T cells acted directly on breast cancer cells the effect of CD4+ cells in NOD/SCID/β2m− mice bearing breast cancer tumor in the absence of human immune cells (no CD34+HPCs transplant) was assessed. As shown in
CD4+T cells are polarized to secrete IL-13. Next, CD4+T cells were isolated from breast cancer tumors in humanized mice. It was found that the CD4+T cells were isolated from breast cancer tumors were polarized towards secretion of type 2 cytokines. CD4+T cells were sorted from tumors and their draining lymph nodes at day 15, activated with PMA and Ionomycin and cytokines were assessed in the supernatants. CD4+T cells secreted large amounts (>10 ng/ml) of IL-2 and IFN-γ but also IL-4, IL-13 and TNF (
DCs infiltrating breast cancer tumors polarize CD4+T cells to secrete type 2 cytokines. To further investigate the influence of tumor associated DCs on CD4+T cell function, human DCs from NOD/SCID/β2m− mice transplanted with CD34+HPCs and implanted with Hs578T breast cancer tumors were studied. DCs were isolated from breast cancer tumors, their draining lymph nodes, spleen and bone marrow 4 days after tumor implantation and tested for their ability to polarize naive allogeneic CD4+T cells in vitro. After 5 days, CD4+T cells were activated with PMA and lonomycin and cytokines were assessed in the supernatants. DCs isolated from all analyzed tissues induced allogeneic CD4+T cells to secrete large amounts of IL-2 and IFN-γ (>10 ng/ml, not shown). Furthermore, DCs isolated from both breast cancer tumors and particularly from their draining lymph nodes primed CD4+T cells to secrete high levels of TNF, IL-13, and IL-4 (mean concentration±SEM=4491±1599 pg/ml TNF; 7961±1342 pg/ml IL-13 and 3345±1508 pg/ml IL-4;
CD4+T cells promote tumor development via IL-13. It was found that human breast cancer tumors developed faster in the microenvironment of human CD4+T cells and skewed them to secrete IL-13. This together with earlier reports on an immunoregulatory role of IL-13 in cancer (Terabe et al., 2000) suggested that IL-13 might be involved. To establish this, humanized mice bearing Hs587T breast cancer tumors were treated with IL-13 antagonists, an antibody neutralizing IL-13 and a soluble IL-13R. It was found that mice reconstituted with T cells, and treated with isotype control, showed accelerated tumor development throughout three weeks follow up (
The presence of mature DCs outside lymphoid organ is associated with inflammation either septic as for example in infections or aseptic as for example in autoimmune diseases (Blanco et al., 2001; Radstake et al., 2005; Thomas et al., 1999). The present inventors had previously found infiltration of breast cancer tumors with mature DCs in patients (Bell et al., 1999). The immunological consequences of the presence of DCs in breast cancer tumor microenvironment was analyzed. It was found that CD4+T cells secreting type 1 and type 2 cytokines (predominantly IL-13) in tumor samples from patients with breast cancer. Because limited amount of tumor material that can be obtained from patients hampers causative studies, NOD/SCID β2m−/− mice engrafted with human CD34+ hematopoietic progenitor cells (HPCs) and implanted with human breast cancer cell lines were used to demonstrate the link between breast cancer, DCs and CD4+T cell polarization. Therefore, it is demonstrated herein that breast cancer cells attract human DCs and imprint them to polarize naïve CD4+T cells to secrete type 2 cytokines including IL-13. CD4+T cells promote early tumor development, and this is associated with enhanced angiogenesis. This pro-cancer effect can be prevented with IL-13 antagonists.
To established whether IL-13 is autocrine, as is the case in Hodgkin's disease (Kapp et al., 1999), or paracrine secreted by CD4+T cells and perhaps accessory cells such as mast cells, the source(s) of the IL-13 were explored. The source of IL-13 could influence the mechanism through which it would regulate tumor development in vivo (Fichtner-Feigl et al., 2006). For example, IL-13 could have a direct effect on breast cancer cells. Surprisingly, it was found that breast cancer cells in tumors from patients express IL-13. Earlier in vitro studies using breast cancer cell lines and recombinant IL-13 (as well as IL-4) demonstrated inhibition of estrogen-induced proliferation and acquisition of breast cancer marker, gross cystic disease fluid protein-15 (GCDFP-15) (Blais et al., 1996; Serve et al., 1996). IL-13 has also been indicated in the control of sex steroid biosynthesis from adrenal precursors (Gingras et al., 1999). In line with possible direct effect of IL-13 in vivo are recent findings on the expression of IL13Rα2 in highly aggressive variants of breast cancer with propensity to from lung metastasis (Minn et al., 2005). An indirect pathway may involve IL-13-mediated polarization of tumor infiltrating macrophages towards M2 cells (Sinha et al., 2005). These in turn promote angiogenesis (Mantovani et al., 2005) and/or secrete factors inhibiting anti-cancer effector function of CD8+T cells, for example TGF-β (Ghiringhelli et al., 2005; Li et al., 2005; Terabe et al., 2003) thereby amplifying tumor development.
Two patterns of cytokine expression by CD4+T cells were observed. One, with the presence of single positive CD4+T cells expressing either IL-13 or IFN-γ, consistent with the classical definition of type 2 polarization (Mosmann and Coffman, 1989). The pattern of expression suggests the bona fide Th1 and Th2 cells in breast cancer tumor microenvironment. The second pattern showed actually two subsets of IL-13-expressing CD4+T cells: single positive (bone fide Th2) and double positive IL-13 and IFN-γ. The latter pattern is reminiscent of IL-4 and IFN-γ expression observed in adult NKT cells (Kadowaki et al., 2001). These results raise a possibility that breast cancer tumors are infiltrated with NKT cells. However, since depletion of Vα24-expressing cells did not abolish induction of IL-13 in the vitro studies, a possible role of non-classical Vα24-negative NKT cells must be considered. Nevertheless, these results demonstrate the presence of two types of CD4+T cells secreting type 2 cytokines in breast cancer microenvironment.
The presence of type 2 cytokines together with TNF producing CD4+T cells resembles the pro-inflammatory type 2 responses induced by TSLP-primed DCs (Soumelis et al., 2002) and mediated via OX40 ligand (Ito et al., 2005). These molecules may be present in the microenvironment of breast cancer. An alternative pathway could include MUC-1 whose potential role in the attraction of myeloid DCs and Th2 polarization has been suggested in the in vitro studies (Carlos et al., 2005). Nevertheless, the priming of these CD4+T cells in vivo is dependent on antigen presentation by autologous DCs as transfer of CD4+T cells only did not promote tumor development in humanized mice. Other studies by the present inventors suggest that the effector phase, i.e., accelerated tumor development upon transfer of in vivo primed CD4+T cells to naïve mice, also depends on the presence of tumor infiltrating DCs autologous to T cells.
Prostate cancer tumors were established using PC3 cell line and reconstituted at days 3 and 6 with T cells (a mixture of CD4+ and CD8+T cells). Control mice received PBS at day 20 post-T cell transfer, tumors were harvested and put in overnight culture with PMA and lonomycin. As shown in
Breast cancer cells show IL-13 and phosphorylated STAT6 staining. To determine IL-13 expression in situ, breast cancer tissue sections were stained with specific mAb and analyzed by immunofluorescence. A very prominent and homogenous IL-13 staining of cancer cells was found (
Phosphorylation of STAT6 is considered as signature of IL-13 (and/or IL-4). All analyzed breast cancer tumor samples showed expression of STAT6 by cancer cells (
a and 13b shows lung tissue from tissue section from a lung tumor and
In conclusion, combining the studies of human cancer using ex vivo analysis of patient samples and in vivo analysis in the model of the human immune system and human cancer it is demonstrated herein that breast cancer attracts human DCs and imprints them to prime CD4+T cells into pro-cancer type 2 immunity. This can be prevented with IL-13 antagonists as target for therapy in cancers that depend on IL-13 production, e.g., breast cancer.
The model can be used at both basic and clinical level. At the basic level it will permit to determine the mechanisms tumors use to escape the immune system and to identify molecules the targeting of which might be used for therapy. At the clinical level the OncoHumouse will eventually permit us to design strategies to eliminate tumor cells through the manipulation of the immune system such as vaccination, antibody therapy, and adoptive transfer coupled or not to traditional chemotherapy regimens.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
This application claims priority to U.S. Provisional Application Ser. No. 60/831,984, filed Jul. 19, 2006, is a continuation-in-part of U.S. patent application Ser. No. 11/544,229 filed Oct. 6, 2006, the contents of each of which are incorporated by reference herein in their entirety. This application is related to U.S. Provisional Application Ser. No. 60,724,316, filed Oct. 6, 2005, the contents of which is incorporated by reference herein in its entirety.
This invention was made with U.S. Government support under Contract No. RO-1 CA89440, R21AI056001, U19 AIO57234, RO-1 CA78846 and CA85540 awarded by the NIH. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60831984 | Jul 2006 | US | |
| 60724316 | Oct 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11544229 | Oct 2006 | US |
| Child | 11687641 | Mar 2007 | US |
