The combination of GM-CSF and inhibition of CCL22-CCR4 signaling in the treatment of cancer

Abstract

The invention relates to therapies comprising the combination of GM-CSF and the inhibition of CCL22-CCR4 signaling for use in enhancing immune response, e.g. for the treatment of immunogenic cancers. Accordingly, the invention provides methods comprising the administration of GM-CSF and the inhibition of CCL22-CCR4 signaling (e.g. by administration of a CCL22 inhibitor and / or a CCR4 inhibitor) (and the use of the therapeutics) for the treatment or amelioration of one or more symptoms of cancer in a subject in need thereof. The combination therapies provided herein may be combined with other anti-cancer therapies such as immunotherapy and / or treatments reliant on activation / elicitation of the immune response, e.g. vaccination therapies.

Description

[0001] The combination of GM-CSF and inhibition of CCL22-CCR4 signaling in the treatment of cancer

[0002] The invention relates to therapies comprising the combination of GM-CSF and the inhibition of CCL22-CCR4 signaling for use in enhancing immune response, e.g. for the treatment of immunogenic cancers. Accordingly, the invention provides methods comprising the administration of GM-CSF and the inhibition of CCL22-CCR4 signaling (e.g. by administration of a CCL22 inhibitor and / or a CCR4 inhibitor) (and the use of the therapeutics) for the treatment or amelioration of one or more symptoms of cancer in a subject in need thereof. The combination therapies provided herein may be combined with other anti-cancer therapies such as immunotherapy and / or treatments reliant on activation / elicitation of the immune response, e.g. vaccination therapies.

[0003] 1. BACKGROUND

[0004] Cancer is a major public health concern worldwide and the second leading cause of death in the United States. Approximately 1,762,450 cancer cases were diagnosed in 2019, the equivalent of more than 4,800 new cases each day, with an estimated 606,880 deaths (almost 1,700 per day). The lifetime probability of being diagnosed with invasive cancer is 39.3 % for men and 37.7 % for women (Siegel CA Cancer J Clin (2019) 69(1) :7-34). Although extensive research relating to cancer has been conducted and pharmaceuticals have been found there is still a significant need in the development of new treatment strategies.

[0005] Immunotherapy has revolutionized the treatment of cancer in recent years. Immunotherapy encompasses therapies designed to modify the endogenous immune response, for example, the use of checkpoint inhibitors, e.g. targeting immune suppressive signaling cascades (such as CTLA-4, PD-1 and PD-L1 signaling pathways), and / or the use of bispecific molecules (e.g. specific for a cancer antigen and an immune cell such as a T cell) to (re-)direct the immune response. However, immunotherapy also encompasses strategies based on recombinant modification of autologous or exogenous immune cells, e.g. chimeric antigen receptor (CAR) T cells in efforts to precisely control immune response. Nevertheless, many different mechanisms exists that can lead to the suppression of the (e.g. tumor-directed) immune responses, such that immunotherapy often fails or is insufficiently therapeutic.

[0006] Accordingly, there remains a need for the provision of further tools and methods for the treatment of cancers, in particular that may operate in conjunction with current immunotherapies and / or that may be considered immunogenic alone, e.g. that result in the elicitation of a desired, therapeutic immune response targeting the cancer.

[0007] 2. SUMMARY

[0008] The invention is based on the surprising finding that the combination of GM-CSF and inhibition of CCL22-CCR4 signaling promotes the endogenous immune response, e.g. as elicited by immunogenic cancers or as present in subjects suffering from immunogenic cancers, which immune response is effective in the treatment or amelioration of the cancer and / or or one or more symptoms thereof.

[0009] The promotion of the immune response is achieved by a therapy comprising the combination of GM-CSF and the inhibition of CCL22-CCR4 signaling. As used herein, "combination", "combination therapy" and analogous terms indicate that a first component (which term is used herein interchangeably with "modality") of the combination (e.g. the inhibition of CCL22- CCR4 signaling) is administered in addition to the second component / modality of the combination (e.g. GM-CSF) to the same individual. As such, the first component may be administered before, during, or after administration of the second component to the individual. The terms "first" and "second" with reference to components / modalities of the described combination treatments and therapies are used herein merely for convenience to differentiate the component / modalities and do not imply any order. For example the combination treatments and therapies provided herein encompass a first component (whether the inhibition of CCL22-CCR4 signaling or GM-CSF) administered before, concurrently with, or after the second component (whether the inhibition of CCL22-CCR4 signaling or GM-CSF); alternately worded, the combination treatments and therapies provided herein encompass a second component (whether the inhibition of CCL22-CCR4 signaling or GM-CSF) administered before, concurrently with, or after the first component (whether the inhibition of CCL22-CCR4 signaling or GM-CSF).

[0010] It is understood in this context that the combination therapy comprises a therapeutically effective combination of both components. Accordingly, the invention comprises the therapeutic combination of inhibition of CCL22-CCR4 signaling and GM-CSF in a single subject. It is understood that the combination of both components must be therapeutically effective. Thus, the combination therapy of the invention may comprise the combination of individually therapeutically effective (amounts of) components / modalities, i.e. the combination of (1) a therapeutic inhibition of CCL22-CCR4; and (2) a therapeutically effective amount of GM-CSF. However, the amounts of implementation of the individual components / modalities in the combination of the invention need not necessarily be therapeutically effective when considered alone. That is, the invention also encompasses the combination of amounts or implementations of components that individually exhibit no detectable activity (e.g. no therapeutic activity), but which combination does exhibit therapeutic activity.

[0011] The therapeutically effective combination of the invention may be achieved where the inhibition of CCL22-CCR4 signaling, and GM-CSF (e.g. exogenously administered GM-CSF) are administered / implemented concurrently (e.g. using a single pharmaceutical composition or separate pharmaceutical compositions administered together) or where they are administered / implemented separately (e.g. temporally separate administrations as described herein). Activity of the combination is demonstrated, e.g. by the increased or otherwise improved anti-tumor activity on implementation of the combination relative to the implementation of one component / modality alone, e.g. increased or improved activity associated with the inhibition of CCL22-CCR4 signaling additional to GM-CSF therapy (e.g. administration of exogenous GM-CSF) relative to activity of the GM-CSF therapy alone; and / or e.g. increased or improved activity associated with GM-CSF therapy additional to the inhibition of CCL22-CCR4 signaling relative to activity of the inhibition of CCL22-CCR4 signaling alone.

[0012] Therefore, provided are methods for the treatment of cancer in a subject in need thereon comprising the inhibition of CCL22-CCR4 signaling and the administration of GM-CSF, wherein said cancer is immunogenic and / or responsive to immunotherapy. Similarly, provided is the use of a first component / modality of the combination as described herein for the treatment of a cancer that is immunogenic and / or responsive to immunotherapy, which first component / modality is to be administered in combination with a second component / modality. Thus, the invention also provides at least (a) the inhibition of CCL22- CCR4 signaling for use in the treatment of cancer, wherein said inhibition is in combination with GM-CSF and wherein the cancer is immunogenic and / or responsive to immunotherapy; (b) GM-CSF for use in the treatment of cancer, wherein said GM-CSF is to be administered in combination with inhibition of CCL22-CCR4 signaling and wherein the cancer is immunogenic and / or responsive to immunotherapy; (c) a combination therapy comprising the inhibition of CCL22-CCR4 signaling and GM-CSF for use in the treatment of cancer, wherein the cancer is immunogenic and / or responsive to immunotherapy; and (d) a pharmaceutical composition comprising GM-CSF and an inhibitor of CCL22-CCR4 signaling for use in the treatment of cancer, wherein the cancer is immunogenic and / or responsive to immunotherapy.

[0013] The combination therapeutic methods and uses provided herein may be further combined with additional anti-cancer therapies as known in the art or described herein. Such additional anti-cancer therapy may include standard therapies (including but not limited to administration of one or more chemotherapeutics and / or implementation of one or more radiotherapies before, concurrently with or subsequent to the combination therapies as provided herein), as well as anti-cancer immunotherapies. In the context of one or more additional anticancer immunotherapies, it is preferred that the combination of (1) the combination therapeutic methods and uses of the invention and (2) the one or more additional anticancer therapies, improves anti-cancer activity relative to either treatment alone. The degree of improvement is not limited provided that the improvement is relative to either treatment alone, e.g. as determined in a similarly situated subject or group of subjects treated with one therapy alone (i.e. a reference standard), or as determined in the subject where treated with the one or more additional anti-cancer therapies or the method / use of the invention alone. The improvement of the combination of (1) the combination therapeutic methods and uses of the invention and (2) the one or more additional anticancer therapies may be additive or synergistic. The skilled person, e.g. treating physician, scientist or team of physicians and scientists, is readily capable of choosing additional treatments based on subject and cancer parameters based on knowledge in the art and standard medical practice.

[0014] A non-limiting exemplary embodiment of cancer immunotherapy that may be used in the context of the invention is adoptive immune cell transfer, including but not limited to dendritic cell vaccination or adoptive T cell transfer. Such cancer immunotherapy may also comprise administration of one or more additional cell stimulating agents as known in the art, such as Alum, CpG oligonucleotides, synthetic ligands forthe Toll-like receptor ?, Interleukin 4, ligands for RIG-1, ligands for MDA-5, ligands for STING, and PGE2.

[0015] Without being bound to any specific theory or hypothesis, the provided therapeutic combination methods and uses promote desirable immunogenic responses but are otherwise not cancer antigen or cancer specific. Thus, any cancer may be treated or targeted (e.g. to provide amelioration thereof or amelioration of one or more symptom thereof) provided that the cancer is immunogenic or otherwise responsive to immunotherapy. Non-limiting examples of immunogenic cancers include but are not limited to adrenal gland tumors, biliary cancer, bladder cancer, brain cancer, breast cancer, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer, hematopoietic cancer, esophageal cancer, gastrointestinal cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumors, ovarian cancer, pancreatic cancer, pancreatic islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, urogenital cancer, and uterine cancer. Preferred examples of immunogenic cancers include melanomas, hepatocellular carcinomas, pancreatic cancers, lung cancers, renal cancers, colon cancers, breast cancers, prostate cancers, bladder cancers, lymphomas, cancer with microsatellite instability, and cancers characterized by PDL- 1 expression.

[0016] The invention relates to exogenous GM-CSF as one component / modality of the therapeutic combination methods and uses of the invention. Any source or type of GM-CSF known in the art and / or described herein can be used as the exogenous GM-CSF in the context of the invention. As used herein, exogenous GM-CSF is understood to include any form or type of GM-CSF that is administered or delivered to the subject, and includes, for example, not only GM-CSF that is exogenously administered to the patient (e.g. via parenteral methods (such as intravenous, intramuscular, or subcutaneous administration) or enteral methods) but also GM-CSF delivered to the patient by recombinant / genetic means (e.g. via an administered cell that expresses GM-CSF (such as has been recombinantly modified to express GM-CSF)). Accordingly, the GM-CSF (exogenous GM-CSF) may be recombinantly produced GM-CSF (whether administered as the molecule or administered as a cell expressing it). The exogenous GM-CSF (e.g. (recombinant GM-CSF) may be human GM-CSF or may be a variant GM-CSF as known in the art.

[0017] The therapeutic inhibition of CCL22-CCR4 inhibition is also one component / modality of the therapeutic combination methods and used of the invention. Without being bound by any particular theory, in the absence of inflammatory or allergic processes, the CCL22-CCR4 axis is believed to positively regulate the recruitment and migration of regulatory T cells, leading to the inhibition of anti-tumor immunity. It is demonstrated for the first time that inhibition of this signaling pathway in combination with GM-CSF restores, increases and / or promotes antitumor activity, e.g. that otherwise elicited by the immunogenic cancer. The inhibition of CCL22-CCR4 signaling may comprise the use of a CCL22-CCR4 signaling inhibitor. The inhibitor may be a CCL22 antagonist, a CCR4 antagonist or a combination of a CCL22 antagonist and a CCR4 antagonist, preferably a CCL22 and / or a CCR4 antagonist that inhibits the binding of CLL22 to CCR4. Because CCR4 can also exhibit immuno-stimulatory activity via interaction with a second ligand, CCL17, it is further preferred that the inhibitor of CCL22-CCR4 signaling is a CCL22 antagonist or is an antagonist of CCR4 specific for CCL22, i.e. that does not or only minimally inhibits the binding of CCR4 to CCL17. Such antagonists may be antibodies or antigen binding fragments thereof, or may be small molecules.

[0018] It is further contemplated that the invention can also be applied in the context of exogenous cell therapies for the treatment of cancer, e.g. anti-cancer CAR T cell therapies. In non-limiting embodiments, the therapeutic methods and uses provided herein comprise the component / modality of inhibition of CCL22-CCR4 signaling wherein said inhibition is the inhibition of CCL22 expression or activity in a cell to be administered in connection with an anti-cancer therapy. The inhibition of CCL22-CCR4 signaling may comprise reducing or eliminating the expression of the CCL22 gene(s) in such a cell, including but not limited to recombinant methods such as knockout of the encoding or controlling gene(s) as well as RNA- based methods, including but not limited to RNA silencing, RNAi and crispr-cas9 based strategies. The inhibition of CCL22 activity in a cell to be administered in connection with cancer therapy may be any cell known in the art or described herein. Non-limiting examples as known in the art include autologous or exogenous cells, which include but are not limited to autologous or exogenous T cells or dendritic cells.

[0019] The herein described combination uses and methods comprise the administration of GM-CSF and / or the inhibitor of CCL22-CCR4 signaling in a weekly schedule, or a schedule of every 2, 3 or 4 weeks. The schedule may be maintained as readily determined by the skilled person according to standard medical practice, e.g. from about 1 to 100 weeks or for the lifetime of the subject. The first and second components / modalities of the provided therapeutic methods and uses can be administered or otherwise implemented concurrently (using the same or separate pharmaceutical compositions) or can be administered sequentially in any order. Where sequentially administered / implemented, the administration and / or implementation may be separated by at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least one week (i.e. at least about 7 days), or at least two weeks (i.e. at least about 14 days). It is preferred that where the components / modalities are separately administered / implemented at different time points, the administrations / implementations are separated by 12 hours to 2 days. Any suitable administration routes known in the art or described herein may be used. In nonlimiting examples, an inhibitor of CCL22-CCR4 signaling and / or the GM-CSF may be administered systemically, including parenterally (such as intravenously), subcutaneously, intramuscularly or intradermally. The inhibitor of CCL22-CCR4 signaling and / or the GM-CSF may also be administered intratumorally, e.g. direct injection into the tumor(s). Again, the administration / implementation regimen of the components / modalities will be determined by the skilled person according to the standard practice in the art pursuant to patient specifics so as to achieve the therapeutically effective combination. Further provided is a kit comprising a medicament comprising an inhibitor of CCL22-CCR4 signaling and a package insert comprising instructions for administration of the medicament in combination with GM-CSF; a kit comprising a medicament comprising GM-CSF and a package insert comprising instructions for administration of the medicament in combination with an inhibitor of CCL22-CCR4 signaling; and a kit comprising (a) a medicament comprising an inhibitor of CCL22-CCR4 signaling, (b) second medicament comprising GM-CSF and a package insert comprising instructions for administration of the two medicaments in combination. The herein described kits are for use in the treatment of cancer, preferably wherein the cancer is immunogenic and / or responsive to immunotherapy.

[0020] The invention also relates to, inter alia, the following items:

[0021] 1. Inhibition of CCL22-CCR4 signaling for use in the treatment of cancer, wherein said inhibition is to be in combination with exogenous GM-CSF and wherein the cancer is immunogenic and / or responsive to immunotherapy.

[0022] 2. GM-CSF for use in the treatment of cancer, wherein GM-CSF is to be administered in combination with inhibition of CCL22-CCR4 signaling and wherein the cancer is immunogenic and / or responsive to immunotherapy.

[0023] 3. A combination comprising the inhibition of CCL22-CCR4 signaling and exogenous GM- CSF for use in the treatment of cancer, wherein the cancer is immunogenic and / or responsive to immunotherapy.

[0024] 4. A pharmaceutical composition comprising GM-CSF and an inhibitor of CCL22-CCR4 signaling for use in the treatment of cancer, wherein the cancer is immunogenic and / or responsive to immunotherapy.

[0025] 5. The inhibition of CCL22-CCR4 signaling for the use according to item 1, the GM-CSF for the use according to item 2, or the combination for the use according to item 3, wherein said inhibition of CCL22-CCR4 signaling comprises the inhibition of CCL22 activity in an autologous or exogenous cell to be administered. The inhibition of CCL22-CCR4 signaling for the use according to item 5, the GM-CSF for the use according to item 5, or the combination for the use according to item 5, wherein said inhibition of CCL22 activity in an autologous or exogenous cell to be administered comprises recombinant knockout of the CCL22 gene. The inhibition of CCL22-CCR4 signaling for the use according to item 5 or 6, the GM-CSF for the use according to item 5 or 6, or the combination for the use according to item 5 or 6, wherein said autologous or exogenous cell to be administered is a dendritic cell. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 7, the GM-CSF for the use according to any one of items 2 and 5 to 7, the combination for the use according to any one of items 3 and 5 to 7, or the pharmaceutical composition for the use according to item 4, wherein at least one additional pharmaceutically active substance is to be administered. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 7, the GM-CSF for the use according to any one of items 2 and 5 to 7, the combination for the use according to any one of items 3 and 5 to 7, or the pharmaceutical composition for the use according to item 4, wherein said inhibition, GM-CSF, combination or pharmaceutical composition is combined with chemotherapy. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 7, the GM-CSF for the use according to any one of items 2 and 5 to 7, the combination for the use according to any one of items 3 and 5 to 7, or the pharmaceutical composition for the use according to item 4, wherein said inhibition, GM-CSF, combination or pharmaceutical composition is combined with radiotherapy. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 7, the GM-CSF for the use according to any one of items 2 and 5 to 7, the combination for the use according to any one of items 3 and 5 to 7, or the pharmaceutical composition for the use according to item 4, wherein said inhibition, GM-CSF, combination or pharmaceutical composition is combined with cancer immunotherapy. The inhibition of CCL22-CCR4 signaling for the use according to item 11, the GM-CSF for the use according to item 11, the combination for the use according to item 11, or the pharmaceutical composition for the use according to item 11, wherein said cancer immunotherapy comprises adoptive T cell transfer. The inhibition of CCL22-CCR4 signaling for the use according to item 11 or 12, the GM- CSF for the use according to item 11 or 12, the combination for the use according to item 11 or 12, or the pharmaceutical composition for the use according to item 11 or

[0026] 12, wherein the cancer immunotherapy comprises administration of dendritic cells. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 11 to

[0027] 13, the GM-CSF for the use according to any one of items 11 to 13, the combination for the use according to any one of items 11 to 13, or the pharmaceutical composition for the use according to any one of items 11 to 13, wherein said cancer immunotherapy comprises administration of one or more dendritic cell stimulating agents, such as Alum, CpG oligonucleotides, synthetic ligands for the Toll-like receptor 7, Interleukin 4, ligands for RIG-1, ligands for MDA-5, ligands for STING, or PGE2. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 14, the GM-CSF for the use according to any one of items 2 and 5 to 14, the combination for the use according to any one of items 3 and 5 to 14, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 14, wherein the cancer is selected from the group of melanoma, hepatocellular carcinoma, pancreatic cancer, lung cancer, renal cancer, colon cancer, breast cancer, prostate cancer, bladder cancer, lymphoma, cancer with microsatellite instability, and cancer characterized by PDL-1 expression The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 15, the GM-CSF for the use according to any one of items 2 and 5 to 15, the combination for the use according to any one of items 3 and 5 to 15, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 15, wherein the GM-CSF is recombinant GM-CSF. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 16, the GM-CSF for the use according to any one of items 2 and 5 to 16, the combination for the use according to any one of items 3 and 5 to 16, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 16, wherein the GM-CSF is human GM-CSF The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 17, the GM-CSF for the use according to any one of items 2 and 5 to 17, or the combination for the use according to any one of items 3 and 5 to 17, wherein said inhibition of CCL22-CCR4 signaling comprises the use of an inhibitor of CCL22-CCR4 signaling. The inhibition of CCL22-CCR4 signaling for the use according to item 18, the GM-CSF for the use according to item 18, the combination for the use according to item 18, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 17, wherein said GM-CSF and / or said inhibitor of CCL22-CCR4 signaling is to be administered in a weekly schedule, or a schedule of every 2, 3 or 4 weeks, and wherein said schedule is maintained from about 1 to 100 weeks or for the lifetime of the subject. The inhibition of CCL22-CCR4 signaling for the use according to item 18, the GM-CSF for the use according to item 18, the combination for the use according to item 18, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 17, wherein the inhibitor of CCL22-CCR4 signaling is administered before the GM-CSF. The inhibition of CCL22-CCR4 signaling for the use according to item 18, the GM-CSF for the use according to item 18, the combination for the use according to item 18, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 17, wherein the inhibitor of CCL22-CCR4 signaling is administered after the GM-CSF. The inhibition of CCL22-CCR4 signaling for the use according to item 18, the GM-CSF for the use according to item 18, the combination for the use according to item 18, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 17, wherein said inhibitor of CCL22-CCR4 signaling is administered concurrently with said GM-CSF, either as separate formulations or in the same formulation. The inhibition of CCL22-CCR4 signaling for the use according to item 20 or 21, the GM- CSF for the use according to item 20 or 21, the combination for the use according to item 20 or 21, or the pharmaceutical composition for the use according to item 20 or 21, wherein the time between the administration of said inhibitor of CCL22-CCR4 signaling and the administration of said GM-CSF is from about 12 hours to about 14 days, preferably from 12 hours to 2 days. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 23, the GM-CSF for the use according to any one of items 2 and 5 to 23, the combination for the use according to any one of items 3 and 5 to 23, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 23, wherein the inhibitor of CCL22-CCR4 signaling and / or the GM-CSF are administered systemically. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 23, the GM-CSF for the use according to any one of items 2 and 5 to 23, the combination for the use according to any one of items 3 and 5 to 23, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 23, wherein the inhibitor of CCL22-CCR4 signaling and / or the GM-CSF are injected into a solid tumor. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and

[0028] 5 to 23, the GM-CSF for the use according to any one of items 2 and 5 to 23, the combination for the use according to any one of items 3 and 5 to 23, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 23, wherein the inhibitor of CCL22-CCR4 signaling and / or the GM-CSF are administered subcutaneously. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 1 and 5 to 23, the GM-CSF for the use according to any one of items 2 and 5 to 23, the combination for the use according to any one of items 3 and 5 to 23, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 23, wherein the inhibitor of CCL22-CCR4 signaling and / or the GM-CSF are administered intradermally or intramuscularly. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 18 to

[0029] 27, the GM-CSF for the use according to any one of items 18 to 27, the combination for the use according to any one of items 18 to 27, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 27, wherein the inhibitor of CCL22-CCR4 signaling is a CCL22 antagonist, a CCR4 antagonist or a combination of a CCL22 antagonist and a CCR4 antagonist. The inhibition of CCL22-CCR4 signaling for the use according to item 28, the GM-CSF for the use according to item 28, the combination for the use according to item 28, or the pharmaceutical composition for the use according to item 28, wherein the CCL22 antagonist inhibits the binding of CCL22 to CCR4. The inhibition of CCL22-CCR4 signaling for the use according to item 28, the GM-CSF for the use according to item 28, the combination for the use according to item 28, or the pharmaceutical composition for the use according to item 28, wherein the CCR4 antagonist inhibits the binding of CCL22 to CCR4. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 18 to

[0030] 28, the GM-CSF for the use according to any one of items 18 to 28, the combination for the use according to any one of items 18 to 28, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 28, wherein said inhibitor of CCL22- CCR4 signaling is an anti-CCL22 antibody, an antigen binding fragment thereof or a small molecule. The inhibition of CCL22-CCR4 signaling for the use according to any one of items 18 to 28, the GM-CSF for the use according to any one of items 18 to 28, the combination for the use according to any one of items 18 to 28, or the pharmaceutical composition for the use according to any one of items 4 and 8 to 28, wherein said inhibitor of CCL22- CCR4 signaling is an anti-CCR4 antibody, an antigen binding fragment thereof or a small molecule. A method for treatment of cancer in a subject in need thereof comprising inhibiting CCL22-CCR4 signaling and administering GM-CSF in a therapeutically effective combination, wherein the cancer is immunogenic and / or responsive to immunotherapy. The method according to item 33, wherein said inhibition of CCL22-CR4 signaling comprises the administration of an inhibitor of CCL22-CCR4 signaling. The method according to item 34, wherein the inhibitor of CCL22-CCR4 signaling is administered before, after or concurrently with said GM-CSF, and wherein said inhibitor of CCL22-CCR4 signaling and said GM-CSF are in separate formulations or the same formulation. The method according to item 35, wherein said inhibitor of CCL22-CCR signaling and said GM-CSF are in separate formulations, and wherein the time between the administration of said inhibitor of CCL22-CCR4 signaling and said GM-CSF is from about 12 hours to about 14 days, preferably from 12 hours to 2 days. The method according to any one of items 34 to 36, wherein said GM-CSF and or / said inhibitor of CCL22-CCR4 signaling are administered in a weekly schedule, or a schedule of every 2, 3 or 4 weeks, and wherein said schedule is maintained from about 1 to about 100 weeks or for the lifetime of the subject. The method according to any one of items 34 to 37, wherein the inhibitor of CCL22-CCR4 signaling inhibits the binding of CCL22 to CCR4. The method according to any one of items 34 to 38, wherein the inhibitor of the CCL22- CCR4 signaling is a CCL22 antagonist, a CCR4 antagonist or a combination of a CCL22 antagonist and a CCR4 antagonist. The method according to any one of items 34 to 38, wherein the inhibitor of the CCL22- CCR4 signaling is an anti-CCL22 antibody or antigen binding fragment thereof; an anti- CCR4 antibody or antigen binding fragment thereof; or a small molecule. The method according to item 40, wherein the inhibitor of the CCL22-CCR4 signaling is an anti-CCL22 antibody or antigen binding fragment thereof, which antibody or fragment is a CCL22 antagonist antibody or fragment. The method according to item 40, wherein the inhibitor of the CCL22-CCR4 signaling is an anti-CCR4 antibody or antigen binding fragment thereof, which antibody or fragment is a CCR4 antagonist antibody or fragment. The method according to any one of items 34 to 42, wherein the inhibitor of CCL22-CCR4 signaling and / or the GM-CSF are administered systemically, subcutaneously, intramuscularly, intradermally, or are injected into a solid tumor. The method according to item 33, wherein said inhibition of CCL22-CCR4 signaling comprises the administration of an autologous or exogenous cell in which CCL22 activity has been inhibited. The method according to item 44, wherein said inhibition of CCL22 activity in an autologous or exogenous cell comprises recombinant knockout of the CCL22 gene. The method according to item 44 or 45, wherein said autologous or exogenous cell is a dendritic cell. The method according to any one of items 33 to 46, wherein at least one additional pharmaceutically active substance is administered. The method according to any one of items 33 to 47, wherein said method further comprises chemotherapy. The method according to any one of items 33 to 48, wherein said method further comprises radiotherapy. The method according to any one of items 33 to 49, wherein said method further comprises cancer immunotherapy. The method according to item 50, wherein the cancer immunotherapy comprises administering dendritic cells. The method according to item 50 or 51, wherein said cancer immunotherapy comprises administering one or more additional dendritic cell stimulating agents. The method according to any one of items 50 to 52, wherein said cancer immunotherapy comprises adoptive T cell transfer. The method according to any one of items 33 to 53, wherein the cancer is selected from the group of melanoma, hepatocellular carcinoma, pancreatic cancer, lung cancer, renal cancer, colon cancer, breast cancer, prostate cancer, bladder cancer, lymphoma, and cancer with microsatellite instability. The method according to any one of items 33 to 54, wherein the GM-CSF is recombinant GM-CSF. 56. The method according to item 55, wherein the GM-CSF is human GM-CSF

[0031] 57. The method according to any one of items 33 to 56, wherein the method enhances immune function in said subject.

[0032] 58. A kit comprising a medicament comprising an inhibitor of CCL22-CCR4 signaling and a package insert comprising instructions for administration of the medicament in combination with GM-CSF.

[0033] 59. A kit comprising a medicament comprising GM-CSF and a package insert comprising instructions for administration of the medicament in combination with an inhibitor of CCL22-CCR4 signaling.

[0034] 60. A kit comprising a first medicament comprising an inhibitor of CCL22-CCR4 signaling and a second medicament comprising GM-CSF.

[0035] 61. The kit according to any one of items 58 to 60 for use in the treatment of cancer, wherein the cancer is immunogenic and / or responsive to immunotherapy.

[0036] The therapeutic methods and combinations of the invention promote and enhance immune response, and thus, it is also contemplated that the herein disclosed methods and uses can be used as to enhance immune response generally, e.g. for use in combination with a vaccination or immunization regimen. Without being bound by any particular scientific theory or hypothesis, the simultaneous positive stimulation effected by GM-CSF (e.g. a therapeutically effective amount of GM-CSF) and suppression of negative immune regulation by inhibition of CCL22-CCR4 signaling may lead to an enhanced immune response against an antigen administered in connection with a vaccination or immunization regimen, e.g. improved immune response relative to the vaccination or immunization regimen alone. Accordingly, also provided is inhibition of CCL22-CCR4 signaling for use in enhancing an induced immune response in response to a vaccination or immunization protocol, wherein said inhibition is in combination with GM-CSF. Also provided is GM-CSF for use in enhancing an induced immune response in response to a vaccination or immunization protocol, wherein said GM-CSF is to be administered in combination with inhibition of CCL22-CCR4 signaling. Further provided is a combination of inhibition of CCL22-CCR4 signaling and GM-CSF for use in enhancing an induced immune response in response to a vaccination or immunization protocol. The invention also relates to a method for the enhancement of an induced immune response in response to a vaccination or immunization protocol in a subject in need thereof, said method comprising the therapeutically effective combination of inhibition of CCL22-CCR4 signaling and administration of GM-CSF.

[0037] 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1: Tumor growth and survival of GM-CSF treated Panc02-OVA tumor-bearing WT and CCL22- / - mice.

[0039] Wild-type (WT) or CCL22- / - mice were subcutaneously injected with 2 x 106Panc02-OVA cells. Starting at an average tumor size of 25 mm2(arrow), WT mice (n = 10) and CCL22- / - mice (n = 10) received 0.5 pg GM-CSF subcutaneously and peritumorally twice per week. As controls, groups of tumor-bearing WT mice (n = 5) and CCL22- / - mice (n = 5) were treated with PBS. Tumor growth (Figure 1A) and survival (Figure IB) were followed over time. Tumor size was determined every 2-3 days, and mice were sacrificed when termination criteria were reached (e.g. tumor size > 225 mm2, tumor exulceration, behavioral abnormalities, or tumor cachexia). Values are presented as mean + SEM (**** p < 0.0001, ns: not significant) and are representative of three independently performed experiments.

[0040] Figure 2: Tumor growth and survival of GM-CSF treated B16-F10-OVA tumor-bearing WT and CCL22- / - mice.

[0041] WT mice or CCL22- / - mice were subcutaneously injected with 0.5 x 106B16- F10-OVA cells. Starting at an average tumor size of 25 mm2(arrow), WT mice (n =8) and CCL22- / - mice (n = 8) received 0.5 pg GM-CSF subcutaneously and peritumorally twice per week. As controls, groups of tumor-bearing WT mice (n = 8) and CCL22- / - mice (n = 8) were treated with PBS. Tumor growth (Figure 2A) and survival (Figure 2B) were followed over time. Tumor size was determined every 2-3 days, and mice were sacrificed when termination criteria were reached (tumor size > 225 mm2, tumor exulceration, behavioral abnormalities, tumor cachexia). Values are presented as mean + SEM (*p < 0.05, ns: not significant)

[0042] Figure 3: Tumor growth and survival of GM-CSF treated Panc02 tumor-bearing WT and

[0043] CCL22- / - mice.

[0044] WT mice or CCL22- / - mice were subcutaneously injected with 2 x 106Panc02 cells. Starting at an average tumor size of 25 mm2(arrow), WT mice (n = 8) and CCL22- / - mice (n = 8) received 0.5 pg GM-CSF subcutaneously and peritumorally twice per week. As controls, groups of tumor-bearing WT mice (n = 8) and CCL22- / - mice (n = 8) were treated with PBS. Tumor growth (Figure 3A) and survival (Figure 3B) were followed over time. Tumor size was determined every 2-3 days, and mice were killed when termination criteria were reached (tumor size > 225 mm2, tumor exulceration, behavioral abnormalities, tumor cachexia). Values are presented as mean + SEM and are representative of two independently performed experiments. No significant difference in tumor growth among the groups was observed.

[0045] Figure 4: Tumor growth and survival of GM-CSF treated Panc02-OVA tumor-bearing

[0046] CCL22- / - mice upon CD8 T cell depletion.

[0047] CCL22- / - mice were subcutaneously injected with 2 x 106Panc02-OVA cells. Upon a tumor size of 25 mm2mice received either an anti-CD8 antibody (500 pg in 100 pl PBS) (n = 8) or an isotype antibody of the same concentration (n = 8), the procedure was repeated once after 7 days. 2 days after the first antibody application the CCL22- / - mice received 0.5 pg GM-CSF subcutaneously and peritumorally twice per week. Tumor growth (Figure 4A) and survival (Figure 4B) were followed over time. Tumor size was determined every 2-3 days, and mice were killed when termination criteria were reached (tumor size > 225 mm2, tumor exulceration, behavioral abnormalities, tumor cachexia). Values are presented as mean + SEM (**** p < 0.0001).

[0048] 4. DETAILED DESCRIPTION

[0049] GM-CSF has been suggested to be an important maturation and activation signal for dendritic cells (DCs) (Mach et al., Cancer research 60,12 (2000). 3239-46). In the context of tumor diseases, activated DCs are the basic prerequisite for effective T-cell priming and thus for the initiation of an antitumor immune response (Fu and Jiang, Front Immunol 9(2018), 3059). Because of these properties, GM-CSF has been intensively evaluated as a potential immunostimulant for antitumor immunotherapies in preclinical models as well as in clinical trials (Yan et al., Immunotherapy 9(2017), 347-360). While some of these studies have reported initial responses, no definitive or significant survival benefit has yet been demonstrated (Jinushi and Tahara, Cancer Sci 100(2009), 1389-1396; Metcalf, Cancer Immunol Res 1(2013), 351-356).

[0050] CCL22 belongs to the group of chemokines that are both constitutively expressed under homeostatic conditions and inducible on inflammation. Several types of immune cells, such as macrophages, dendritic cells (DCs), B cells, and T cells, secrete CCL22 upon activation. The only yet known receptor of CCL22 is CCR4, which is expressed, inter alia, by activated Th2-biased CD4+T cells (D'Ambrosio et al., J Immunol 161(1998), 5111-5115) and by regulatory T cells (Tregs) (lellem et al., J Exp Med 194(2001). 847-854). CCL22 is recognized in certain conditions to act as an immunosuppressive. For example, it has been reported that the immune response in CCL22-deficient mice is increased (Rapp et al., J Exp Med 216(2019), 1170-1181) and that agents that inhibit CCL22 or the interaction with the receptor CCR4 enhance T cell immunity (Robles et al., J Med Chem 63(2020), 8584-8607). It has also been demonstrated that the expression of CCL22 can be strongly enhanced by the addition GM-CSF (Piseddu et al., J Immunol 205(2020), 2056-2065). Thus, the immunostimulatory capacity of GM-CSF may be counteracted by its property to induce CCL22, which is, again, known to act as an immunosuppressant. However, the inventors surprisingly found that combinations of inhibition of CCL22-CCR4 signaling and exogenous GM-CSF are able to enhance, improve or otherwise promote immune responses. In particular, it was surprisingly demonstrated that the combination of GM-CSF and inhibition of CCL22-CCR4 signaling promotes endogenous immune response allowing the treatment of immunogenic cancers.

[0051] Thus, provided are the combination of a first component / modality that is the inhibition of CCL22-CCR4 signaling and a second component / modality that is exogenous GM-CSF for the enhancement, improvement or promotion of immune response. The combination encompasses (a) the combination for use in therapy; as well as (b) the first or second component / modality for use in therapy , which first or second component or modality is to be administered / implemented in combination with the second or first component / modality, respectively. The invention also encompasses methods for use in therapy in a subject in need thereof comprising the administration / implementation of the first and second component / modality in combination. The first and second component / modality may be administered / implemented in any order and together or separately (e.g. by means of a single or separate pharmaceutical compositions). Further, where separately administered / implemented, the first and second component modality may be temporally separated, e.g. administered / implemented in any order at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least one week (i.e. at least about 7 days), or at least two weeks (i.e. at least about 14 days).

[0052] The disclosed methods and uses are of benefit in any therapy where the enhancement, improvement or promotion of an immune response is desired (such as in combination with a vaccine or immunization regimen), and are, in particular, of use for the treatment or amelioration of an immunogenic cancer or a cancer that is responsive to immunotherapy, or the treatment of amelioration of one or more symptom thereof. The combination of the first and second component / modalities is therapeutically effective to enhance, improve or promote an immune response for therapy. Thus, the combination may comprise a therapeutic first component (e.g. a therapeutic inhibition of CCL22-CCR4 signaling) and a therapeutic second component (e.g. administration of a therapeutically effective amount of GM-CSF), however, the individual components / modalities of the combination need not be necessarily therapeutic individually. That is, the therapeutic combination may comprise a first component that is inhibition of CCL22-CCR4 that does not alone exhibit therapeutic efficacy and a second component that is exogenous GM-CSF that is present or administered at a level / dose that alone is not therapeutically effective.

[0053] The terms "combination therapy", "in combination", "combination" and analogous terms as used herein denote any form of concurrent or parallel treatment with the two described components / modalities of the combination, i.e. the inhibition of CCL22-CCR4 signaling and GM-CSF, e.g. exogenously administered GM-CSF. The combination uses and methods described herein encompass co-administration / co-implementation of the first and second components / modalities in a substantially simultaneous manner, or in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the combination therapy. The terms do not imply that the first and second components / modalities must be physically mixed or administered / implemented at the same time and / or formulated for delivery / implementation together, although these methods and uses, and corresponding formulations, are within the scope described herein. The first and second component / modality of the described combination(s) can be administered concurrently with, prior to, or subsequent to, each other, in any order and may further be administered / implemented concurrently with, prior to, subsequent to, or interspersed among one or more other additional therapies or therapeutic agents, e.g. anti-cancer therapies or anti-cancer agents. In general, each component / modality will be administered / implemented at a dose and / or on a time schedule determined for that agent such that the therapeutic effect is obtained when combination with the other component / modality. In general, it is expected that the components / modalities of the disclosed combination(s) are utilized at levels that do not exceed the levels at which they may utilized individually. In some embodiments, the levels utilized in the combination are lower than those utilized as single-agent therapeutics. Where the first and second component / modality are administered / implemented separately, the term "in combination" includes sequential or concomitant administration. For example, when the first component / modality (inhibition of CCL22-CCR4 signaling) is administered / implemented before administration of one or more doses of GM-CSF, it may be may be administered / implemented at least about 4 weeks, at least about 3 weeks, at least about 2 weeks, at least about 1 week, at least about 6 days, at least about 5 days, at least about 4 days, at least about 3 days, at least about 2 days, at least about 24 hours, at least about 20 hours, at least about 18 hours, at least about 16 hours, at least about 14 hours, at least about 12 hours, at least about 10 hours, at least about 9 hours, at least about 8 hours, at least about 7 hours, at least about 6 hours, at least about 5 hours, at least about 4 hours, at least about 3 hours, at least about 2 hours, at least about 1 hour, at least about 45 minutes, at least about 30 minutes or at least about 20 minutes prior to the administration of one or more doses of GM-CSF. When the first component / modality (inhibition of CCL22-CCR4 signaling) is administered / implemented after the administration of one or more doses of GM- CSF, it may be administered at least about 4 weeks, at least about 3 weeks, at least about 2 weeks, at least about 1 week, at least about 6 days, at least about 5 days, at least about 4 days, at least about 3 days, at least about 2 days, at least about 24 hours, at least about 20 hours, at least about 18 hours, at least about 16 hours, at least about 14 hours, at least about 12 hours, at least about 10 hours, at least about 9 hours, at least about 8 hours, at least about 7 hours, at least about 6 hours, at least about 5 hours, at least about 4 hours, at least about 3 hours, at least about 2 hours, at least about 1 hour, at least about 45 minutes, at least about 30 minutes or at least about 20 minutes after the administration of the one or more doses of GM-CSF.

[0054] Where the first and second component / modality are administered / implemented concurrently, it may indicate that the first and second component / modality are administered / implemented with the same pharmaceutical composition or formulation. Concurrent administration also may indicate the administration / implementation of the first and second modality using separate pharmaceutical compositions, formulations or dosage forms within less than 2 hours, less than 1.5 hours, less than 1 hour, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes or less than 10 minutes of each other, in any order.

[0055] By "therapeutically effective amount", "therapeutic" and analogous terms with reference to an individual component / modality, other substance or the combination as disclosed herein, is meant that a given substance or combination is administered to or implemented in a subject suffering from a condition, in a sufficient amount, in a single or multiple doses to alleviate or partially arrest the condition or one or more of its symptoms, i.e. in a treatment of the condition. The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and / or may be therapeutic in terms of partially or completely curing the disease or condition, and / or adverse effect attributed to the disease or condition. The term "treatment" as used herein covers any treatment of a disease or condition in a subject and includes: (a) preventing and / or ameliorating a proliferative disease (preferably cancer) from occurring in a subject that may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development, such as inhibition of cancer progression; (c) relieving the disease, i.e. causing regression of the disease, such as the repression of cancer; (d) preventing, inhibiting or relieving any symptom or adverse effect associated with the disease or condition; (e) preventing, inhibiting, relieving or otherwise decreasing one or more disease markers; and / or (f) increasing the frequency or duration or symptom-free periods. Preferably, the term "treatment" as used herein relates to medical intervention of an already manifested disorder, e.g. the treatment of a diagnosed cancer.

[0056] The exact amount and level of a component / modality of the disclosed combination, e.g. the amount of GM-CSF to be administered or have expressed in a subject and / or the level of inhibition of CCL22-CCR4 signaling (such as obtained by the administration of a CCL22-CCR4 signaling inhibitor) will vary depending on the particular compound or composition used, its mode of administration, patient specifics and the like. The appropriate effective amount can be determined by one of ordinary skill in the art informed by the present disclosure and according to common general knowledge and standard medical practice. Therapeutic efficacy or toxicity of any component / modality, other substance or the combination (e.g. sufficient for any treatment as defined herein) can be determined by standard pharmaceutical procedures, e.g. using cell cultures or experimental animals to determine ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). Treatment also includes prophylaxis and treatment of relapse, as well as the alleviation of acute or chronic signs, symptoms and / or malfunctions associated with the indication targeted by therapy. Treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, over a medium term, or can be a long-term treatment, such as, forexample within the context of a maintenance therapy. Administrations of the first component / modality, the second component modality and / or the combination of the first and second component / modality (in any order or concurrently) may be intermittent, periodic, or continuous so as to maintain a therapeutically effective combination.

[0057] As used herein, the term "subject" may be interchangeably used with the term "patient", "subject in need thereof" or "patient in need thereof". The expressions indicate a non-human mammal, but preferably a human, that exhibits one or more symptoms or indications of a disease or condition that would benefit from the enhancement, improvement or promotion of an immune response. Such subject in need thereof include subjects undergoing vaccination or immunizations protocols or regimens, but preferably indicates a subject who has been diagnosed with cancer or who had cancer or a cancer diagnosis previously. The subject, preferably human, may be diagnosed with a primary or a metastatic tumor and / or with one or more symptoms or indications including, but not limited to, enlarged lymph node(s), swollen abdomen, chest pain / pressure, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, enlargement of spleen, itching. The expression includes patients who have received one or more cycles of anti-cancer therapy (including chemotherapy or radiotherapy). The expression "a subject in need thereof" may also include patients with cancer that has been treated but which has subsequently relapsed or metastasized. For example, patients that may have received treatment with one or more anticancer agents leading to tumor regression; however, subsequently have relapsed with cancer resistant to the one or more anti-cancer agents (e.g., chemotherapy-resistant cancer) may be treated with the uses and methods described herein. 4.1 Inhibition of CCL22-CCR4 signaling

[0058] The methods and uses described herein comprise the inhibition of CCL22-CCR4 signaling. In this context, inhibition refers to inhibiting, blocking, abrogating or interfering with the activity of the CCL22-CCR4 signaling pathway in any manner. The term "CCL22-CCR4 signaling" or "CCL22-CCR signaling pathway" may be used interchangeably with "CCL22-CCR4 signaling cascade", "CCL22-CCR4 axis", "CCL22-CCR4 signaling axis", "CCL22-CCR4 pathway" and analogous terms refers to the complete biochemical cascade upstream and downstream of the CCR4 receptor. As is known in the art, CCL22 binds to and activates the CCR4 receptor leading to a signaling cascade in the CCR-expressing cell ultimately resulting in the modification of the expression of certain genes and / or proteins. The modified expression of these genes and / or proteins is recognized in the art as part of the CCL22-CCR4 signaling cascade, in particular, "downstream of the CCR4 receptor". Thus the invention not only encompasses direct action on CCL22 and / or CCR4 for inhibition, but also encompasses the inhibition of any factor downstream of CCR4 that is known or suspected to act in the CCL22- CCR4 signaling pathway. Thus, inhibition of CCL22-CCR4 signaling also encompasses the inhibition any factor downstream of CCR4 mediating CCL22 induced signaling, including but not limited to the genes or proteins having expression that is induced or modified by CCL22- CCR4 signaling. Similarly, inhibition of CCL22-CCR4 signaling also encompasses the inhibition of any factor "upstream" of CCL22 expression. Accordingly, any factor upstream of CCL22 may be inhibited to modify or inhibit CLL22 expression or activity and, thus, inhibit CCL22-CCR4 signaling. For example, any factor mediating / inducing the release or production of CCL22 may be inhibited or modified to inhibit CCL22-CCR4 signaling.

[0059] Accordingly, inhibition of CCL22-CCR4 signaling may be direct, i.e. encompass the use of molecules that directly target (e.g. bind to) CCL22 and / or CCR4 and inhibit their ability to interact with one another. However, the invention also encompasses the inhibition of CCL22- CCR4 signaling indirectly, i.e. encompasses the use of molecules that target any factor upstream of CCL22 to inhibit its expression activity, or molecules that target any factor downstream of CCR4 to inhibit the propagation of CCL22 mediated CCR4 signaling. Thus, the invention encompasses all suitable molecules that may interfere with or inhibit the physiological / biochemical action of CCL22, CCR4 or upstream or downstream factors that mediate, e.g. their expression or signaling activity. Non-limiting examples of such molecules, include small molecules and biological macromolecules, e.g. including nucleic acids (such as RNAi molecules) or polypeptides / proteins. Preferred examples of polypeptides / proteins include dominant negative proteins and antibodies / antibody-derived or antibody-like molecules (including antibody derived antigen binding fragments and chimeric constructs comprising antibody antigen binding domains).

[0060] Non limiting examples of CCR4 inhibitors / antagonists that may be used include C021 dihydrochloride (Chemical name: 2-[l,4'-Bipiperidin]-l'-yl- / \ / -cycloheptyl-6,7-dimethoxy-4- quinazolinamine dihydrochloride) available from e.g. Tocris; CCR4-351 (Chemical name 1H- Pyrazolo[3,4-b]pyrazine-3-carbonitrile, l-[(lR)-l-(2,4-dichlorophenyl)ethyl]-6-[3-[(3R)-l-(2- hydroxyethyl)-3-piperidinyl]-l-azetidinyl]) available from e.g. MCE MedChemExpress; Chemical name: 2-[(3R)-3-[l-[l-[(lR)-l-(2,4-dichlorophenyl)ethyl]-3-

[0061] (trifluoromethyl)pyrazolo[3,4-b]pyrazin-6-yl]azetidin-3-yl]piperidin-l-yl]ethanol; AZD-2098 (Chemical name: 2,3-Dichloro-N-(3-methoxy-2-pyrazinyl)benzenesulfonamide) as available from e.g. SigmaAldrich; Compound 22 / BMS-397 (Bristol-Meyers Squibb, disclosed in, e.g. Purandare et al., Bioorg Med Chem Lett 17(2007), 679-682); SP50 / AF399 (as disclosed in, e.g. Bayry et al., PNAS USA 105(2008), 10221-10226; Bozza et al., Biomolecules 11(2021), 351; Pere et al., Blood 118(2011), 4853-4862; Klein et al., Oncotarget 8(2017), 31079-31091; and EP-A1 2303324); AF399 / 42018025 (as disclosed in, e.g. Bayry et al., PNAS USA 105(2008), 10221-10226; Bosschem et al., PLoS One 10(2015), e013164; Bayry et al., VirusDisease 25(2013), 18-25); AF399 / 420018078 (as disclosed in, e.g. Bayry et al., PNAS USA 105(2008), 10221-10226); Co21 (as disclosed in, e.g. Yokoyama et al., Bioorg Med Chem 17(2009), 64-73); GSK2239633 (GSK, as disclosed in, e.g. Cahn et al., BMC Pharmacol Toxicol 14(2013), article 14); AZD-2098 and AZD-1678 (AstraZeneca, as disclosed in, e.g. Kindon et al., ACS Med Chem Lett 8(2017), 981-986); RPT193 / Zelnecirnon (RAPT Therapeutics, as disclosed in, e.g. Bissonnette et al., Allergy 79(2024), 924-936, epub Nov. 20, 2023); Compound 31 (RAPT Therapeutics, as disclosed in, e.g. Jackson et al., J Med Chem 62 (2019), 6190-6213); FLX475 (RAPT Therapeutics, as disclosed in, e.g. WO2018 / 022992; NCT04894994; NCT04768686; NCT03674567); CCR4-351 (RAPT Therapeutics, as disclosed in, e.g. Jorapur et al., Pios Pathog 18(2022), el010200; Marshall et al., J Immunother Cancer 8(2020), e000764); and mogamulizumab (as disclosed in, e.g. Ishii et al., Clin Cancer Res 16(2010), 1520-1531; 1 Yamamoto et al., J Clin Oncol 28(2010), 1591-1598; Ogura et al., J Clin Oncol 32(2014), 1157- 1163).

[0062] As known in the art, CCR4 also exhibit immuno-stimulatory activity mediated by interaction with CCL17. Thus it is preferred that the inhibition of CCL22-CCR4 signaling is CCL22 specific, i.e. not impacting or minimally impacting CCL17 mediated CCR4 signaling. A non-limiting example of such an inhibitor is a CCL22 antagonist or is an antagonist of CCR4 specific for CCL22, i.e. that does not or only minimally inhibits the binding of CCR4 to CCL17. The CCL22 antagonist and the CCL22 specific CCR4 antagonist may or may not referto the same molecule, e.g. a molecule that inhibits the binding of CCL22 to CCR4 and / or CCL22 mediated CCR4 signaling in any manner. The term "antagonist" is used herein in the broadest sense and relates generally to a molecule that interferes with or inhibits the physiological / biochemical action of another substance. Thus, a CCL22 antagonist or CCL22 specific CCR4 antagonist may interfere with or inhibit the physiological / biochemical activity of CCL22 (including its expression). The CCL22 antagonist or CCL22 specific CCR4 antagonist may also bind to CCR4 and prevent the binding of CCL22 or the eliciting of the CCL22 mediated signal cascade, but, preferably does not substantially interfere with the activity of CCL17 or the CCL17 mediated CCR4 signal cascade.

[0063] In some embodiments, the CCL22 antagonist or CCL22 specific CCR4 antagonist is an antibody or antibody-like / antibody-derived molecule. The term "antibody," as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (i.e., "full antibody molecules"), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof.

[0064] As used herein, the terms "antigen-binding fragment" of an antibody and the like include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex derived from an antibody. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phageantibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0065] Non-limiting examples of antigen-binding fragments include Fab fragments; F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; dAb fragments; and minimal recognition units, e.g. comprising amino acid residues that mimic the hypervariable region of an antibody such as an isolated complementarity determining region (CDR), e.g. a CDR3 peptide). Other antibody-derived or antibody-like molecules include but are not limited to domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0066] In embodiments of the therapeutic combination methods and uses described comprising the use of antibody therapeutics, the antibodies or antibody derived molecules may be human antibodies or derived from human antibodies. As used herein, the term "human antibody" refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The human antibody may be a recombinant human antibody. As used herein, the term "recombinant human antibody" includes all human antibodies that are prepared, expressed, created or isolated by recombinant means as known in the art, e.g. humanized antibodies; chimeric antibodies; antibodies isolated from a recombinant, combinatorial antibody library (e.g. a recombinant, combinatorial human antibody library); antibodies isolated from animals comprising human antibody genes, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.

[0067] The CCL22-CCR4 signaling inhibitors of the methods and uses disclosed herein may be antibodies (or antigen-binding fragments thereof) or antibody derived molecules that specifically bind CL22 and / or CCR4 as described herein. The term "specifically binds" or the like means that the molecule forms a complex with an antigen that is stable under physiologic conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody, antibody fragment or antibody derived molecule that "specifically binds" CCL22 or CCR4 includes molecules that bind CTLA4 antibody or a portion thereof with a KD of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or less than about 0.5 nM, e.g. as measured by surface plasmon resonance assay. As understood in the art, the term specifically binds does not exclude that the molecules may have crossreactivity with other antigens.

[0068] Non-limiting examples of known anti CCL22 antibodies that may be used according to the methods and uses herein include MAB366 (R&D Systems, disclosed in, e.g. Kbtting et al., Cancers (Basel) 16(2021), 1335; Yang et al., Oncol Lett 16(2018), 5389-5397; Mizukami er al., Int J Cancer 15(2008), 2286-2293), AF336 (R&D Systems, disclosed in e.g. Sarkar et al., Diabetes 17(2012), 436-446; Castriconi et al., J Immunol 190(2013), 5321-5328), MAB4391 (R&D Systems, disclosed in, e.g. Shaniel et al., J Exp Med 188(1998), 451; Chang et al., J Biol Chem 272(1997), 25229); AF439 (R&D Systems, disclosed in, e.g. Araujo-Pires et al., J Bone Miner Res 30(2015), 412-422; Li et al., eLife (2023), June 2;12:e80443; Montane et al., J Clin Invest 121(2011), 3024); and AF15083D (BioLegend, e.g. AB_2941631 (Cat. No. 694402), AB_2650730, (Cat. Nos. 694403 and 694404), AB_3097162 (Cat. No. 694405)). The inhibition of CCL22-CCR4 signaling may also be achieved using nucleic acid molecules including, but not limited to, small hairpin RNA (shRNA), siRNA, antisense nucleotides and the like. A small hairpin RNA or short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules of about 20-25 base pairs in length. siRNA interferes with the expression of specific genes with complementary nucleotide sequences. siRNA functions by causing mRNA to be broken down after transcription resulting in no translation.

[0069] Inhibition of CCL22-CCR4 signaling may also be achieved by gene knockout. "Knockout" refers to the partial or complete reduction of expression of at least a portion of a polypeptide encoded by a gene, e.g., the CCL22 gene, where the gene is endogenous to a cell, such as a dendritic cell. "Knockout" also encompasses situations where biological function is reduced, but where expression is not necessarily reduced, e.g., an expressed CCL22 polypeptide that contains an inserted inactivating peptide, oligopeptide, or polypeptide. Disruptions in a coding sequence or a regulatory sequence are encompassed by the knockout technique. The "knockout" may be a "heterozygous knockout", where one allele of the endogenous gene has been disrupted. Alternatively, the "knockout" may be a "homozygous knockout" where both alleles of the endogenous gene have been disrupted. "Homozygous knockout" is not intended to limit the disruption of both alleles to identical techniques or to identical outcomes at the genome. All suitable techniques may be employed for a knockout such as CrispR / Cas and the like.

[0070] 4.2 GM-CSF

[0071] The methods and uses described herein comprise the use of GM-CSF, e.g. the administration of exogenous GM-CSF. GM-CSF is known to have considerable effect on the function of mature myeloid cells, including enhanced antibody production, phagocytosis and cytotoxicity. In the context of the invention, it is preferred that GM-CSF is present (e.g. exogenously administered via parenteral or enteral means or via expression by an administered cell, e.g. recombinantly administered cell) in an amount sufficient to increase circulating neutrophils.

[0072] Any source of GM-CSF can be used in accordance with the therapeutic combination methods and uses herein, e.g. whether administered directly or via an administered cell that recombinantly produces GM-CSF. The GM-CSF may be derived from non-human mammals, but is preferably GM-CSF of the species to be treated. Thus, where the subject is a human, it is preferred that the GM-CSF is human GM-CSF. Sources of GM-CSF and GM-CSF encoding sequences are known in the art. For example, production of recombinant GM-CSF (rGM-CSF) by culturing a host cell transformed with a vector comprising a gene coding for a primate GM- CSF protein is disclosed in EP-B1 0 188 479. Procedures for the production of recombinant human GM-CSF have also been described in U.S. Pat. No. 4,810,643 and by Burgess et al. Blood 69(1987), 43-51. rGM-CSF is also commercially available, e.g. Molgramostim, sold by Schering-Plough under the trade name Leucomax®; and Sargramostirn, sold by Schering AG in Germany under the trade name Leukine®.

[0073] GM-CSF proteins in accordance with the therapeutic methods and uses disclosed herein may also be modified by changing the (native) amino acid sequence. For example, from 1 to 5 amino acids in the sequences may be changed, or the sequences may be lengthened, without changing the fundamental character thereof and provide modified proteins which are the full functional equivalents of the native proteins. A GM-CSF differing by a single amino acid from the common native sequence is disclosed in U.S. Pat. No. 5,229,496 and has been clinically demonstrated to be a biological equivalent of native GM-CSF (known as GM-CSF (Leu-23)).

[0074] The natural or recombinantly prepared proteins, and their functional equivalents are preferably purified and substantially cell-free, which may be accomplished by known procedures. Additionally or alternately the exogenously administered GM-CSF may be produced by cells, e.g. tumor targeting cells, engineered to express and secrete GM-CSF. Without being bound by any specific theory, GM-CSF secretion at the tumor site or at the site of vaccination / immunization is expected to recruit and differentiate dendritic cells (DCs) and / or otherwise enhance immune response at the target site. For example, activated and antigen-loaded DCs can migrate to the draining lymph nodes and activate tumor antigenspecific T cells (CD4 and CD8) that can recirculate to metastatic sites and kill tumor cells. Accordingly, in some embodiments, the combination as disclosed herein comprises recombinantly modified cells that are a source of the GM-CSF, for example, an MHC negative cell line. A non-limiting embodiment of such a cell line is the K562 cell line (available from the American Type Culture Collection, Manassas, Va.). As recognized in the art, the use of such cells reduces the incidence of allogeneic responses to the cells on introduction into individuals.

[0075] 4.2 Immunogenic cancers

[0076] The combination therapy of the invention is of particular relevance in the context of an existing or induced immune response. With respect to cancer, the combination therapy may be used to treat immunogenic cancers or cancers otherwise responsive to immunotherapy. For example it is demonstrated that administration of GM-CSF in mice deficient in CCL22-CCR4 signaling was most efficient when the model tumors expressed the highly immunogenic OVA antigen (Figure 3).

[0077] The term "cancer" or "proliferative disease" as used herein means any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art. In particular, the herein described uses and methods are particularly suitable for the treatment of immunogenic cancers. The term "immunogenic cancer" as used herein means that the cancer induces an immune response. A cancer may induce an immune response when it expresses or presents cancer antigens that are recognized by the immune system as foreign antigens. When these antigens are presented in the context of MHC molecules, T cells (including T helper cells and CD8+ cytotoxic T cells) may be activated and an immune response is induced. Whether the cancer is immunogenic is readily determined by the skilled person using standard procedures routine in the art. An immunogenic cancer as used herein may comprise one or more of the following characteristics:

[0078] (a) tumor infiltrating lymphocytes (TIL), for example but not limited to, 5 or more TIL, 6 or more TIL, 7 or more TIL, 8 or more TIL, 9 or more TIL, or 10 or more TIL per 1000 tumor cells; (b) mutations, for example but not limited to 1 or more, 10 or more, 100 or more, 200 or more, 300 or more, or 400 or more somatic mutations per megabase of tumor genomic DNA;

[0079] (c) neoantigens, for example but not limited to., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, neoantigen(s) with one or more endogenous T cell receptor(s) and / or one or more idiotype clone that recognizes a processed and presented moiety of the neoantigen;

[0080] (d) tertiary lymphoid structures;

[0081] (e) high expression of inflammatory gene expression, for example but not limited to, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold increased expression of a cytokine above baseline expression in non-cancerous tissue; and / or

[0082] (f) immune cells exhibiting an immunosuppressive phenotype, for example but not limited to dendritic cells lacking cytokine expression.

[0083] Methods for assessing the immunogenic characteristics of cancers are known to the person skilled in the art; e.g. as disclosed in Eerola et al., Clin Cancer Res 6(2000), 1875-81; Alexandrov et al., Nature 500(2013), 415-21; Gubin et al., Nature. 515(2014), 577-81; Dieu-Nosjean et al., Trends Immunol 35(2014), 571-80; Dudek et al., Front Immunol 4(2013), 438; Eisenhauer et al., Eur J Cancer 45(2009), 228-47.

[0084] The cancer may also be characterized as a cancer that is responsive to immunotherapy, e.g. PD-1 inhibition, PD-L1 inhibition, CTLA-4 inhibition, and tumor vaccination. The determination of whether a given cancer is responsive to immunotherapy is known in the art and may be performed by the skilled person using established and routine procedures; e.g. as disclosed in Yarchoan et al., N Engl J Med. 377(2017), 2500-2501; Lawlor et al., Cancers 13(2021), 3119; Strickler et al., Clin Cancer Res 27(2021), 1236-1241.

[0085] Nonlimiting examples of immunogenic cancers and / or cancers that are responsive to immunotherapy that can be treated according to the methods and uses disclosed herein include but are not limited to adrenal gland tumors, biliary cancer, colon cancer, endocrine or neuroendocrine cancer, hematopoietic cancer, liver cancer, meningioma, multiple neuroendocrine type I and type II tumors, osteogenic sarcoma tumors, parathyroid cancer, pheochromocytoma, pituitary tumors, testicular cancer, tracheal cancer, gastrointestinal cancer (such as colorectal cancer, rectal cancer, pancreatic cancer, pancreatic islet cell cancer, gastric cancer, esophageal cancer, stomach cancer, hepatocellular cancer, and cholangiocellular cancer); urogenital cancer (such as hormone sensitive prostate cancer, hormone refractory prostate cancer, renal cell cancer, bladder cancer, and penile cancer); gynecological cancer (such as ovarian cancer, uterine cancer, cervical cancer, and endometrial cancer); lung cancer (such as small-cell lung cancer and non-small-cell lung cancer); head and neck cancer (such as squamous cell cancer, oral cancer, lip cancer, nasopharyngeal cancer, oropharyngeal cancer); central or peripheral nervous system tissue cancer (such as malignant glioma, astrocytoma, retinoblastoma and brain metastasis); malignant mesothelioma; breast cancer (such as hormone refractory metastatic breast cancer); skin cancer (such as malignant melanoma, basal cell skin cancer, squamous cell skin cancer, Merkel cell carcinoma, lymphoma of the skin, and Kaposi Sarcoma); thyroid cancer; bone or soft tissue sarcoma; hematologic neoplasia (such as multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, or Hodgkin's lymphoma). Preferred cancers to be treated according to the herein provided methods and uses are melanoma, hepatocellular carcinoma, pancreatic cancer, lung cancer, renal cancer, colon cancer, breast cancer, prostate cancer, bladder cancer lymphoma, cancer with microsatellite instability and cancer characterized by PDL-1 expression.

[0086] In the context of treatment of cancer, the treatment may produce a therapeutic effect selected from one or more of delay in tumor growth, reduction in tumor cell number, tumor regression, increase in survival, partial response, complete response and / or amelioration of one or more symptoms associated with the cancer. In some embodiments, the tumor growth is delayed by at least 10 days as compared to tumor growth in an untreated patient. In some embodiments, the tumor growth is inhibited by at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%) as compared to an untreated patient. 4.3 Pharmaceutical compositions

[0087] The term "pharmaceutical composition" is used interchangeably with the term "medicament" and relates to a composition suitable for administration to a patient, preferably a human patient. The (pharmaceutical) compositions as described herein may be in solid / lyophilized or liquid form and may be, e.g., in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). Furthermore, it is envisaged that the (pharmaceutical) compositions as described herein may comprise further biologically active agents, depending on the intended use of the pharmaceutical composition. The (pharmaceutical) compositions as described herein may further comprise a pharmaceutically acceptable carrier, excipient and / or diluent. The carrier, excipient and / or diluent must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. Examples of suitable pharmaceutical carriers, excipients and / or diluents are well known in the art and include but are not limited to phosphate buffered saline solutions or other buffer solutions, water, emulsions, such as oil / water emulsions and various types of wetting agents. Compositions comprising such carriers can be formulated by well-known conventional methods.

[0088] Suitable carriers may comprise any material as long as GM-CSF and / or the inhibitor or compound effecting inhibition of CCL22-CCR4 signaling described herein retain their biological and / or pharmaceutical activity upon contact with said carriers. Pharmaceutical compositions may include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solutions include but are not limited to propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous solutions include water, alcoholic / aqueous solutions, including saline and buffered media. Pharmaceutical compositions may further include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride or lactated Ringer's. Pharmaceutical compositions may include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. The pharmaceutical compositions as described herein may also comprise preservatives and other additives including but not limited to antimicrobials, antioxidants, chelating agents and / or inert gases and the like. In addition, a pharmaceutical composition as described herein may comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.

[0089] The methods and uses disclosed herein encompass the combination of the individual components / modalities implemented by administration of (exogenous) components, i.e. administration of (exogenous) GM-CSF and / or the administration of an inhibitor of CCL22- CCR4 signaling as defined herein. Such GM-CSF and / or inhibitor(s) may be administered concurrently (in the same or separate pharmaceutical compositions), or temporally separated in any order, e.g. by at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least one week (i.e. at least about 7 days), or at least two weeks (i.e. at least about 14 days).

[0090] The individual components of the combination according to the method and uses disclosed herein may be administered together or separately on the same or different schedules, e.g. together or individually hourly, daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, weekly (i.e. about every 7 days), every 2 weeks, every 3 weeks or every 4 weeks. As recognized in the art, the required dosage of a component / modality need not be administered in a single administration. For example, a daily dosage may be divided into multiple smaller dosages to be administered throughout the day. The timings of the administrations need not be coincident, e.g. one component can be administered every two weeks while the other is administered every three weeks. It is within the common skill in the art to determine the treatment regimen (dosage and timing) to treat a cancer and / or a subject. In a non-limiting example, the skilled person can consult the treatment specifications forthe separate components / modalities when used as individual therapies. For example, with respect to GM-CSF, the treatment specifications for the GM-CSF medicament LEUKINE® (sargramostin). As appreciated in the art, the dosing regimen will depend on the inhibitor used, e.g. a small molecule inhibitor may be required to be administered up to 3 times daily, whereas an antibody may be required to be administered only once every 4 weeks.

[0091] The combination therapy as disclosed herein may be administered from at least about 1 to

[0092] 100 weeks, e.g. at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 weeks. The combination therapy as disclosed herein may also be administered as a maintenance therapy, i.e. for the lifetime of the subject.

[0093] 4.4 Additional therapies

[0094] The methods, uses and / or pharmaceutical compositions as disclosed herein may be used or implemented in a treatment or therapy alone (comprising the only active therapeutic compounds or actions) or may be used or implemented in combination with appropriate treatment protocols for the particular disease or condition as known in the art. Non-limiting examples of such protocols in the context of cancer include but are not limited to, administration of pain medications, administration of chemotherapeutics, therapeutic radiation, and surgical handling of the disease, conditions or symptoms thereof. Accordingly, the treatment regimens disclosed herein encompass inhibition of CCL22-CCR4 signaling and GM-CSF administration together with none, one, or more than one treatment protocol suitable for the treatment or prevention of a disease, condition or a symptom thereof, either as described herein or as known in the art. The herein described uses, methods and pharmaceutical compositions may be administered / implemented alone or in combination with other therapies or treatments during periods of active disease, or during a period of remission or less active disease. The skilled person can readily determine suitable other anticancer therapies and regimens for a given cancer using standard medical practice.

[0095] Nonlimiting examples of chemotherapeutic agents that may be of use in connection with the invention include anti-metabolites / anti-cancer agents, such as pyrimidine analogs (5- fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, epidipodophyllotoxins (teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, iphosphamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, paclitaxel, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes (e.g., dacarbazinine (DTIC)); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); immunosuppressives (cyclosporine, tacrolimus (FK-5O6), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors, epidermal growth factor (EGF) inhibitors); anti-sense oligonucleotides; antibodies (trastuzumab); mTOR inhibitors; topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan); corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers; caspase activators; chromatin disruptors; and combinations thereof.

[0096] Nonlimiting examples of radiotherapies that may be of use in connection with the invention include, but are not limited to, fractioned external involved field irradiation, brachytherapy and whole body irradiation approaches.

[0097] The invention may also be used in the context of immunotherapy. Immunotherapy can include any of a variety of interventions by which the activity levels of one or more cells of the adaptive immune system are altered (e.g. up- or downregulated). For example, the intervention can be used with known methods of inducing, recruit, enhance or otherwise potentiate an adaptive immune response, which may be an antigen-specific immune response. Immunotherapy as used herein may comprise administration of one or more specific antibodies that recognize adaptive immune system cells to alter the immunological activity of such cells. Other immunotherapeutic approaches may include the use of cytokines that similarly can directly or indirectly alter an immune response. Immunotherapy may also comprise inhibition of negative regulators of adaptive immune responses, such as inhibition of CTLA4, LAG3, and / or PD-1. Furthermore, in context of immunotherapy, vaccines are envisaged that elicit adaptive immune responses such as antigen-specific responses to tumor-associated antigens.

[0098] Immunotherapy as used herein may also comprise adoptive cell therapy, such as adoptive T cell transfer (including CAR T cell transfer). The terms "adoptive cell therapy" and "adoptive cell transfer" may be used interchangeably herein. Adoptive cell therapy may refer to the transfer of cells, most commonly immune-derived cells, back into the same patient or into a new recipient host with the goal of transferring a new or an enhanced immunologic functionality and / or characteristics into the patient or the new recipient host. The use of autologous cells may minimize problems regarding graft-versus-host disease. The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) (Zacharakis et al., Nat Med (2018) 24(6):724-730; Besser et al., Clin Cancer Res (2010) 16 (9) 2646-55; Dudley et al., Science (2002) 298 (5594):850-4; and Dudley et al., J Clin Oncol (2005) 23(10):2346-57.) or genetically re-directed peripheral blood mononuclear cells (Johnson et al., Blood (2009) 114 (3):535-46; and Morgan et al., Science (2006) 314(5796):126-9) has been used to successfully treat patients with advanced solid tumors, including melanoma, metastatic breast cancer and colorectal carcinoma, as well as patients with CD19-expressing hematologic malignancies (Kalos et al., Sci Transl Med (2011) 3 (95):95ra73) and is envisaged herein in context of immunotherapy.

[0099] In context of adoptive cell therapy, cells of the immune system are transferred, such as T cells, preferably specific for selected antigens, such as tumor associated antigens or tumor specific neoantigens (see, e.g., Maus et al., Annu Rev Immunol (2014) 32:189-225; Rosenberg and Restifo, Science (2015) 348(6230):62-68; Restifo et al., Nat Rev Immunol (2015) 12(4):269- 281; Jenson and Riddell, Immunol Rev (2014) 257(1):127-144; and Rajasagi et al., Blood (2014) 124(3):453-62). Non-limiting examples of the antigen to be targeted include, but are not limited to MR1; B cell maturation antigen (BCMA); PSA (prostate-specific antigen); prostatespecific membrane antigen (PSMA); PSCA (Prostate stem cell antigen); Tyrosine-protein kinase transmembrane receptor ROR1; fibroblast activation protein (FAP); Tumor-associated glycoprotein 72 (TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2 (ERBB2 (Her2 / neu)); Prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1 receptor (IGF-1R); gplOO; BCR-ABL (breakpoint cluster region-Abelson); tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO-1); MAGE (melanoma antigen); Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain; Human papillomavirus (HPV) E6; HPV E7; prostein; survivin; PCTA1 (Galectin 8); Melan-A / MART-1; Ras mutant; TRP-1 (tyrosinase related protein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2 / INT2 (TRP-2 / intron 2); RAGE (renal antigen); receptor for advanced glycation end products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1, RU2); intestinal carboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant; thyroid stimulating hormone receptor (TSHR); CD123; CD171; CD19; CD20; CD22; CD26; CD30; CD33; CD44v7 / 8 (cluster of differentiation 44, exons 7 / 8); CD53; CD92; CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); Tn antigen (Tn Ag); Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (I L-13Ra2); Interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR- beta); stage-specific embryonic antigen-4 (SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16); epidermal growth factor receptor (EGFR); epidermal growth factor receptor variant III (EGFRvlll); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); Proteasome Subunit LMP2; ephrin type-A receptor 2 (EphA2); Ephrin B2; Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l- l)Cer); TGS5; high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); G protein- coupled receptor class C group 5, member D (GPRCSD); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placentaspecific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT (cancer / testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53; p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; Cyclin DI; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma Antigen Recognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocytespecific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRLS); mouse double minute 2 homolog (MDM2); livin; alphafetoprotein (AFP); transmembrane activator and CAML Interactor (TACI); B-cell activating factor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP (707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4 cells); BAGE (B antigen; b-catenin / m, b-catenin / mutated); CAMEL (CTL-recognized antigen on melanoma); CAP1 (carcinoembryonic antigen peptide 1); CASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated); CDK4 / m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM (differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2); EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein); fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (G antigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicose antigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ring tumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR / FUT (low density lipid receptor / GDP L-fucose: b-D-galactosidase 2-a- L fucosyltransferase); L1CAM (LI cell adhesion molecule); MC1R (melanocortin 1 receptor); Myosin / m (myosin mutated); MUM-1, -2, -3 (melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patient M88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen (h5T4); pl90 minor bcr-abl (protein of 190KD bcr-abl); Pml / RARa (promyelocytic leukaemia / retinoic acid receptor a); PRAME (preferentially expressed antigen of melanoma); SAGE (sarcoma antigen); TEL / AML1 (translocation Ets-family leukemia / acute myeloid leukemia 1); TPI / m (triosephosphate isomerase mutated); CD70; and any combination thereof.

[0100] Immunotherapy as used herein also encompasses dendritic cell (DC) vaccination as known in the art, for example as disclosed in Schwarze et al., Curr Opin Oncol 2(2023), 87-93; Hernandez et al., Int J Mol Sci 19(2022), 11397; Santos et al., J Immunol 2(2018), 443-449; and Sabado et al., Cell Res 1(2017), 74-95. Thus, the therapeutic methods and uses disclosed herein are also contemplated to combined with administration of one or more dendritic cell stimulating agents. Non-limiting examples of dendritic cell stimulating agents include Alum, CpG oligonucleotides, synthetic ligands for the Toll-like receptor 7, Interleukin 4, ligands for RIG-1, ligands for MDA-5, ligands for STING (Pan et al., Biochem Pharmacol 213(2023), 115596) and PGE2.

[0101] In the context of adoptive cell transfer, the invention further encompasses inhibition of CCL22- CCR signaling, wherein the inhibition is the recombinant modification of the cells to be transferred such that they do not promote or induce CCL22-CCR4 signaling (e.g. do not express CCL22 and / or CCR4) and / or do not exhibit CCL22 mediated CCR4 signaling (e.g. by recombinant modification to remove or inhibit CCL22 induced activity of CCR4 and / or one or more necessary component of the CCL22-CCR4 signaling cascade). Accordingly, inhibition of CCL22-CCR4 signaling as used herein include systemic inhibition of the signaling cascade, inhibition at the tumor or vaccination / immunization site, and also includes inhibition (exclusively) in cells to be administered as part of adoptive cell therapy as known in the art.

[0102] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. For example, the (genetically engineered) lymphocytes may be administered to the subject at a dose of 104to IO10cells / kg body weight, preferably 105to 106cells / kg body weight. The lymphocytes may be administered in such a way that an upscaling of the cells to be administered is performed by starting with a subject dose of about 105to 106cells / kg body weight and then increasing to dose of IO10cells / kg body weight.

[0103] The therapeutic methods and uses disclosed herein need not be implemented concurrently with the one or more additional anti-cancer agents / therapies. For example, it is envisaged that a patient is, e.g. first treated with chemotherapy / radiotherapy / immunotherapy and several days or even weeks later treated according to the disclosed combination therapy of the invention. Accordingly, the herein described uses and methods may be performed / executed / employed before, after or concurrently to an additional treatment such as chemotherapy, radiotherapy or radiotherapy.

[0104] However, in the context of immunotherapy and without necessarily being bound by scientific theory it is envisaged that the herein described uses and methods enhance / improve / prolong / extend the efficiency / activity of a cancer immunotherapy. Accordingly, in some embodiments the herein described uses and methods are performed / executed / employed in temporal proximity to a cancer immunotherapy.

[0105] 4.5 Kits

[0106] The invention also provides kits that are applicable for the therapeutic uses and methods disclosed herein. It is envisaged that the kit comprises instructions for the treatment of an immunogenic cancer in a subject in need thereof. The kit may comprise one or both the first and second component / modalities of the therapeutic combination methods and uses described herein. Thus, the kit may comprise (i) a first component / modality (e.g. allowing / implementing / effecting inhibition of CCL22-CCR4 signaling) together with a package insert (e.g. comprising instructions) for use in combination with the second component / modality (e.g. GM-CSF); (ii) a second component / modality (e.g. allowing / implementing / effecting administration of GM-CSF) together with a package insert (e.g. comprising instructions) for use in combination with the first component / modality (e.g. inhibition of CCL22-CCR4 signaling); or (iii) a first component / modality (e.g. allowing / implementing / effecting inhibition of CCL22-CCR4 signaling) and a second component / modality (e.g. allowing / implementing / effecting administration of GM-CSF) together with a package insert (e.g. comprising instructions) that the first and second components / modalities be used in combination. It is preferred that the disclosed kits are for use in the treatment of cancer, preferably wherein the cancer is immunogenic and / or responsive to immunotherapy.

[0107] In the foregoing detailed description of the invention, a number of individual elements, characterizing features, techniques and / or steps are disclosed. It is readily recognized that each of these has benefit not only individually when considered or used alone, but also when considered and used in combination with one another. Accordingly, to avoid exceedingly repetitious and redundant passages, this description has refrained from reiterating every possible combination and permutation. Nevertheless, whether expressly recited or not, it is understood that such combinations are entirely within the scope of the presently disclosed subject matter.

[0108] All technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. 5. EXAMPLES

[0109] The following materials and methods are applicable to all examples below

[0110] 5.1 Materials and Methods

[0111] Mice

[0112] Female and male C57BL / 6J WT mice were purchased from Janvier (Saint-Berthevin Cedex, France). CCL22- / - mice on C57BL6 background were obtained from the Knockout Mouse Project (KOMP), a project of the National Institutes of Health (NIH). All experimental animals were between 4 and 10 weeks old at the start of the experiment, and were age-matched for each experiment. All animal experiments were approved by the government of Upper Bavaria.

[0113] Tumor cells

[0114] The murine pancreatic cell line PancO2 was aquired from ATCC. The PancO2-OVA cell line was generated by recombinantly modifying PancO2 cells (via retrovirus transduction) to express the chicken derived Ovalbumin antigen (OVA). Both Panc02 and PancO2-OVA have been previously described, e.g., in Jacobs et al., Int J Cancer 128(2011), 897-907. The murine pancreatic cell line B16-F1 (further referred to as B16) was aquired from ATCC. B16-OVA cell line was generated by retroviral transduction of B16 to express the chicken derived Ovalbumin antigen (OVA). Both B16 and B16-OVA have been widely used as immunological tumor models (e.g. Ji et al., Hum Vaccin Immunother 17(2021), 1910-1922; Van Damme et al., J Immunother Cancer 9(2021), e001749).

[0115] Tumor cells were maintained in culture flasks in full medium containing RPMI, 10% FCS, 2mM L-glutamin, 100 lU / ml penicillin and 100 pg / ml streptomycin. Confluent cultures were passaged using standard techniques and trypsin-EDTA at 1:10, approximately 3 times per week.

[0116] Tumor induction and monitoring

[0117] For tumor induction, tumor cell cultures were split 1:2 one day before the planned inoculation. On the day of the inoculation, adherent cells were solubilized with trypsin-EDTA and washed three times to remove FCS residues. 2xl06tumor cells were suspended in 100 pl PBS and injected subcutaneously into the left flank of the mouse under isofluorane short anesthesia. Tumor growth was monitored three times per week using an electronic caliper (length X width in mm). Mice were euthanized if tumor size exceeded 225 mm2, tumor exulceration occurred, tumor-related deterioration of general condition was observed, or tumor cachexia occurred.

[0118] GM-CSF treatment

[0119] GM-CSF was administered as 0.5 pg murine recombinant GM-CSF (Miltenyi Biotech, Bergisch Gladbach) in 100 pl PBS injected subcutaneously into the left flank.

[0120] 5.2 Treatment of Panc02-OVA tumor bearing CCL22 deficient mice with GM-CSF

[0121] WT mice or CCL22 / _mice were subcutaneously injected with 2 x 106Panc02-OVA cells. Starting at an average tumor size of 25 mm2(arrow), WT mice (n = 10) and CCL22- / - mice (n = 10) received 0.5 pg GM-CSF subcutaneously and peritumorally twice per week. As controls, groups of tumor-bearing WT mice (n = 5) and CCL22- / - mice (n = 5) were similarly treated with PBS. Tumor growth and survival were followed over time. Tumor size was determined every 2-3 days. Figure 1 represents the tumor growth and survival in three independently performed experiments. It was shown that GM-CSF treatment was highly effective in inhibiting tumor growth and prolonging survival in CCL22-deficient but not in wild-type mice. These findings demonstrate the significant anti-cancer activity of the combination of inhibition of CCL22 signaling and GM-CSF. Without being bound by any therapy, it is believed that inhibiting the CCL22-CCR4 pathway increases the limited efficacy of GM-CSF as yet observed in cancer therapy.

[0122] 5.3 Treatment of B16-OVA tumor-bearing CCL22-deficient mice with GM-CSF

[0123] In order to evaluate the anti-tumor efficacy of GM-CSF in the setting of CCL22-deficiency in another tumor model, the same experiment was performed in B16-OVA tumor-bearing mice. WT mice as well as in CCL22- / - mice were subcutaneously injected with 0.5 x 106B16-F10-OVA cells. Starting at an average tumor size of 25 mm2(arrow), WT mice (n =8) and CCL22- / - mice (n = 8) received 0.5 pg GM-CSF subcutaneously and peritumorally twice per week. As controls, groups of tumor-bearing WT mice (n = 8) and CCL22- / - mice (n = 8) were treated with PBS. Tumor growth and survival were followed over time. The enhancement of GM-CSF efficacy in the setting of CCL22 deficiency could also be shown in this model (Figure 2).

[0124] 5.4 Treatment of Panc02 tumor bearing CCL22 deficient mice with GM-CSF

[0125] In the Panc02-OVA and the B16-OVA tumor models used above the cancer cells express the antigen OVA, that may promote immunogenicity of these tumors. To test whether the therapeutic efficacy of GM-CSF in CCL22-deficient mice is applicable in non-OVA expressing tumors the same experiment as in (1) and (2) was performed using the Panc02 tumor cell line. WT mice as well as CCL22- / - mice were subcutaneously injected with 2 x 106Panc02 cells. Starting at an average tumor size of 25 mm2(arrow), WT mice (n = 8) and CCL22- / - mice (n = 8) received 0.5 pg GM-CSF subcutaneously and peritumorally twice per week. As controls, groups of tumor-bearing WT mice (n = 8) and CCL22- / - mice (n = 8) were treated with PBS. Tumor growth and survival were followed over time (Figure 3). Here, no benefit of CCL22- deficiency was observed for GM-CSF treatment. This demonstrates that GM-CSF treatment may be most efficient in immunogenic tumors or upon a tumor vaccination (Figure 3).

[0126] 5.5 Treatment of Panc02-OVA tumor bearing CCL22 deficient mice with GM-CSF and anti- CD8 antibody

[0127] To evaluate whether GM-CSF efficiency in CCL22 deficiency depends on cytotoxic T cells a similar experiment as in (1) was performed using CD8-depleting antibodies. CCL22- / - mice were subcutaneously injected with 2 x 106Panc02-OVA cells. Upon a tumor size of 25 mm2mice received either an anti-CD8 antibody (500 pg in 100 pl PBS) (n = 8) or an isotype antibody of the same concentration (n = 8), the procedure was repeated once after 7 days. 2 days after the first antibody application the CCL22- / - mice received 0.5 pg GM-CSF subcutaneously and peritumorally twice per week. Tumor growth and survival were followed over time (Figure 4). Indeed, it was demonstrated that the anti-tumor effect of GM-CSF was dependent on CD8 T cells. This also shows that GM-CSF induces an anti-tumor immune response and fits with the hypothesis that immunogenic tumors may benefit most from this therapeutic approach (Figure 4). 5.6 Conclusions

[0128] GM-CSF has been proposed as a treatment for cancer due to its activity in the activation of dendritic cells. However, it has as yet been demonstrated to have no or very limited efficacy in clinical studies.

[0129] It has now been surprisingly demonstrated that the combination of inhibition of CCL22-CCR4 signaling and GM-CSF provides significant anti-tumor activity in immunogenic cancers. The anti-tumor effects have been demonstrated in murine tumor models expressing the OVA protein as a model for a strong tumor antigen. Therefore, it is believed that CCL22-CCR4 blockade in combination with GM-CSF is effective in promoting or enhancing immune response and, thus, that the combination therapy will be effective in immunogenic tumors as well as in any setting were enhancement of immune response is desired, e.g. in the context of vaccination or immunization protocols.

Claims

CLAIMS1. A combination comprising the inhibition of CCL22-CCR4 signaling and GM-CSF for use in the treatment of cancer, wherein the cancer is immunogenic and / or responsive to immunotherapy.

2. The combination for the use according to claim 1, wherein said inhibition of CCL22-CCR4 signaling comprises or is obtained by (a) the inhibition of CCL22 activity in an autologous or exogenous cell to be administered; and / or (b) an inhibitor of CCL22-CCR4 signaling.

3. The combination for the use according to claim 2, wherein the inhibitor of CCL22-CCR4 signaling is a CCL22 antagonist, a CCR4 antagonist or a combination of a CCL22 antagonist and a CCR4 antagonist.

4. The combination for the use according to any one of claims 1 to 3, wherein the GM-CSF is recombinant GM-CSF; or is to be administered via a recombinantly modified autologous or exogenous cell to be administered.

5. The combination for the use according to any one of claims 2 to 4, wherein the inhibitor of CCL22-CCR4 signaling and the GM-CSF are to be administered in a single or separate pharmaceutical formulations.

6. The combination for the use according to any one of claims 2 to 4, wherein the inhibitor of CCL22-CCR4 signaling is administered before or after the GM-CSF; or wherein the inhibitor of CCL22-CCR4 signaling is administered concurrently with GM-CSF, either in separate formulations or in the same formulation.

7. The combination for the use according to claim 6, wherein the inhibition of CCL22-CCR4 signaling and the GM-CSF are to be administered in separate formulations and the time between the administration of said inhibitor of CCL22-CCR4 signaling and theadministration of said GM-CSF is from about 12 hours to about 14 days, preferably from 12 hours to 2 days.

8. The combination for the use according to any one of claims 4 to 7, wherein the administration is systemic (such as intravenous), injection into a solid tumor, subcutaneous, intradermal, intramuscular, or a combination thereof.

9. The combination for the use according to any one of claims 1 to 8, wherein said combination is to be combined with chemotherapy or cancer immunotherapy.

10. The combination for the use according to claim 8 to be combined with cancer immunotherapy, wherein said cancer immunotherapy comprises adoptive T cell transfer or administration of dendritic cells.

11. The combination for the use according to any one of claims 1 to 10, wherein said combination enhances immune function in said subject.

12. A kit comprising a first medicament comprising an inhibitor of CCL22-CCR4 signaling and a package insert comprising instructions for administration of the first medicament in combination with GM-CSF; or comprising a second medicament comprising GM-CSF and a package insert comprising instructions for administration of the second medicament in combination with an inhibitor of CCL22-CCR4 signaling.

13. A kit comprising a first medicament comprising an inhibitor of CCL22-CCR4 signaling and a second medicament comprising GM-CSF; or comprising a first medicament comprising an inhibitor of CCL22-CCR4 signaling and GM-CSF.

14. The kit according to claims 12 or 13 for use in the treatment of cancer, wherein the cancer is immunogenic and / or responsive to immunotherapy.

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