Patent Description:
The present invention generally relates to compositions for use in treating cancer.

Cancer is the deadliest disease that kills millions of people every year in this world. Understanding mechanisms by which cancer cells escape death and identifying the associated therapeutic target are important areas of research. Suppression of cell-mediated inflammation (<NUM>) is believed to be one of the major reasons for the persistence and progression of this fatal disease. However, mechanisms by which this high level of suppression is maintained and tumor cells escape death are poorly understood. Since IL-<NUM> heterodimer is the most important cytokine in terms of cell-mediated immunity (<NUM>), this molecule is always under scanner for the treatment of cancer (<NUM>, <NUM>). IL-<NUM> family of cytokines has four different members including IL-12p40 monomer (p40), IL-12p40 homodimer (p40<NUM>), IL-<NUM> heterodimer (p40:p35), and IL-<NUM> heterodimer (p40:p19). In this era of science, where heterodimers rule, only IL-<NUM> heterodimer and IL-<NUM> heterodimer were thought to have biological functions. As a result, IL-12p40 monomer and IL-12p40 homodimer were considered as nonfunctional members of the IL-<NUM> family (<NUM>). However, we have demonstrated the proinflammatory property of IL-12p40 homodimer (<NUM>-<NUM>) and delineated that biological functions of IL-12p40 homodimer are different from that of IL-<NUM> heterodimer and IL-<NUM> heterodimer (<NUM>, <NUM>). Furthermore, after raising separate functional blocking monoclonal antibodies (mAb) and ELISA against each of mouse IL-12p40 homodimer and IL-12p40 monomer (<NUM>), we have delineated that mAb against IL-12p40 homodimer protects mice against EAE (<NUM>).

<NPL>, discloses antibody C17. <NUM> as able to inhibit liver metastasis of a mouse TK-<NUM> tumour model.

Here, we demonstrate that different forms of cancer cells except the lung cancer one are associated with specific elevation of IL-12p40 monomer. Selective ablation of IL-12p40 monomer by mAb stimulates cell death in different cancer cells and in vivo in TRAMP tumor tissue. Furthermore, IL-12p40 monomer suppresed the caveolin-mediated internalization of IL-12Rβ1 and associated IL-<NUM> heterodimer signaling that were neutralized by IL-12p40 monomer mAb. These results delineate a novel pathogenic role of IL-12p40 monomer, in which it helps cancer cells to evade cell death.

The present invention provides a composition comprising a therapeutically effective amount of an antibody against IL-12p40 monomer for use in treating cancer, wherein the antibody neutralizes the IL-12p40 monomer, does not significantly neutralize the action of IL-12p40 homodimer, and does not bind IL-<NUM> heterodimer, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, liver cancer, colon cancer, ovarian cancer and pancreatic cancer. In one embodiment, the antibody suppresses inhibition of IL-<NUM> heterodimer signaling. In another embodiment, the antibody upregulates production of IFN-γ. In another embodiment, the antibody thereof at least reduces the internalization of IL-12Rβ1 via a caveolin-mediated pathway.

The antibody thereof may be a monoclonal antibody or an immunologically active fragment of a monoclonal antibody. In other embodiments, the antibody is a polyclonal, monoclonal, human, humanized, and chimeric antibody; a single chain antibody or an epitope-binding antibody fragment of such an antibody. In another embodiment, the antibody is a humanized antibody or an immunologically active fragment thereof.

The cancer may be, for example, prostate cancer, breast cancer or liver cancer. In another embodiment, the cancer is characterized by excess production of IL-12p40 monomer.

In one embodiment, the composition also includes at least one pharmaceutically acceptable carrier. The composition may be administered orally. In other embodiments, the composition is administered by a subcutaneous, intra-articular, intradermal, intravenous, intraperitoneal or intramuscular route. In yet other embodiments, the subject is a human subject.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

The uses of the terms "a" and "an" and "the" and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as", "for example") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

The term "therapeutic effect" as used herein means an effect which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder, for example restenosis, of a human or veterinary patient. The term "therapeutically effective amount" as used with respect to a drug means an amount of the drug which imparts a therapeutic effect to the human or veterinary patient.

The term "internalization" as used herein means a process in which molecules, such as proteins, are engulfed by the cell membrane and drawn into the cell.

The term "antibody" herein is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments etc., so long as these fragments exhibit the desired immunological activity.

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to embodiments, some of which are illustrated in the drawings, and specific language will be used to describe the same. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates. In the discussions that follow, a number of potential features or selections of assay methods, methods of analysis, or other aspects, are disclosed. It is to be understood that each such disclosed feature or features can be combined with the generalized features discussed, to form a disclosed embodiment of the present invention.

One aspect of the invention provides a composition comprising a therapeutically effective amount of an antibody against IL-12p40 monomer for use in treating cancer, as defined in the present claims, for example prostate cancer. Prostate cancer is the most common form of male cancer that develops in the prostate in elderly people. Since the impairment of immune system is also very common among elderly population, several immunotherapies including activation of tumor-specific T cells, inflammatory cytokine production, and the activation of antigen-presenting cells are possible approaches to fight against this deadly disease (<NUM>).

However, the present composition for use in treating cancer is not limited to the treatment of prostate cancer. The composition for use is applicable to the treatment of ovarian cancer, prostate cancer, liver cancer, colon cancer, breast cancer and pancreatic cancer. In preferred embodiments, the method is applicable to cancers characterized by an overproduction of IL-12p40 monomer.

IL-<NUM> heterodimer is the most important cytokine that triggers cell-mediated immune response. IL-<NUM> heterodimer consists of a heavy chain (p40) and a light chain (p35) linked covalently by disulfide bonds to give rise to the so-called bioactive heterodimeric (p70) molecule (<NUM>). It is produced mainly by antigen presenting cells upon activation through Toll-like receptors and by interactions with CD4+ T cells (<NUM>, <NUM>). Eventually, IL-12p40 monomer has been shown to pair with p19 to form a newly discovered cytokine, IL-<NUM> heterodimer (<NUM>). Either p19 or p35 is constitutively expressed in many cell types. It is known that dendritic cells and macrophages, cells which are able to secrete heterodimeric IL-<NUM> or IL-<NUM>, always produce an excess of IL-12p40 as monomer (p40) or homodimer (p40<NUM>) (<NUM>). However, the biological functions of IL-12p40 homodimer and p40 monomer have remained unknown.

The results presented herein demonstrate that, in many different cancers, cancer cells produce excess IL-12p40 monomer as compared to IL-12p40 homodimer, IL-<NUM> heterodimer and IL-<NUM> heterodimer and that IL-12p40 monomer is involved in cancer cell survival. This conclusion is based, in part, on the following observations: First, the level of IL-12p40 monomer was much higher in TRAMP, 4T1 and Hepa cells as compared to IL-12p40 homodimer, IL-<NUM> heterodimer and IL-<NUM> heterodimer. This selective increase in IL-12p40 monomer was not observed in this specific KLN lung cancer cell line, indicating the specificity of this finding. However, this lack of a selective increase in IL-12p40 monomer may not be a general feature of lung cancer. Second, neutralization of IL-12p40 monomer, but not IL-12p40 homodimer, induced the release of LDH in TRAMP, 4T1, and Hepa cells. Alternatively, mAb against IL-12p40 monomer, but not IL-12p40 homodimer, reduced MTT metabolism in TRAMP, 4T1 and Hepa cells. Again, IL-12p40 monomer mAb had no effect on LDH and MTT in KLN cells. Third, t-type calcium influx, a growth supportive event in cancer cells, was significantly reduced in TRAMP, 4T1 and Hepa, but not KLN, cells when treated with IL-12p40 monomer neutralizing antibody. Fourth, TUNEL and Annexin-V staining experiments displayed more death in TRAMP, 4T1 and Hepa, but not KLN, cells after treatment with IL-12p40 monomer mAb. Finally, intraperitoneal injection of IL-12p40 monomer mAb, but not IgG, significantly reduced the size of the prostate tumors grown in the flank of male C57BL/<NUM> mice. This is an unexpected result, demonstrating a biological role of IL-12p40 monomer, the so-called non-functional member of the IL-<NUM> family, in cancer cell survival. Furthermore, these results indicate the possible therapeutic prospect of the IL-12p40 monomer neutralization in various cancers.

While investigating mechanisms behind IL-12p40 monomer-mediated killing of tumor cells, the inventors observed upregulation of IFNγ, a major cytotoxic inflammatory cytokine (<NUM>-<NUM>), by IL-12p40 monomer mAb in pure TRAMP cells. This observation is striking as T lymphocytes (<NUM>) and natural killer cells (<NUM>) are considered as primary sources of IFN-γ. However, previous literatures demonstrate that it can be produced by murine macrophages (<NUM>) as well as epithelial cells (<NUM>, <NUM>), prompting the inventors to examine IFN- γ production in TRAMP epithelial cells. TRAMP cells expressed very low amount of IFNγ in unstimulated condition and IL-12p40 monomer mAb treatment stimulated the production of IFNγ by several fold. Moreover, cancer cells with epithelial origin such as 4T1 and hepatocellular origin such as Hepa also expressed significant amount of IFNγ when treated with IL-12p40 monomer mAb further suggesting that functional blocking of IL-12p40 monomer could stimulate IFNγ production in a wide range of cancer cells. Consistent with the cytotoxic nature of IFNγ, neutralization of this molecule abrogated IL-12p40 monomer mAb-mediated death of TRAMP, 4T1 and Hepa cells, demonstrating that IL-12p40 monomer mAb induces death of cancer cells via IFNγ.

Activation of the IL-<NUM> signaling pathway plays a critical role in the induction of IFNγ in various cells (<NUM>). Interaction of IL-<NUM> heterodimer and its receptor IL-12R in the plasma membrane triggers the activation of Janus family of tyrosine kinases that in turn phosphorylates the tyrosine residues of signal transducer and activator of transcription <NUM> and <NUM> (STAT3 and STAT4). These tyrosine phosphorylations are responsible for the formation of STAT4/ STAT4 homodimer and STAT3/ STAT4 heterodimers. These dimers then translocate to the nucleus and bind to IFNγ promoter for the transcription of IFNγ (<NUM>).

Accordingly, IL-12p40 monomer mAb stimulated the production of IL-<NUM> heterodimer in TRAMP cells, suggesting that the absence of IL-12p40 monomer may favor the interaction of IL-<NUM> heterodimer with IL-12R to turn on the positive autoregulatory effect of IL-<NUM> (<NUM>) in these cells. A successful interaction between IL-<NUM> heterodimer and IL-12R transmits the downstream signal and then internalizes the receptor inside the cell. On the other hand, an unsuccessful interaction between IL-<NUM> heterodimer and IL-12R leaves the receptor arrested in the membrane, which is unable to transmit any downstream signaling cascade. IL-12p40 monomer treatment increased the membrane localization of IL-12Rβ1 and IL-12p40 monomer mAb decreased the level of IL-12Rβ1 in the membrane. On the other hand, either IL-12p40 monomer or IL-12p40 monomer mAb did not have any effect on the internalization of IL-12Rβ2. These results demonstrate that IL-12p40 monomer is involved in the membrane arrest of IL-12Rβ1, but not IL-12Rβ2. To further explore the mechanism, the inventors examined whether IL-12p40 monomer mAb-mediated internalization of IL-12Rβ1 in TRAMP cells was dependent on clathrin or caveolin. In this case, caveolin, but not clathrin, was found to be involved in IL-12p40 monomer mAb mediated membrane internalization of IL-12Rβ1.

It has been reported that IL-12p40 homodimer is an antagonist of IL-<NUM> heterodimer as the former competes with the latter for binding to IL-12Rβ1 (<NUM>). On the other hand, IL-12p40 monomer reportedly does not have any IL-<NUM> heterodimer-antagonizing activity and binds IL-12Rβ1 very weakly (<NUM> to <NUM> times less potent compared to p402) (<NUM>). In contrast to these reports, the results presented herein from TRAMP cells suggest that it is IL-12p40 monomer, but not IL-12p40 homodimer, that antagonizes IL-<NUM> heterodimer signaling via suppressing caveolin-mediated internalization of IL-12Rβ1. Therefore, this is a paradigm shift of knowledge. Since neutralization of IL-12p40 monomer by IL-12p40 monomer mAb reinstalls the internalization of IL-12Rβ1 and induces death via IL-<NUM>-mediated production of IFN-γ, IL-12p40 monomer mAb may have therapeutic efficacy in prostate and other cancers, which are associated with excessive production of IL-12p40 monomer.

In one aspect, the invention provides a composition for use in treating a cancer in a human or veterinary subject, as defined in the present claims. In various embodiments, the antibody is, for example, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a Fab fragment, a Fab' fragment, a F(ab)<NUM> fragment, or a single chain Fv (scFv) fragment. In other embodiments, the antibody is linked to another molecule to form an immunoconjugate molecule. For example, the antibody may be linked to a cytotoxic agent, possibly through a linker. The antibody encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Also disclosed herein are pharmaceutical compositions including at least one antibody to IL-12p40 monomer. For example, the pharmaceutical composition may include <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more of such antibodies. The antibody can be administered in combination with another therapeutic substance. For example, it may be combined with a cytotoxic drug or other anti-cancer agent.

The pharmaceutical compositions can be in the form of, for example, tablets, pills, dragees, hard and soft gel capsules, granules, pellets, aqueous, lipid, oily or other solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, alixiers, solid emulsions, solid dispersions or dispersible powders. In pharmaceutical compositions for oral administration, the agent may be admixed with commonly known and used adjuvants and excipients, for example, gum arabic, talcum, starch, sugars (such as, e.g., mannitose, methyl cellulose, lactose), gelatin, surface-active agents, magnesium stearate, aqueous or non-aqueous solvents, paraffin derivatives, cross-linking agents, dispersants, emulsifiers, lubricants, conserving agents, flavoring agents (e.g., ethereal oils), solubility enhancers (e.g., benzyl benzoate or benzyl alcohol) or bioavailability enhancers (e.g. GELUCIRE). In the pharmaceutical composition, the agent may also be dispersed in a microparticle, e.g. a nanoparticulate, composition.

For parenteral administration, the agent or pharmaceutical compositions of the agent can be dissolved or suspended in a physiologically acceptable diluent, such as, e.g., water, buffer, oils with or without solubilizers, surface-active agents, dispersants or emulsifiers. As oils for example and without limitation, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil may be used. More generally, for parenteral administration the agent or pharmaceutical compositions of the agent can be in the form of an aqueous, lipid, oily or other kind of solution or suspension or even administered in the form of liposomes or nano-suspensions.

The IL-12p40 monomer antibody or pharmaceutical compositions including the antibody can be administered by any method that allows for the delivery of a therapeutic effective amount of the agent to the subject. Modes of administration can include, but are not limited to oral, topical, transdermal and parenteral routes, as well as direct injection into a tissue and delivery by a catheter. Parenteral routes can include, but are not limited to subcutaneous, intradermal, intra-articular, intravenous, intraperitoneal and intramuscular routes. In one embodiment, the route of administration is by topical or transdermal administration, such as by a lotion, cream, a patch, an injection, an implanted device, a graft or other controlled release carrier. Routes of administration include any route which directly delivers the composition to the systemic circulation (e.g., by injection), including any parenteral route. Alternatively, administration can be by delivery directly to the affected tissue.

One embodiment of the composition for use according to the invention comprises administering at least one IL-12p40 monomer antibody, in a dose, concentration and for a time sufficient to prevent the development of, or to lessen the extent of a cancer, for example, any of the cancers mentioned above. Certain embodiments include administering systemically the IL-12p40 monomer antibody in a dose between about <NUM> micrograms and about <NUM> milligrams per kilogram body weight of the subject, between about <NUM> micrograms and about <NUM> milligrams per kilogram body weight of the subject, between about <NUM> micrograms and about <NUM> milligram per kilogram body weight of the subject. In practicing this method, the IL-12p40 monomer antibody or therapeutic composition containing the agent can be administered in a single daily dose or in multiple doses per day. Treatment may require administration over extended periods of time. The amount per administered dose or the total amount administered will be determined by the physician and will depend on such factors as the mass of the patient, the age and general health of the patient and the tolerance of the patient to the compound.

Embodiments of the invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Animals and Regents: All murine cancer cell lines were purchased from ATCC. Male C57 BL/<NUM> mice (Harlan) were used for this study. Mouse IL-12p40 monomer (cat# <NUM>) and p70 (cat# <NUM>) were purchased from BD Biosciences. Mouse IL-12p40 homodimer (cat# <NUM>-ML) was purchased from R&D. Hamster IgG (cat# IR-HT-GF) was obtained from Innovative Research. Mouse IgG (cat# sc-<NUM>) was purchased from Santa Cruz Biotechnology. Chloropromazine (cat # C8138), filipin (cat# F9765), MTT assay kit (cat# CGD1), and LDH assay kit (cat# TOX7) were purchased from Sigma. IFNγ neutralizing antibody (cat# <NUM>-<NUM>-<NUM>) was purchased from eBioscience. TUNEL assay kit (cat# QIA39) was purchased from Calbiochem and Annexin V assay kit (cat# K101-<NUM>) was purchased from Biovision.

Sandwich ELISA: Sandwich ELISA was used to quantify mouse IL-12p40 homodimer and IL-12p40 monomer as described by us (<NUM>, <NUM>). Briefly, for IL-12p40 homodimer, mAb a3-1d (<NUM>/mL) was diluted <NUM>:<NUM> and added to each well (<NUM>µL/well) of a <NUM>-well ELISA plate for coating. The biotinylated IL-12p40 homodimer mAb d7-12c (<NUM>/mL) was diluted <NUM>:<NUM> and used as detection antibody. Similarly for IL-12p40 monomer, mAb a3-3a (<NUM>/mL) and biotinylated IL-12p40 monomer mAb a3-<NUM> (<NUM>/mL) were also diluted <NUM>:<NUM> and used as coating and detection antibodies, respectively (<NUM>). Concentrations of IFN-γ, IL-<NUM> heterodimer and IL-<NUM> were measured in serum free supernatants of different treatment groups by ELISA (eBioscience), according to the manufacturer's instructions.

MTT and LDH assays: These assays were performed as described by Jana. (<NUM>) and Khasnavis.

Tumor development and measurement: Animal maintaining and experiments were in accordance with National Institute of Health guidelines and were approved by the Institutional Animal Care and Use committee (IACUC#<NUM>-<NUM>) of the Rush University of Medical Center, Chicago, IL. Tumors were generated subcutaneously in male C57 BL/<NUM> mice. Mice were injected with <NUM> ×<NUM> TRAMP-C2 cells in their flank for tumor generation. Mice were maintained in our temperature-controlled animal vivarium with adequate food and water. Tumor growth was measured with a caliper and tumor cross-sectional area was determined with the formula (mm2 = longest diameter X shortest diameter). Treatment with IL-12p40 monomer mAb started when the tumor sizes reached <NUM>-1cm2. The IL-12p40 monomer mAb a3-3a was injected once a week intraperitonially in <NUM> volume of sterile PBS-<NUM>% normal mouse serum. The tumors were then measured to determine regression or progression. Infrared dye (Alexa <NUM>-conjugated 2DG dye; Licor) was injected via tail-vein on the day before imaging analysis. Mice were sacrificed at the end of the study and tumor tissues were collected appropriately for western blot, mRNA expression and immunohistochemical analysis.

Tissue preparation and Immunohistochemistry: Paraffin embedded tissue sections were prepared and tissue sections were cut <NUM> micron in size. To eliminate endogenous peroxidase activity, tissue sections were deparaffinized, rehydrated and incubated with <NUM>% H<NUM>O<NUM> in methanol for <NUM> at room temperature. Antigen retrieval was performed at <NUM> for <NUM> by placing the slides in <NUM> sodium citrate buffer (pH <NUM>). After blocking, the slides were then incubated with the primary antibodies (Table <NUM>) at <NUM> room temperature followed by washing and incubation with Cy2, Cy3 or Cy5 (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies at RT for <NUM>. Mouse IgG was used as an isotype control (<NUM>).

TUNEL assay: Following treatments with mAb against either IL-12p40 monomer or IL-12p40 homodimer, TUNEL assays were performed as described by Corbett GT et al.

Semi-quantitative RT-PCR: Total RNA was isolated and semi-quantitative RT-PCR analyses for IFNγ, IL-<NUM>, T-bet, GATA3, FoxP3, and GAPDH were performed as described by Brahmachari S et al. (<NUM>); Jana M et al. (<NUM>) and Corbett GT et al. (<NUM>) using primers (Table <NUM>).

Real-time quantitative PCR: The mRNA quantification was performed using the ABI-Prism7700 sequence detection system (Applied Biosystems) using SYBR GREEN (Applied Biosystems) as described by Brahmachari S et al. (<NUM>); Jana M et al. (<NUM>) and Corbett GT et al. The mRNA expressions of respective genes were normalized to the level of GAPDH mRNA. Data were processed by the ABI Sequence Detection System <NUM> software and analyzed by ANOVA.

FACS: Surface expression of IL-12Rβ1 and IL-12Rβ2 were monitored as described by Brahmachari S et al. Briefly, after treatment, cells were incubated for <NUM> with Accutase (BD Bioscience) for detachment of adherent cells. After washing with FACS buffer, cells were incubated with PEtagged IL-12Rβ1 and IL-12Rβ2 antibodies at <NUM> for <NUM>. For intracellular staining, permeabilization was done before incubation with IL-12p40 monomer and IL-12p40 homodimer mAbs. APC-conjugated antihamster secondary antibodies were used. After washing, the cells were analyzed through FACS (BD Biosciences, San Jose, CA). Cells were gated based on morphological characteristics. Apoptotic and necrotic cells were not accepted for FACS analysis.

Membrane isolation: After treatment, cells were scraped in PBS and cell pellets were dissolved in homogenization buffer (<NUM> sucrose, <NUM> EDTA, <NUM> Tris-HCl, pH <NUM>, protease inhibitors and phosphatase inhibitors) and then homogenized with hand homogenizer. Cell debris was removed by centrifugation at <NUM> for <NUM> at <NUM> followed by centrifugation of supernatant at <NUM>,<NUM> for <NUM>. Supernatants were discarded and the pellet containing membrane fractions were dissolved in SDS-PAGE sample buffer.

Immunoblot analyses: Immunoblot analyses were performed as described by Jana M et al. (<NUM>) Khasnavis S (<NUM>) and Corbett GT et al. (<NUM>) using different primary antibodies (Table <NUM>).

Statistical Analysis: For tumor regression, quantitative data were presented as the mean ± SEM. Statistical significances were accessed via one-way ANOVA with Student-Newman-Keuls posthoc analysis. Other data were expressed as means ± SD of three independent experiments. Statistical differences between means were calculated by the Student's t-test. A p- value of less than <NUM> (p<<NUM>) was considered statistically significant.

To understand the role of IL-<NUM> family of cytokines in cancer, at first, we monitored the level of these cytokines in different cancer cell lines. It was not possible to examine the role of IL-12p40 monomer and IL-12p40 homodimer in the pathogenesis of any disease due to the unavailability of specific functional blocking monoclonal antibodies (mAb). Therefore, we have generated neutralizing mAbs against each of IL-12p40 monomer and IL-12p40 homodimer and developed ELISA to monitor these cytokines separately (<NUM>). The quantification analyses were performed in different adherent mouse cancer cells including squamous (KLN), prostate (TRAMP), breast (4T1), and liver hepatoma (Hepa) cell lines. Cells were cultured under serum free condition for <NUM> hrs followed by measuring the levels of IL-12p40 monomer, IL-12p40 homodimer, IL-<NUM> heterodimer, and IL-<NUM> heterodimer by sandwich ELISA. In general, the levels of IL-<NUM> heterodimer and IL-<NUM> heterodimer were very low as compared to IL-12p40 monomer and IL-12p40 homodimer in each of these cell lines (<FIG>). The level of IL-12p40 monomer was much higher than IL-12p40 homodimer, IL-<NUM> heterodimer or IL-<NUM> heterodimer in TRAMP, 4T1 and Hepa cells (<FIG>). However, levels of IL-12p40 monomer and IL-12p40 homodimer were almost same in KLN lung cancer cells (<FIG>), suggesting the specificity of the effect. To confirm the presence of IL-12p40 monomer in cancer cells, we adopted different techniques. First, we monitored the level of IL-12p40 monomer in the supernatants of TRAMP cells by native PAGE analysis (<FIG>). Coomassie staining of native PAGE and comparing the band with pure monomeric IL-12p40 monomer protein (extreme left lane) clearly indicates the presence of a <NUM> kDa protein as the major secretory molecule in TRAMP cells (<FIG>). Second, we performed native immunoblot analysis of supernatants of TRAMP cells with our specific IL-12p40 monomer monoclonal antibody (p40 mAb) a3-3a and found the presence of IL-12p40 monomer in supernatants of TRAMP cells (<FIG>). Finally, intracellular FACS analyses with IL-12p40 monomer mAb a3-3a and IL-12p40 homodimer mAb a3-1d show that the level of IL-12p40 monomer was significantly higher than IL-12p40 homodimer in TRAMP cells (<FIG>).

Next, we measured the level of IL-12p40 monomer in different human cancer cell lines. Interestingly, our ESI-MS analyses (<FIG>) in the supernatants clearly indicated that both human hepatoma Hep3B and prostate LNCaP cells expressed significantly higher level of IL-12p40 monomer than IL-<NUM> heterodimer (<FIG>). Furthermore, native PAGE analyses followed by coomassie staining of supernatants along with IL-12p40 monomer standard protein demonstrated that Hep3B, LNCaP and human breast MCF-<NUM> cancer cells produced significant level of IL-12p40 monomer (<FIG>). Immunoblot analyses of different supernatants with anti-human pan IL-12p40 monomer/p70 antibody also showed that all three human cancer cells produced a higher level of IL-12p40 monomer compared to other IL-<NUM> cytokines (<FIG>). Together, our results suggest that IL-12p40 monomer is produced in excess by a wide spectrum of cancer cells.

Since among the IL-<NUM> family members, the level of IL-12p40 monomer was the highest in most of the cancer cells, we examined its role in growth and survival of cancer cells. It is often quite straightforward to consider a knock out mouse model to investigate the role of a molecule in any disease process. However, we cannot use IL-12p40 monomer (-/-) mice in this case because knocking out the p40 gene will knock down IL-<NUM> heterodimer, IL-<NUM> heterodimer, IL-12p40 homodimer, and IL-12p40 monomer. Therefore, to investigate the role of IL-12p40 homodimer and IL-12p40 monomer in life and death of different cancer cells, the only feasible approach is to use neutralizing monoclonal antibodies against these molecules. The IL-12p40 monomer mAb a3-3a, but not IL-12p40 homodimer mAb a3-1d, increased release of LDH <FIG> (A-D) and decreased MTT <FIG> (E-H) in TRAMP <FIG> (B & F), 4T1 <FIG> (C & G) and Hepa <FIG> (D & H) cells. On the other hand, IL-12p40 monomer mAb had not effect on either LDH or MTT in KLN lung cancer cells, indicating the specificity of the effect. To monitor death in tumor cells from another angle, we measured calcium influx through t-type calcium channel. Treatment of different cancer cells with IL-12p40 monomer, but not IL-12p40 homodimer, mAb displayed a reduced t-type calcium influx in TRAMP (<FIG>), 4T1 (<FIG>) and Hepa (Fig. <NUM>) cells. Again, IL-12p40 monomer mAb remained unable to modulate t-type calcium influx in KLN cancer cells (<FIG>). Accordingly, TUNEL (<FIG>) and Annexin V-labeling (<FIG>) followed by the quantitative analyses (<FIG>) reiterated that neutralization of IL-12p40 monomer, but not IL-12p40 homodimer, stimulated death in TRAMP, 4T1 and Hepa cancer cells. However, p40 mAb had no effect on the apoptosis of KLN cancer cells. Together, these results suggest that specific ablation of IL-12p40 monomer, but not IL-12p40 homodimer, stimulates the death response in prostate, breast and liver tumor cells, without altering the survival of lung cancer cells.

Specific neutralization of IL-12p40 monomer induces the regression of tumor growth and stimulates the death response in vivo in TRAMP tumor tissues. Next, we examined the effect of IL-12p40 monomer mAb on tumor size and the death of tumor tissue in vivo when TRAMP cells were grown as a tumor in the flank of male C57BL/<NUM> mice. Once tumor reached <NUM> to <NUM> size, mice were treated with IL-12p40 monomer mAa3-3a at a dose of <NUM>/Kg body weight via intraperitoneally twice a week for <NUM> weeks. The tumor size was recorded every alternate day after treatment of IL-12p40 monomer mAb. Animals that received IgG were analyzed as negative controls. Control animals did not receive any antibody. After two weeks, tumors were labeled with infrared dye <NUM> conjugated <NUM> deoxy D glucose (IRDye800 2DG) via tail vein injection and then imaged in Licor Odyssey infrared scanner. Interestingly, we observed that administration of IL-12p40 monomer mAb significantly reduced the size of tumors as evident from whole animal infrared images (<FIG>) and pictures of excised tumors (<FIG>).

From the tumor regression curve, it was clear that the size of tumors in IL-12p40 monomer mAb-treated group decreased steadily and significantly as compared to both control and IgG-treated group (<FIG>). Next, we monitored apoptosis in these tumor tissues. Our TUNEL results clearly showed that the population of TUNEL-positive dead cells in the IL-12p40 monomer mAb-treated tumors was higher than either control or IgG-treated tumors (<FIG>), suggesting that the neutralization of IL-12p40 monomer by IL-12p40 monomer mAb is capable of inducing apoptosis in tumor tissues. To further confirm this finding, we monitored the mRNA expression of different apoptotis-related genes in treated and untreated tumor tissues using custom gene array. Gene array (<FIG>) followed by real-time PCR analysis of individual genes (<FIG>) clearly indicated that p40 mAb treatment significantly elevated the expression of apoptotis-related different genes such as caspases <NUM>, caspase <NUM>, caspase <NUM>, caspase <NUM>, BAD, BID, cytochrome C, BAK, and p53. Taken together, these results suggest that the neutralization of IL-12p40 monomer induces apoptosis in vivo in prostate tumor cells.

Next, we investigated mechanism by which IL-12p40 monomer mAb induced death response in cancer cells. Induction of IFN-γ production is a proven therapeutic strategy to induce cytotoxicity in cancer cells (<NUM>). Therefore, we examined if IL-12p40 monomer mAb treatment is capable of upregulating the expression of IFN-γ in TRAMP tumor cells. We observed that IL-12p40 monomer mAb, but not IgG, significantly upregulated the mRNA expression of IFN-γ in cultured TRAMP cells (<FIG>). Although IFN-γ is a Th1 cell cytokine, our ELISA results (<FIG>) and immunocytochemical analysis (<FIG>) clearly indicated that IL-12p40 monomer mAb-treatment increased the level of IFNγ in TRAMP cells. On the other hand, IL-12p40 monomer mAb treatment (<FIG>) decreased the level of IL-<NUM>, an anti-inflammatory cytokine that is known to support the growth of cancer cells (<NUM>). Next, we investigated if the elevated expression of IFNγ in p40 mAb-treated TRAMP cells was indeed involved in the cell death. Therefore, we treated TRAMP cells with IFNγ neutralizing antibody either alone or together with p40 mAb. TUNEL (<FIG>), LDH (<FIG>), and MTT (<FIG>) assays revealed that IFNγ neutralizing antibody abrogated IL-12p40 monomer mAb mediated cell death in TRAMP cells. These results were specific as IgG was unable to protect the IL-12p40 monomer mAb-mediated cell death in TRAMP cells (<FIG>). Other tumor cells including Hepa (<FIG>) and 4T1 (Fig. S2C-D) also displayed upregulated expression of IFNγ. Similar to TRAMP cells, our MTT viability assay (<FIG> & <FIG>) and LDH release assay (<FIG> & <FIG>) revealed that IL-12p40 monomer mAb, but not IgG, significantly stimulated death in both Hepa and 4T1 tumor cells, suggesting that neutralization of IL-12p40 monomer could be crucial in inducing death of different tumor cells.

When analyzing the level of IFNγ in tumor tissue, we also observed that similar to cell culture data, IL-12p40 monomer mAb-treated tumor tissue expressed more IFNγ mRNA (<FIG>) and protein (<FIG> & <FIG>) compared to control and IgG-treated tumors. Moreover, the expression of T-bet, IFNγ inducing transcription factor, was also found to be upregulated in the tumor of IL-12p40 monomer mAb-treated, but not control and IgG-treated, mice (<FIG>). Since the upregulation of IL-<NUM> (<NUM>) and the regulatory T cell marker Foxp3 (<NUM>) are believed to inhibit the cytotoxic effects in cancer cells, we also monitored these molecules in tumor tissue. Interestingly, the expression of IL10, GATA-<NUM> and Foxp3 decreased in IL-12p40 monomer mAb treated tumors as compared to control and IgG treated tumor (<FIG>). These results suggest that neutralization of IL-12p40 monomer is capable of inducing cell-mediated immunity and down-regulating humoral immunity and Tregs in cultured TRAMP cells and in vivo in TRAMP tumor tissue.

The upregulation of IFN-γ is achieved by the activation of IL-<NUM> signaling pathway (<NUM>, <NUM>). Since IL-12p40 monomer mAb increased the production of IFN-γ and induced death in TRAMP cells via IFN-γ, we investigated the involvement of IL-<NUM> heterodimer in these processes. The production of IL-<NUM> heterodimer markedly increased in IL-12p40 monomer mAb-treated TRAMP cells (<FIG>) and in vivo in tumor tissue (<FIG>) as compared to control and IgG-treatment, suggesting that IL-<NUM> signaling pathway could be involved in L-12p40 monomer mAb-mediated IFN-γ production and cell death. We found that neutralization of IL-<NUM> heterodimer by functional blocking antibodies suppressed IL-12p40 monomer mAb-induced production of IFN-γ in TRAMP cells (data not shown). Furthermore, neutralizing antibodies against IL-<NUM> abrogated IL-12p40 monomer mAb-mediated death of TRAMP cells as indicated MTT (<FIG>) and LDH release (<FIG>). These results suggest that neutralization of IL-12p40 monomer induces IFN-γ and cell death in cancer cells via IL-<NUM> signaling pathway.

The IL-<NUM> signaling pathway is initiated by the interaction between IL-<NUM> heterodimer and IL-<NUM> receptor, which is a heterodimer of IL-12Rβ1 and IL-12Rβ2. A functional IL-<NUM> receptor has been reported to be internalized after successful binding with its ligand IL-<NUM> heterodimer (<NUM>), otherwise it stays arrested in the membrane. Therefore, we examined if IL-12p40 monomer was involved in the arresting of IL-<NUM> receptor in TRAMP cells in order to negate the IL-<NUM> signaling pathway. Our FACS analyses revealed that the treatment with IL-12p40 monomer, but neither IL-12p40 homodimer nor p70, increased the surface expression of IL-12Rβ1 in TRAMP cells (<FIG>). On contrary, IL-12p40 monomer did not have any effect on the surface expression of IL-12Rβ2 (<FIG>). Furthermore, treatment with IL-12p40 monomer mAb, but not IgG, down-regulated the membrane level of IL-12Rβ1 (<FIG>), suggesting the involvement of IL-12p40 monomer in the arresting of IL-12Rβ1 on the membrane. To further confirm, we performed immunoblot analyses of IL-12Rβ1 in the membrane fraction of TRAMP cells treated with IL-12p40 monomer, IL-12p40 homodimer, or p70, separately. Interestingly, we found that treatment with IL-12p40 monomer, but neither IL-12p40 homodimer nor IL-<NUM> heterodimer, increased the presence of IL-12Rβ1 in the membrane (<FIG>). Pan-cadherin was analyzed to check the purity of the membrane fraction (<FIG>; top panel). Surprisingly, we found increased level of β-actin in membrane fractions of IL-12p40 homodimer- and p70-treated TRAMP cells, suggesting that the treatment with IL-12p40 homodimer or p70 is possibly associated with increased formation of endocytic vesicles in the membrane (<FIG>; bottom panel). In contrast, we did not observe increased membrane level of β-actin in IL-12p40 monomer-treated TRAMP cells (<FIG>). These results suggest that IL-12p40 monomer, but neither IL-12p40 homodimer nor p70, may be involved in the arresting of IL-12Rβ1 in the membrane.

However, there was no difference in IL-12Rβ1 in whole cell extract when TRAMP cells were treated with these cytokines (<FIG>), negating the possibility of induction of IL-12Rβ1 level by IL-12p40 monomer. Consistently, the IL-12p40 monomer mAb abrogated IL-12p40 monomer-mediated increase in IL-12Rβ1 in membrane of TRAMP cells (<FIG>), suggesting that IL-12p40 monomer is indeed involved in the membrane arrest of IL-12Rβ1. Pan-Cadherin was analyzed to monitor the purity of the membrane fraction (<FIG>; top panel). The level of β-actin was higher in IL-12p40 monomer mAb-treated cells, suggesting that the absence of IL-12p40 monomer may induce the formation of endocytic vesicles in TRAMP cells (<FIG>; bottom panel). However, again, there was no difference in the level of total IL-12Rβ1 between (IL-12p40 monomer + IL-12p40 monomer mAb)-treated cells and IL-12p40 monomer-treated cells, suggesting that IL-12p40 monomer mAb treatment does not down-regulate the expression of IL-12Rβ1 in TRAMP cells. Together, these results suggest that excess IL-12p40 monomer released by TRAMP cells inhibit IL-<NUM> signaling by suppressing the internalization or endocytosis of IL-12Rβ1.

Next, we investigated mechanisms by which neutralization of IL-12p40 monomer induced the internalization of IL-12Rβ1. Receptor internalization primarily happens via two mechanisms - clathrin-dependent and caveolin-dependent. To examine the involvement of clathrin or caveolin, we used two pharmacological inhibitors filipin and chlorpromazine (CPM). Interestingly, pre-treatment with filipin, but not CPM, significantly inhibited the membrane internalization of IL-12Rβ1in IL-12p40 monomer mAb-treated TRAMP cells (<FIG> &5I), suggesting that IL-12p40 monomer-mediated internalization of IL 12Rβ1 occurs via caveolin-sensitive and clathrin-independent pathway. Immunofluorescence analysis further confirmed that the IL-12p40 monomer mAb-mediated internalization of IL-12Rβ1 in TRAMP cells is caveolin-dependent (<FIG>) as neutralizing IL-12p40 monomer with IL-12p40 monomer mAb was unable to internalize IL-12Rβ1 when TRAMP cells were pretreated with filipin (<FIG>).

Table <NUM> shows serum levels of IL-12p40 monomer, IL-12p40 homodimer and IL-<NUM> heterodimer measured using ELISA in <NUM> prostate cancer patients and <NUM> control subjects. Concentrations of IL-12p40 monomer and IL-12p40 homodimer were measured in serum of prostate cancer patients and healthy controls by a sandwich ELISA as described in <NPL>; <NPL>. Briefly, for quantifying p40, we used mAb a3-3a for coating and mAb a3-<NUM> for detection. Similarly, for measuring IL-12p40 homodimer, mAb a3-1d and mAb d7-12c were used for coating and detection, respectively. Levels of IL-<NUM> heterodimer in serum were measured using the IL-<NUM> ELISA kit from eBioscience (San Diego, CA <NUM>).

Each level reported in Table <NUM> is an average of three measurements. The serum levels of IL-12p40 monomer are higher in the prostate cancer patients as compared to the control subjects. The results suggest that excess IL-12p40 monomer may play a role in the pathogenesis of prostate cancer and that treatment of cancer patients with the monoclonal antibody against IL-12p40 monomer may inhibit/stop the progression of the prostate cancer.

Claim 1:
A composition comprising a therapeutically effective amount of an antibody against IL12 p40 monomer for use in treating cancer, wherein the antibody neutralizes the IL12 p40 monomer. does not significantly neutralize action of IL12 p40 homodimer, and does not bind IL-<NUM> heterodimer wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, liver cancer, colon cancer, ovarian cancer and pancreatic cancer.