WO2012121679A1

WO2012121679A1 - Method of modulating phenotype of a renal cell cancer-associated monocyte or macrophage

Info

Publication number: WO2012121679A1
Application number: PCT/SG2012/000081
Authority: WO
Inventors: Subhra Kumar BISWAS; Seng Cheong Alvin WONG
Original assignee: Agency For Science, Technology And Research; National University Hospital
Priority date: 2011-03-09
Filing date: 2012-03-09
Publication date: 2012-09-13

Abstract

Methods and uses relating to the identification and modulation of the pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage are provided, including a method of modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage, the method comprising exposing the monocyte or macrophage to an interleukin-1 blocker.

Description

METHOD OF MODULATING PHENOTYPE OF A RENAL CELL CANCER- ASSOCIATED MONOCYTE OR MACROPHAGE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of, and priority from, U.S. provisional patent application No. 61/451,092, filed on March 9, 201 1, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of modulating the phenotype of monocytes or macrophages associated with renal cell cancers.

BACKGROUND OF THE INVENTION

[0003] Interleukin 1 (IL-1) is a cytokine that is released during inflammation and plays a role in regulating the inflammatory response. IL-1 is produced in 2 forms: IL-1 a and IL-Ιβ. Both IL-1 a and IL-Ιβ are secreted. IL-1 receptor antagonist (IL-lra) is another member of the IL-1 family of cytokines. IL-lra binds tightly to the IL-1 receptor type I (IL-1RI) and blocks the activity of either IL-1 a or IL-1 (8. IL-lra is also secreted.

[0004] IL-1, upon binding to IL-IRI on the surface of target cells, activates binding of IL-1R1 to an accessory protein, IL-lRAcP, which results in transduction of downstream signal within the target cell. Binding of IL-1 to IL-IR Type 2 (IL-IRII) does not appear to result in transduction of signal; thus, IL-RII is thought to be involved in down- regulating IL-1 signal and is considered as a decoy receptor.

SUMMARY OF THE INVENTION

[0005] In one aspect, there is provided a method of modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage, the method comprising: exposing the monocyte or macrophage to an interleukin- 1 blocker. The exposing may be performed in vitro or in vivo.

[0006] In another aspect, there is provided a method of treating or preventing renal cell cancer tumor growth in a subject, the method comprising: administering an interleukin-1 blocker to a subject in whom renal cell cancer tumor growth promoted by a renal cell cancer-associated monocyte or macrophage is to be treated or prevented.

Administering may comprise injection or surgical implantation at or near a site of a renal cell cancer tumor, or may comprise intravenous, intradermal, subcutaneous or intramuscular injection.

[0007] In the methods, the IL-1 blocker may be formulated for delivery with a liposome, a nanoparticle or a microcapsule.

[0008] In the methods, the IL-1 blocker may be a molecule that binds IL-1, an IL-1 receptor antagonist or an antibody directed against IL-1, including a soluble IL-1 receptor or rilonacept, anakinra, canakinumab or XOMA-052.

[0009] The methods may further include exposing the monocyte or macrophage to an additional anti-tumor treatment or administering the interleukin-1 blocker in combination with the additional anti-tumor treatment. For example, the exposing to the IL-1 blocker may be performed prior to tumor resection (neoadjuvant treatment), following tumor resection (adjuvant treatment) or in metastatic disease in combination with an additional anti-tumor treatment. The additional anti-tumor treatment may comprise tumor resection, chemotherapy, radiation therapy, an anti-angiogenic agent, a kinase inhibitor, a tumor vaccine or a therapeutic antibody.

[0010] In another aspect, and in keeping with the methods described above, there is provided a method of determining whether a subject having renal cell cancer may benefit from treatment with IL-1 blocker comprising: determining the phenotype of a renal cell cancer-associated monocyte or macrophage from a sample from the subject, wherein a pro-tumoral phenotype is indicative that the subject may benefit from treatment with IL-1 blocker.

[0011] In another aspect, and in keeping with the methods described above, there is provided a method of determining the phenotype of a renal cell cancer-associated monocyte or macrophage comprising: providing a sample containing a renal cell cancer- associated monocyte or macrophage; determining the gene or protein expression profile of the renal cell cancer-associated monocyte or macrophage; and comparing the gene or protein expression profile of the renal cell cancer-associated monocyte or macrophage with the gene expression profile from a monocyte or macrophage that is not associate with a renal cell cancer, wherein an increase in expression of one or more genes or proteins that promotes tumor progression and/or a decrease in expression of one or more genes or proteins that promotes anti-tumor immune response in the renal cell cancer- associated monocyte or macrophage is indicative of a pro-tumoral phenotype.

[0012] In another aspect, there is provided use of an interleukin-1 blocker for modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage.

[0013] In another aspect, there is provided use of an interleukin-1 blocker in the manufacture of a medicament for modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage.

[0014] In another aspect, there is provided an interleukin-1 blocker for use in the modulation of pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage.

[0015] In another aspect, there is provided use of an interleukin-1 blocker for treating or preventing renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer-associated monocyte or macrophage.

[0016] In another aspect, there is provided use of an interleukin-1 blocker in the manufacture of a medicament for treating or preventing renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer- associated monocyte or macrophage.

[0017] In another aspect, there is provided an interleukin-1 blocker for use in the treatment or prevention of renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer-associated monocyte or macrophage.

[0018] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In the figures, which illustrate, by way of example only, embodiments of the present invention:

[0020] Figure 1. Monocytes from RCC subjects show a pro-tumoral phenotype.

(A) qPCR analysis of gene expression from RCC subjects (RCC-Mo) as compared to normal monocytes from healthy donors; (B) assessment of in vitro angiogenesis, measure by induction of endothelial tube formation on MATRIGEL™; (C) assessment of in vitro invasion of RCC4 tumor cells as induced by RCC-Mo compared to normal Mo.

[0021] Figure 2. Pro-tumoral phenotype of monocytes from RCC subjects is mediated by tumor-derived IL-lbeta and can be blocked by IL-lra treatment. (A) measurement of expression levels of IL-lbeta by different RCC tumor cell lines, in comparison with normal kidney cells or normal monocytes; (B) measurement of effect of IL-1 receptor antagonist on expression of proumoral genes in RCC co-cultured monocytes, compared with normal monocytes.

[0022] Figure 3. In vivo treatment with EL-lra in a mouse model of human RCC slow tumor growth, blocks pro-tumoral phenotype of TAMs and shifts them to anti- tumoral Ml phenotype. (A) kinetics of tumor growth in xenograft tumor model of human RCC in SCID mice and the in vivo effect of treatment with IL-1 receptor antagonist on tumor growth; (B-C) assessment of macrophage pro-tumoral phenotype and switching of phenotype in response to IL-1 receptor antagonist treatment.

[0023] Figure 4. In vivo targeting IL-1R1 in a xenograft model of human RCC attenuates tumor growth and pro-tumoral phenotype of TAMs. (A) kinetics of tumor growth were measured in wildtype or IL-1R1-/- mice; (B) expression of pro-tumoral genes in tumor-associated macrophages from wildtype or IL-1R1-/- mice were assessed.

[0024] Figure 5. Anti-human IL-1 beta attenuates tumor invasion promoting activity of RCC-conditioned monocytes. Human anti-IL-lbeta antibodies were used to treat monocytes (normal or RCC-conditioned); culture supematants from the treated monocytes were then tested for ability to induce RCC invasiveness; monocytes were tested for expression of MMP9, which promotes invasion.

[0025] Figure 6. Anti-human IL-1 beta attenuates angiogenesis activity of RCC- conditioned monocytes. Human anti-IL-Ιβ antibodies were used to treat monocytes (normal or RCC-conditioned); culture supematants from the treated monocytes were then tested for ability to induce endothelial tube formation on MATRIGEL™ as a measure of angiogenesis.

DETAILED DESCRIPTION

[0026] Renal cell cancer is generally not considered to be an inflammation-induced cancer, unlike various other cancer types. To date, it was believed that renal cancer cells did not secrete IL-1, and it had been previously shown that IL-1 plasma levels in renal cell cancer patients had no correlation with disease progression. Since IL-1 is involved in generating an inflammation response, IL-1 inhibitors have not previously been used to treat renal cell cancer tumors.

[0027] However, it has now been surprisingly discovered that monocytes or macrophages associated with renal cell cancer may have what is termed a "pro-tumoral" phenotype, in that these monocytes and macrophages express genes that are associated with tumor promoting activity, and hence contribute to tumor progression. It has also been surprisingly discovered that blocking or down regulating the effects of tumor- derived IL-1 in such monocytes and macrophages inhibits their pro-tumoral activity and slows tumor growth.

[0028] Thus, there is provided a method of modulating pro-tumoral phenotype of a monocyte or macrophage associated with a renal cell cancer, including a renal cell cancer tumor.

[0029] As will be appreciated, a monocyte is mononuclear phagocytic cell produced in the bone marrow from the myeloid progenitors. Monocytes circulate in the blood and differentiate into macrophages upon entry into tissue; a macrophage is a differentiated monocyte, and is typically located in tissue. A renal cell cancer-associated monocyte or macrophage is a monocyte or macrophage that has been conditioned by growing in the presence of a renal cell cancer, including a renal cell cancer in a tumor. For example, the renal cell cancer-associated monocyte may be circulating within a subject that has renal cell cancer. The renal cell cancer-associated monocyte or the renal cell cancer-associated macrophage may be at a tissue site in a subject including at or near a tissue site of a renal cell cancer tumor. The monocyte or the macrophage may be cultured in tissue culture together with a renal cell cancer, including a primary renal cell cancer, a renal cell cancer derived from an established tissue culture cell line or an excised renal cell cancer tumor or portion thereof. The monocyte or the macrophage may be cultured in medium conditioned by previously culturing of a renal cell cancer, including a primary renal cell cancer, a renal cell cancer derived from an established tissue culture cell line or an excised renal cell cancer tumor or portion thereof.

[0030] Renal cell cancer-associated monocytes may be identified or isolated from the blood of a subject having renal cell cancer. Renal cell cancer-associated macrophages may be identified or isolated from tumor tissue from a subject having renal cell cancer. Such monocytes and macrophages may be characterised using lineage cell markers and flow cytometry, by gene expression profiles, and by functional assays, using routine laboratory techniques.

[0031] As used herein, reference to a cell, including a monocyte, a macrophage or a renal cell cancer (RCC) cell, is intended to refer to a single cell as well as a plurality or population of cells, where context allows. Similarly, reference to cells, including monocytes, macrophages or RCC cells, is also intended to refer to a single cell, where context allows. The cell may be a cell grown in batch culture or in tissue culture plates, or may be a cell in vivo. The cell may be an explanted cell from a subject.

[0032] A RCC cell is any cell of renal origin that has been transformed or immortalised, and which undergoes uncontrolled or unregulated growth. As indicated above, the RCC cell may be a primary RCC cell and may be explanted from a tumor or may be contained within a tumor. The RCC cell may be from an established renal cell cancer cell line. A renal cell cancer tumor is a tumor originating from a RCC cell. The methods as described herein relate to the identification and modulation of various phenotypes of monocytes and macrophages that are associated with a renal cell cancer tumor.

[0033] In a first phenotype, termed pro-tumoral or M2, the renal cell cancer- associated monocyte or macrophage has increased expression of one or more genes, including cytokine genes, that results in promotion of renal cell cancer tumor growth and progression. For example, tumor progression may include increased tumor cell proliferation, increased tumor cell migration, increased ability of a rumor cell to invade surrounding tissue such as epithelial tissue, or may include increased angiogenesis within a renal cell cancer tumor.

[0034] In addition, a pro-tumoral renal cell cancer-associated monocyte or macrophage may have increased expression of one or more of anti-inflammatory genes. In the pro-tumoral phenotype, the monocyte or macrophage may have decreased expression of one or more genes, including cytokine genes, that promote antitumor immune response and/or suppress or down regulate cell proliferation or cancer progression. For example, renal cell cancer-associated macrophages may have increased expression of anti- inflammatory cytokine IL-10 or Arginase 1, and may have decreased expression of pro-inflammatory genes such as IL-12p40 and NOS2.

[0035] Increased or decreased expression of a gene or protein in a renal cell cancer- associated monocyte or macrophage is measured in comparison to a monocyte or macrophage that has not grown in the presence of, or been conditioned by, a renal cell cancer. Such levels may be measured using routine laboratory methods, including for example quantitative PCR or gene expression microarrays, gene sequencing, ELISA, Western blotting, flow cytometry or mass spectrometry-based methods.

[0036] In a second phenotype, termed anti-tumoral or Ml, the monocyte or macrophage has decreased expression of one or more genes, including cytokine genes, that promote renal cell cancer tumor growth and progression.

[0037] In addition, an anti-tumoral monocyte or macrophage may have increased expression of one or more of pro-inflammatory genes. In the anti-tumoral phenotype, the monocyte or macrophage may have increased expression of one or more genes, including cytokine genes, that activate antitumor immune response and/or decreased expression of genes that promote cell proliferation or cancer progression. Morever, these monocyte or macrophage may have decreased expression of anti-inflammatory genes. For example, renal cell cancer-associated macrophages may have increased expression of proinflammatory genes such as IL-12p40 and NOS2 and may have decreased expression of anti- inflammatory cytokine IL-10 or Arginase 1.

[0038] In the methods as described herein, exposing the renal cell cancer-associated monocyte or macrophage with an IL-1 blocker blocks, reduces or down regulates the pro- tumoral phenotype of the monocyte or macrophage. For example, such phenotype switching or modulation may be monitored using routine laboratory methods, such as assays to determine gene expression profile and functional assays.

[0039] Thus, modulating the phenotype of the renal cell cancer-associated monocyte or macrophage with an IL-1 blocker includes down regulating, reducing or suppressing expression of one or more pro-tumor genes as well as up regulating or promoting expression of one or more anti-tumor genes.

[0040] Such modulating is achieved by exposing the monocyte or macrophage to an IL-1 blocker. That is, the IL-1 blocker is delivered to the vicinity of the monocyte or macrophage so that the IL-1 blocker prevents IL-1 from binding to the IL-IR on the monocyte or macrophage.

[0041] The IL-1 blocker may be any molecule that blocks the effect of IL-1 binding the IL-1R1 receptor on the surface of the monocyte or macrophage. For example, the IL- 1 blocker may be a molecule that binds to IL-1, including IL-1 β, to prevent the IL-1 from binding to the receptor. Thus, the IL-1 blocker may be a soluble form of an IL-IR, including soluble IL-1R2, or may be rilonacept. The IL-1 blocker may be an anti-IL-1 antibody, for example, canakinumab or XOMA-052. Or, the IL-1 blocker may be an antagonist molecule that binds to the IL-1R1 receptor, on the surface of monocyte or macrophage and prevents binding and activation of the receptor by IL-1, including IL-1 β. For example, the IL-1 blocker may be an IL-IR antagonist such as anakinra.

[0042] Thus, exposing includes delivering the IL-1 blocker to tissue culture medium containing the monocyte or macrophage, or administering the IL-1 blocker to a subject in need of treatment of renal cell cancer in which the tumor growth is promoted by the associated monocyte or macrophage. As well, exposing includes administering at or near the site of a renal cell cancer tumor with which the monocyte or macrophage is associated.

[0043] In order to perform the exposing of the renal cell cancer-associated monocyte or macrophage to the IL-IR blocker, the IL-IR may be formulated for administering to tissue culture or to a subject. For example, the IL-1 blocker may be formulated for delivery with a liposome, a nanoparticle or a microcapsule, or other suitable delivery systems.

[0044] Thus, the methods described herein may be performed in vitro or in vivo.

[0045] The in vivo methods are performed by administering the IL-1 blocker to a subject in need of treatment of renal cell cancer. Administration may be performed by providing the IL-1 blocker at or near the site of a renal cell cancer tumour in the subject, as well as administering systemically.

[0046] The administering of the IL-1 blocker may be performed prior to tumor resection (neoadjuvant treatment), following tumor resection (adjuvant treatment), or in metastatic disease in combination with an additional anti-tumor treatment, as described below.

[0047] The subject is any animal in need of treatment of a renal cell cancer, including renal cell cancer tumor, which cancer or tumor has renal cell cancer-associated monocytes or macrophages having a pro-tumoral phenotype. The renal cell cancer- associated monocyte or macrophage may be involved in promoting growth of the cancer or tumor. The subject includes a mammal, including a human, and includes a subject that is currently undergoing additional anti-tumor treatment.

[0048] Thus, the monocyte or macrophage may be in a culture or in a subject, the culture or subject currently undergoing an additional anti-tumor treatment, meaning that the tissue culture or subject is currently being treated with an anti-tumor regimen, including simultaneously with, overlapping with, or sequentially to the modulation of monocyte or macrophage phenotype.

[0049] The modulation and the anti-tumor treatment each may be achieved in one or more discrete treatments or may be performed continuously for a given time period required in order to achieve the desired result.

[0050] Thus, an additional anti-tumor treatment includes chemotherapy that covers a greater or lesser period of time as the time period for which the monocyte or macrophage phenotype is to be modulated.

[0051 ] Additional anti-tumor treatment for treating a renal cell cancer tumor may comprise tumor resection, chemotherapy, radiation therapy, an anti-angiogenic agent, a kinase inhibitor, a tumor vaccine or a therapeutic antibody. Such anti-tumor treatments, including chemotherapy and chemotherapeutic agents, are known in the art.

[0052] Reference to treating of a renal cell cancer tumor with an anti-tumor treatment refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilization of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset, amelioration or palliation of the disease state, and remission (whether partial or total). "Treating" can also mean prolonging survival of a subject beyond that expected in the absence of treatment. "Treating" can also mean inhibiting the progression of disease, slowing the progression of disease temporarily, although more preferably, it involves halting the progression of the disease pennanently.

[0053] For in vivo methods and uses, the modulation of the phenotype of the monocyte or macrophage may be achieved by administering to the subject the IL-1 blocker at the site of the monocyte or the macrophage, including at or near the site of a tumor, for example by injection, including intratumoral injection, or by surgical implantation. The modulation may also be achieved by administering the IL-1 blocker systemically, for example intravenously, subcutaneously, intradermally or

intramuscularly.

[0054] In the in vitro and in vivo methods described herein, an effective amount of the IL-1 blocker is administered to the cell or the subject. The term "effective amount" as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired result, for example, to decrease the pro-tumoral phenotype or switch the phenotype of the monocyte or macrophage from M2 phenotype to Ml phenotype. Other clinical parameters of disease progression may be observed, including stabilization or reduction of tumor volume, invasiveness and/or angiogenesis, decrease of disease markers, increased patient health or length of survival, or other measures of decreased disease progression.

[0055] The concentration and amount of the IL-1 blocker that modulates the phenotype to be administered to the cell or subject will vary, depending on the type of IL- 1 blocker that is administered, the mode of administration, and, for in vivo approaches, the age and health of the subject.

[0056] In different embodiments, the IL-1 blocker may be administered to a subject topically or by injection (subcutaneously, intravenously, intramuscularly, etc.) directly at the desired site where the monocytes or macrophages in which phenotype is to be modulated are located in the subject.

[0057] The dose of the IL-1 blocker that is to be used depends on the particular condition being treated, the severity of the condition, the individual subject parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and other similar factors that are within the knowledge and expertise of the health practitioner. These factors are known to those of skill in the art and can be addressed with minimal routine experimentation.

[0058] In keeping with the above description, the methods described herein also include a method of determining the phenotype of a renal cell cancer-associated monocyte or macrophage.

[0059] As indicated above, the method includes providing a sample containing a renal cell cancer-associated monocyte or macrophage. The gene or protein expression profile of the renal cell cancer-associated monocyte or macrophage is determined and compared with the gene expression profile from a monocyte or macrophage that is not associate with a renal cell cancer. An increase in expression of one or more genes or proteins that promotes tumor progression and/or a decrease in expression of one or more genes or proteins that promotes anti-tumor immune response in the renal cell cancer- associated monocyte or macrophage is indicative of a pro-tumoral phenotype.

[0060] The observation of pro-tumoral phenotype of renal cell cancer-associated monocytes and macrophages, and the ability to down-regulate or switch a pro-tumoral phenotype to an anti-tumoral phenotype using IL-1 blocker, allows for determination of whether a given subject having renal cell cancer may be treated with IL-1 blocker. Thus, the methods described herein also include determination of whether a subject is in need of treatment with IL-1 blocker.

[0061] As stated above, the subject is any subject having renal cell cancer, including a renal cell cancer tumor. The subject may be undergoing anti-tumor treatment for the renal cell cancer.

[0062] The method is performed on a sample obtained from the subject. The sample may be a blood sample containing monocytes including renal cell cancer-associated monocytes. The sample may be a tumor biopsy sample containing renal cell cancer- associated macrophages.

[0063] The sample is tested to determine the phenotype of the renal cell cancer- associated monocytes or macrophages, as described above. Gene or protein expression profiles may be measured for the monocytes or macrophages, using routine techniques such as quantitative PCR or gene expression microarrays, gene sequencing, ELISA, Western blotting, flow cytometry or mass spectrometry-based methods.

[0064] The phenotype of the monocytes or macrophages from the sample from the subject may be compared to the phenotype of monocytes or macrophages obtained from subjects that do not have renal cell cancer.

[0065] A pro-tumoral phenotype of monocytes or macrophages in the test sample from the subject having renal cell cancer is indicative that the subject may benefit from treatment with IL-1 blocker to attempt to down-regulate or switch the pro-tumoral phenotype of the monocytes or macrophages to an anti-tumoral phenotype, thereby slowing tumor progression.

[0066] In keeping with the methods of the invention, various uses are also contemplated.

[0067] In one aspect of the present invention, there is provided use of an interleukin-1 blocker for modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage.

[0068] In another aspect of the present invention, there is provided use of an interleukin-1 blocker in the manufacture of a medicament for modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage.

[0069] In another aspect of the present invention, there is provided an interleukin-1 blocker for use in the modulation of pro-tumoral phenotype of a renal cell cancer- associated monocyte or macrophage. [0070] In another aspect of the present invention, there is provided use of an interleukin-1 blocker for treating or preventing renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer- associated monocyte or macrophage.

[0071] In another aspect of the present invention, there is provided use of an interleukin-1 blocker in the manufacture of a medicament for treating or preventing renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer-associated monocyte or macrophage.

[0072] In another aspect of the present invention, there is provided an interleukin-1 blocker for use in the treatment or prevention of renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer- associated monocyte or macrophage.

[0073] The present methods and uses are further exemplified by way of the following non-limiting examples.

EXAMPLES

[0074] Example 1: Monocytes from RCC subjects show a pro-tumoral phenotype

[0075] Example 1 shows that circulating monocytes from RCC subjects express higher levels of pro-tumoral molecules compared to circulating monocytes from healthy donors; in vitro, they are better stimulators of angiogenesis and invasion (a marker for metastatic functions) compared to healthy donor monocytes.

[0076] Gene expression in monocytes from RCC subjects (RCC-Mo) as compared to nonnal monocytes from healthy donors (Mo) were assessed by qPCR (Figure 1A). Expression of inflammatory cytokines and pro-tumoral genes in RCC-Mo is represented as fold change with respect to normal monocytes from healthy donors. Data is mean+SEMs for 7 subjects.

[0077] Figure IB shows in vitro angiogenesis as a functional measure of pro-tumoral activity of RCC-Mo. RCC-Mo showed elevated ability of inducing endothelial tube formation on matrigel (a measure of in vitro angiogenesis) as compared to normal Mo. Data shown is mean+SEMs of 6 replicates each from 2 subjects. ***p<0.05, vs Mo. Representative photograph of tube formation assay for each study group is shown in the right panel.

[0078] Figure 1C shows in vitro invasion as a functional measure of pro-tumoral activity of RCC-Mo. RCC-Mo showed elevated ability to induce invasion of RCC4 tumor cells as compared to nonnal Mo. Y-axis represents optical density as a measure of the number of invaded cells. Data is mean± SEM of 6 replicates each from 3 subjects. ***p<0.05, vs Mo. Representative photograph of invading RCC4 cells for each study group is shown in the right panel.

[0079] Example 2: Pro-tumoral phenotype of monocytes from RCC subjects is mediated by tumor-derived IL-Ιβ and can be blocked by IL-1RA treatment

[0080] Example 2 shows that human RCC cell lines are capable of producing high levels of IL-Ι β and that blocking IL-1 in a co-culture of human monocytes and RCC cells results in inhibition of the expression of pro-inflammatory, pro-angiogenic molecules. Since IL-lbeta is the main secreted form of IL-1, the effect is possibly through IL-lbeta.

[0081] Elevated ILIB expression by different RCC tumor cell lines (RCC4, Caki-2, A498) but not by normal kidney cells (HRPTEC) or normal monocytes (Mo) (Figure 2A). ILIB gene expression was detected by qPCR and represented as fold change with respect to normal human renal cells (HRPTEC) in the case of RCC cell lines. ILIB gene expression by RCC monocytes is represented as fold change with respect to normal monocytes.

[0082] IL-IRA treatment blocks the elevated expression of pro-tumoral genes (and hence the pro-tumoral phenotype) in RCC co-cultured monocytes, as compared to normal monocytes (Figure 2B). Normal monocytes (1.5xl0⁶/well) from healthy donors were seeded onto the wells of a 12-well plate and co-cultured with RCC4 cells (0.5xl0⁶ well) in 0.4μηι pore sized transwell inserts for 48 hrs in the presence or absence of 20ng/well IL-1 Receptor antagonist (IL-1RA). After co-culture, inserts containing RCC cells were removed, monocytes washed with PBS and RNA was prepared by trizol extraction. Expression of the indicated genes was assessed by qPCR. Gene expression in all cases is represented as fold change with respect to untreated normal monocytes. Results are mean+SEM representative of three independent experiments.

[0083] In Figure 2B, Mo: Normal Monocytes from healthy donors; RCC+Mo: Normal Monocytes co-cultured with RCC cells; Mo+IL-lRA+RCC cells: Normal monocytes co-cultured with RCC cells in the presence of IL-1 RA.

[0084] Example 3: In vivo treatment with IL-1RA in a mouse model of human RCC slows tumor growth, blocks pro-tumoral phenotype of TAMs and shifts them to anti-tumoral Ml phenotype

[0085] Example 3 demonstrates that in a xenograft model of human RCC, local treatment with an IL-1 blocker results in delayed tumor growth and lower expression of pro-inflammatory, pro-angiogenic molecules by tumor-associated macrophages which also are skewed towards a more anti-tumor phenotype (Ml). In these experiments, recombinant IL-1 receptor antagonist (IL-IRA) is used to inhibit EL-l function. IL-1RA is also an approved drug for treatment of rheumatoid arthritis under the name of

Anakinra, marketed as KINERET™ (Amgen)

[0086] Kinetics of tumor growth in xenograft tumor model of human RCC in SCID mice (Figure 3A). Mice were inoculated subcutaneously (s.c) on the right hind flank with a total of 10⁶ RCC4 cells per mouse. Mice were administered with 3 intratumoral doses of IL-1 RA ^g/Kg Body wt.) on 7^th, 9^th and 11^th day after tumor injection. Tumor take was monitored and the diameter of the growing tumor was measured in millimeters every 2 or 3 days by using a caliper. Results are mean±SEM of representative of three independent experiments.

[0087] Age- and sex-matched SCID mice (6-8 weeks old) were inoculated subcutaneously with RCC4 cells (lxlO⁶ cells/mice). IL-IRA was administered as described above. After 3-4 weeks animals were euthanized. Tumor was excised, tumor associated macrophages (TAMs) isolated and RNA extracted. Expression of the indicated pro-tumoral genes was assessed by qPCR (Figure 3B). Gene expression in all cases is represented as fold change with respect to peritoneal exudate cells (PECs) from non- tumor bearing mice.

[0088] Macrophages are known to exhibit different functional phenotypes. Ml macrophages exhibit an antitumor function and show increased expression of proinflammatory gene IL-12p40 and the NO producing enzyme NOS2. In contrast, M2 macrophages are pro-tumoral, i.e. they support tumor growth, express very little IL- 12p40 or NOS2 but high levels of anti-inflammatory cytokine IL-10 and Arginase 1 (Arg 1). TAMs from in vivo IL- IRA-treated animals show a switch of phenotype from M2 to a Ml state as revealed by the expression of the indicated marker genes (Figure 3C). Results are mean+SEM representative of two independent experiments.

[0089] Example 4: In vivo genetic deletion of IL-1R1 attenuates tumor growth as well as pro-tumoral gene expression in TAMs.

[0090] Example 4 shows that genetic depletion of IL-1 receptor 1(IL-1 1), the receptor responsible for responding to IL- 1 , resulted in slower tumor growth which correlate to lower expression of pro-tumoral genes in the tumor associated macrophages (TAMs) as compared to their wild type counterparts. This evidence complements the data on IL-IRA and further strengthens the rationale of targeting the IL-1R pathway, which is responsible for the tumor promoting functions of macrophages.

[0091] RCC4 xenograft tumors were grown and monitored for tumor kinetics and harvested for TAM isolation using similar conditions as mentioned in Example 3. Kinetics of tumor growth in WT or IL-1R1-/- mice. Data is mean± SEM of 4 mice per group (Figure 4A). Expression of a selected panel of pro-tumoral genes in TAMs, by qPCR (Figure 4B). Data is mean± SEM of triplicate. *p<0.05 wrt WT TAMs

[0092] Example 5: Anti-human IL-Ιβ attenuates tumor invasion promoting activity of RCC 'conditioned' monocytes.

[0093] Example 5 tests another strategy of targeting the IL-1/IL-1R axis, i.e. by using an in-house developed neutralizing antibody against human IL-Ι β to block pro-tumoral action of monocytes/macrophages. Data shows that this antibody could significantly block the capacity of RCC co-cultured monocytes to stimulate tumor cell invasion (a measure of their pro-tumoral functional).

[0094] Normal monocytes were co-cultured for with or without RCC4 cells in a transwell culture system as stated in Example 2 in the presence or absence of the indicated concentration of human anti-IL-Ι β antibody or its isotype control. Thereafter, cell free supernatants were collected and tested for in vitro invasion assays using RCC4 as the target cells. Figure 5A shows representative photographs of invading RCC4 cells attached to the lower side of the transwell insert (signifying invasion) (see left panel). Invasion was also quantified by a colorimetric assay (see right panel). Data is mean± SEM of triplicates from two independent experiments (*p < 0.05 versus Mo+RCC). Figure 5B shows the downregulation of MMP9 gene expression (as measured by quantitative PCR) in RCC-co-cultured monocytes treated with indicated concentration of human anti-IL-lb antibody. Data is mean±SEM of triplicates of a single experiment (*p< 0.05, versus Mo+RCC).

[0095] Example 6: Anti-human IL-Ιβ attenuates angiogenesis promoting activity of RCC 'conditioned' monocytes.

[0096] In keeping with Example 5, Example 6 uses the neutralizing antibody against human IL-Ι β to block pro-tumoral action of monocytes/macrophages. The data show that the antibody also blocks the capacity of RCC co-cultured monocytes to stimulate tumor angiogenesis in vitro. These observations were then correlated to downregulation of pro- tumoral genes like MMP9 and VEGF in the RCC-co-cultured monocytes, upon blocking with human IL- 1 β antibody.

[0097] Normal monocytes were co-cultured with or without RCC4 cells in a transwell culture system as stated in Example 1, in the presence or absence of the indicated concentration of human anti-IL-lb antibody or its isotype control (Isotype Con). Thereafter, cell free supernatants were collected and tested for in vitro endothelial tube formation on matrigel. In Figure 6A, the left panel shows the corresponding bright field images of endothelial tube formation from a representative experiment (xlO magnification). The right panel shows quantitative analysis of tube formation represented as average number of tubes per field. Data is mean± SEM of triplicates from two independent experiments. (*p < 0.05 versus Mo+RCC).

[0098] In Figure 6B, qPCR analysis results show the downregulated VEGFA gene expression in RCC-co-cultured monocytes treated with indicated concentration of human anti-IL-lb antibody. Data is mean±SEM of triplicates of a single experiment (*p< 0.05, versus Mo+RCC).

[0099] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[00100] Concentrations given in this specification, when given in terms of percentages, include weight/weight (w/w), weight/volume (w/v) and volume/volume (v/v) percentages.

[00101] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise. As used in this specification and the appended claims, the terms "comprise",

"comprising", "comprises" and other forms of these terms are intended in the non- limiting inclusive sense, that is, to include particular recited elements or components without excluding any other element or component. Enumerated lists of elements are intended to include sub-lists. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

[00102] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage, the method comprising: exposing the monocyte or macrophage to an interleukin-1 blocker.

2. The method according to claim 1, wherein the exposing is performed in vitro.

3. The method according to claim 1, wherein the exposing is performed in vivo.

4. A method of treating or preventing renal cell cancer tumor growth in a subject, the method comprising: administering an interleukin-1 blocker to a subject in whom renal cell cancer tumor growth promoted by a renal cell cancer-associated monocyte or macrophage is to be treated or prevented.

5. The method according to claim 4, wherein administering comprises injection or surgical implantation at or near a site of a renal cell cancer tumor.

6. The method according to claim 4, wherein administering comprises intravenous, intradermal, subcutaneous or intramuscular injection.

7. The method according to any one of claims 1 to 6, wherein the IL-1 blocker is formulated for delivery with a liposome, a nanoparticle or a microcapsule.

8. The method according to any one of claims 1 to 7, wherein the IL-1 blocker is a molecule that binds IL-1, an IL-1 receptor antagonist or an antibody directed against IL- 1.

9. The method according to claim 8, wherein the molecule that binds IL-1 is soluble IL-1 receptor or rilonacept.

10. The method according to claim 8, wherein the IL-1 receptor antagonist is anakinra.

11. The method according to claim 8, wherein the antibody is canakinumab or XOMA-052.

12. The method according to any one of claims 1 to 11, further comprising exposing the monocyte or macrophage to an additional anti-tumor treatment or administering the interleukin-1 blocker in combination with the additional anti-tumor treatment.

13. The method according to any one of claims 1 to 12, wherein the exposing to the IL-1 blocker is performed prior to tumor resection (neoadjuvant treatment), following tumor resection (adjuvant treatment) or in metastatic disease in combination with an additional anti-tumor treatment.

14. The method according to claim 12 or 13, wherein the additional anti-tumor treatment comprises tumor resection, chemotherapy, radiation therapy, an anti-angiogenic agent, a kinase inhibitor, a tumor vaccine or a therapeutic antibody.

15. A method of determining whether a subject having renal cell cancer may benefit from treatment with IL-1 blocker comprising: determining the phenotype of a renal cell cancer-associated monocyte or macrophage from a sample from the subject, wherein a pro-tumoral phenotype is indicative that the subject may benefit from treatment with IL-1 blocker.

16. A method of determining the phenotype of a renal cell cancer-associated monocyte or macrophage comprising: providing a sample containing a renal cell cancer-associated monocyte or macrophage; determining the gene or protein expression profile of the renal cell cancer- associated monocyte or macrophage; and comparing the gene or protein expression profile of the renal cell cancer- associated monocyte or macrophage with the gene expression profile from a monocyte or macrophage that is not associate with a renal cell cancer, wherein an increase in expression of one or more genes or proteins that promotes tumor progression and/or a decrease in expression of one or more genes or proteins that promotes anti-tumor immune response in the renal cell cancer- associated monocyte or macrophage is indicative of a pro-tumoral phenotype.

17. Use of an interleukin- blocker for modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage.

18. Use of an interleukin-1 blocker in the manufacture of a medicament for modulating pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage.

19. An interleukin-1 blocker for use in the modulation of pro-tumoral phenotype of a renal cell cancer-associated monocyte or macrophage.

20. Use of an interleukin-1 blocker for treating or preventing renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer-associated monocyte or macrophage.

21. Use of an interleukin-1 blocker in the manufacture of a medicament for treating or preventing renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer-associated monocyte or macrophage.

22. An interleukin-1 blocker for use in the treatment or prevention of renal cell cancer tumor growth in a subject, the renal cell cancer tumor growth being promoted by a renal cell cancer-associated monocyte or macrophage.