WO2013083659A1

WO2013083659A1 - Combination treatment comprising ho - 1 inhibitor and immunotherapeutic agent

Info

Publication number: WO2013083659A1
Application number: PCT/EP2012/074570
Authority: WO
Inventors: Douglas Fearon; James Arnold
Original assignee: Cambridge Enterprise Limited
Priority date: 2011-12-05
Filing date: 2012-12-05
Publication date: 2013-06-13
Also published as: GB201120860D0

Abstract

This invention relates to the finding that HO-1 inhibitors reduce the immunosuppressive effects of CD45+ FAP+ stromal cells in tumours and increase the efficacy of immunotherapeutic agents, such as cancer vaccines. Cancer therapies involving combinations of HO-1 inhibitors, such as SnMP, and immunotherapeutic agents are disclosed.

Description

Cancer Immunotherapy

Field of Invention

This invention relates to the field of cancer treatment with immunotherapy .

Background to Invention

Immunotherapy has always been an attractive and potentially effective treatment for cancer patients . The discovery that human melanomas express non-mutated tumour antigens that spontaneously elicit a CD8+ T cell response (Van der Bruggen et al . , 1991 Science Dec 13; 254 (5038) :1643-7) and the subsequent identification and molecular definition of a series of tumour antigens expressed by various human tumours (reviewed in Van den Eynde & Brichard, 1995 Curr . Opin . Immunol . Oct; 7 (5) : 674-81), enabled evaluation of immunotherapeutic strategies aimed at inducing specific anti-tumour immune responses against such antigens . These immunotherapeutic vaccination approaches are currently being evaluated in phase III clinical trials . Despite this and the related clinical success of cancer targeted immunotherapies with the FDA approval of the cell-based cancer vaccine Sipuleucel-T ( Provenge™, Dendreon) and the monoclonal antibody Ipilimumab (Bristol-Myers Squibb) for melanoma, there remains underlying concerns that currently approved cancer immunotherapies demonstrate a limited response rate (e.g. 20% of treated patients ) and only an incremental benefit in cancer patients in modest extension of life .

Immunogenic cancer cells may evade immune destruction by disabling or co-opting components of the immune system that would normally eliminate them. For example , cancer cells may paralyze infiltrating CTLs and NK cells, by secreting TGF-β or other immunosuppressive factors . Additional mechanisms may operate through the recruitment of inflammatory cells that are actively immunosuppressive , including regulatory T cells (Tregs ) and myeloid-derived suppressor cells (MDSCs ) , which can suppress the actions of cytotoxic lymphocytes. Immune suppression by tumour stromal cells within the tumour microenvironment is a maj or determinant of the modest effectiveness of cancer

immunotherapies , such as therapeutic cancer vaccines .

Significant cellular heterogeneity is found within tumour microenvironment as a result of the tumour stromal compartment of recruited, ostensibly normal cells (stromal cells ) which may acquire mechanisms capable of subverting or evading the immune response that tumour antigens elicit . Of the two general types of nonvascular stromal cells , hematopoietic and mesenchymal , the former, which includes myeloid-derived suppressor cells

(J. Exp. Med.181 : 35, 1995; Immunol. Rev. 222 : 162, 2008) , M2 macrophages (Nature, 45 : 436, 2008) , and certain natural killer T cells have been considered more often in this context than mesenchymal cells , which have usually been studied as human carcinoma-associated fibroblasts in xenografted, immune- deficient mice (Cell 121:335, 2005) . Nevertheless , a tumoural stromal cell of apparent mesenchymal origin- identifiable by its expression of the type I I membrane dipeptidyl-peptidase

fibroblast activation protein-alpha (FAP) (Proc . Natl . Acad. Sci . U.S.A. 87:7235, 1990) - is associated with other biological process in which immune suppression may occur (Cancer Immun . 3:10, 2005, Arthritis Res. Ther. 8 :R171, 2006) .

Recently, the present inventors identified that a subset of FAP+ cells which are positive for CD45 (also known as Protein tyrosine phosphatase , receptor type, C or PTPRC) are

immunosuppressive . Depletion or ablation of CD45+, FAP+ stromal cells was shown to lead to tumour regression in an immunogenic LL2 /OVA tumour model by a process involving interferon-gamma and tumour necrosis factor-alpha (Kraman et al 2010 Science 330 827- Summary of Invention

This invention is based on the observation that targeting CD45+, FAP+, HO-1+ tumour stromal cells with an inhibitor of HO-1 slows the growth of an immunogenic tumour . This may be useful for enhancing the efficacy of immunotherapeutic agents such as cancer vaccines in treating cancer .

An aspect of the invention provides a method of treating cancer comprising administering a HO-1 inhibitor and immunotherapeutic agents , such as a cancer vaccine , to an individual in need thereof .

As used herein, HO-1 inhibitors may include any of the following compounds; SnMP, ZnMP, FeMP, CrMP, SnPP, ZnPP, CrPP, Syb-0702, PEG-ZnPP, RTA-403, OB-24, and OB-28. Of particular relevance is the HO-1 inhibitor SnMP.

Other aspects of the invention provide an HO-1 inhibitor for use in combination with an immunotherapeutic agent , such as a cancer vaccine , in the treatment of cancer, and an immunotherapeutic agent , such as a cancer vaccine, for use in combination with an HO-1 inhibitor in the treatment of cancer .

Another aspect of the invention provides the use of an HO-1 inhibitor and an immunotherapeutic agent , such as a cancer vaccine , in the manufacture of a medicament for use in the treatment of cancer .

Other aspects of the invention provide the use of an

immunotherapeutic agent, such as a cancer vaccine, in the manufacture of a medicament for use in combination with an HO-1 inhibitor in the treatment of cancer in an individual and the use of an HO-1 inhibitor in the manufacture of a medicament for use in combination with an immunotherapeutic agent , such as a cancer vaccine, thereof for the treatment of cancer in an individual . Other aspects of the invention provide pharmaceutical

compositions and kits comprising an HO-1 inhibitor and an immunotherapeutic agent, such as a cancer vaccine . These compositions and kits may be useful in the treatment of cancer as described herein .

Brief Description of Figures

Figure 1 shows the expression of heme oxygenase-1 (HO-1 ) by the CD45+ subset of FAP+ tumoural stromal cells using four-color fluorescent confocal microscopy .

Figure 2 shows the effect of an HO-1 inhibitor, SnMP on the growth of immunogenic lewi s lung carcinomas expres sing ovalbumin (LL2/OVA) or non-immunogenic lewis lung carcinoma (LL2 ) .

Figure 3 shows that SnMP causes rapid cell death in an

immunogenic tumour .

Figure 4 shows that SnMP causes acute hypoxia of an immunogenic tumour .

Figure 5 shows that SnMP induces rapid expression of tissue factor by endothelial cells in an immunogenic tumour .

Figures 6 and 7 show confocal microscopy images of frozen sections of a murine LL2 /OVA tumor stained with antibodies against CD4, Foxp3 , and HO-1.

Figure 8 shows the design of experiments to observe the effect of HO-1 inhibition in the P815 model .

Figure 9 shows the progression of P815 tumors in mice treated or not treated with the tumor vaccine (L1210. P1A.B7-1) and SnMP. Mean tumor size ± SEM is shown . Data were analyzed using the Student T-test . Figure 10 shows the design of experiments to observe the effect of HO-1 inhibition in the CT26 model.

Figure 11 shows the mean tumour size of CT26 tumors in mice treated or not with SnMP. Mean tumor size ± SEM is shown .

Figure 12 shows CT26 tumor progression in individual mice (one curve per mouse ) treated or not with SnMP .

Detailed Description of Invention

This invention relates to the use of HO-1 inhibitors in

combination with immunotherapeutic agents , such as cancer vaccines , to reduce or abolish tumour immunosuppression in an individual with cancer . This may increase the effectiveness of the immunotherapeutic agents in eliciting an immune response against cancer cells in the individual .

Individuals suitable for treatment as described herein include mammals , preferably humans .

Provided herein are methods of treating cancer in subj ect (e.g. , a mammal ) in need thereof comprising administering to the subj ect an HO-1 inhibitor and an immunotherapeutic agent as described herein in an amount effective to treat said cancer .

In some cases , the mammalian subj ect is a human subj ect .

Practice of methods described herein in other mammalian

subj ects , especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans

(e.g. murine primate , porcine, canine , or rabbit animals) , is also encompassed. Standard dose-response studies are used to optimize dose and dosing schedule .

The disclosed methods are useful for treating cancer, for example , inhibiting cancer growth, including complete cancer remission, for inhibiting cancer metastasis, and for promoting cancer resistance . The term "cancer growth" generally refers to any one of a number of indices that suggest change within the cancer to a more developed form. Thus , indices for measuring an inhibition of cancer growth include but are not limited to a decrease in cancer cell survival , a decrease in tumor volume or morphology ( for example, as determined using computed

tomographic (CT) , sonography, or other imaging method) , a delayed tumor growth, a destruction of tumor vasculature , improved performance in delayed hypersensitivity skin test , an increase in the activity of cytolytic T-lymphocytes , and a decrease in levels of tumor- specific antigens . The term "cancer resistance" refers to an improved capacity of a subj ect to resist cancer growth, in particular growth of a cancer already had . In other words , the term "cancer resistance" refers to a decreased propensity for cancer growth in a subj ect .

Cancer cells in the individual with cancer may be

immunologically distinct from normal somatic cells in the individual . For example, the cancer cells may express an antigen which is not expressed by normal somatic cells in the individual (i.e. a tumour antigen ) . Tumour antigens are well-known in the art and are described in more detail below .

Various types of cancers are known in the art . The cancer may be metastatic or non-metastatic . The cancer may be familial or sporadic . In some embodiments , the cancer is selected from the group consisting of : leukemia and multiple myeloma . Additional cancers that can be treated using the methods of the invention include , for example , benign and malignant solid tumours and benign and malignant non-solid tumours .

For example , a cancer may comprise a solid tumour, for example, a carcinoma or a sarcoma . Carcinomas include malignant neoplasms derived from epithelial cells which infiltrate , for example, invade, surrounding tissues and give rise to metastases . Adenocarcinomas are carcinomas derived from glandular tissue, or from tissues that form recognizable glandular structures .

Carcinomas that may be treated include adrenocortical , acinar, acinic cell , acinous , adenocystic, adenoid cystic, adenoid squamous cell , cancer adenomatosum, adenosquamous , adnexel , cancer of adrenal cortex, adrenocortical , aldosterone-producing, aldosterone-secreting, alveolar, alveolar cell , ameloblastic , ampullary, anaplastic cancer of thyroid gland, apocrine, basal cell , basal cell , alveolar, comedo basal cell , cystic basal cell , morphea-like basal cell , multicentric basal cell , nodulo- ulcerative basal cell , pigmented basal cell , sclerosing basal cell , superficial basal cell , basaloid, basosquamous cell , bile duct, extrahepatic bile duct , intrahepatic bile duct ,

bronchioalveolar, bronchiolar, bronchioloalveolar,

bronchoalveolar , bronchoalveolar cell , bronchogenic,

cerebriform, cholangiocelluarl , chorionic, choroids plexus , clear cell , cloacogenic anal , colloid, comedo, corpus , cancer of corpus uteri , Cortisol-producing, cribriform, cylindrical , cylindrical cell , duct , ductal , ductal cancer of the prostate, ductal cancer in situ (DCIS) , eccrine, embryonal , cancer en cuirasse, endometrial , cancer of endometrium, endometroid, epidermoid, cancer ex mixed tumor, cancer ex pleomorphic adenoma, exophytic, fibrolamellar, cancer fibro ' sum, follicular cancer of thyroid gland, gastric , gelatinform, gelatinous , giant cell , giant cell cancer of thyroid gland, cancer

gigantocellulare, glandular, granulose cell , hepatocellular, Hurthle cell , hypernephroid, infantile embryonal , islet cell carcinoma, inflammatory cancer of the breast , cancer in situ, intraductal , intraepidermal , intraepithelial , j uvenile

embryonal , Kulchitsky-cell , large cell , leptomeningeal , lobular, infiltrating lobular, invasive lobular, lobular cancer in situ

(LCIS ) , lymphoepithelial , cancer medullare , medullary, medullary cancer of thyroid gland, medullary thyroid, melanotic,

meningeal , Merkel cell , metatypical cell , micropapillary, mucinous , cancer muciparum, cancer mucocellulare ,

mucoepidermoid, cancer mucosum, mucous , nasopharyngeal ,

neuroendocrine cancer of the skin, noninfiltrating, non-small cell , non-small cell lung cancer (NSCLC) , oat cell , cancer ossificans , osteoid, Paget ' s , papillary, papillary cancer of thyroid gland, periampullary, preinvasive, prickle cell , primary intrasseous , renal cell , scar, schistosomal bladder,

Schneiderian, scirrhous , sebaceous , signet-ring cell , cancer simplex, small cell , small cell lung cancer (SCLC) , spindle cell , cancer spongiosum, squamous , squamous cell , terminal duct , anaplastic thyroid, follicular thyroid, medullary thyroid, papillary thyroid, trabecular cancer of the skin, transitional cell , tubular, undifferentiated cancer of thyroid gland, uterine corpus , verrucous , villous , cancer villosum, yolk sac, squamous cell particularly of the head and neck, esophageal squamous cell , and oral cancers and carcinomas . Another broad category of cancers includes sarcomas and

fibrosarcomas , which are tumors whose cells are embedded in a fibrillar or homogeneous substance , such as embryonic connective tissue . Sarcomas that may be targeted include adipose, alveolar soft part, ameloblastic, avian, botryoid, sarcoma botryoides , chicken, chloromatous , chondroblastic, clear cell sarcoma of kidney, embryonal , endometrial stromal , epithelioid, Ewing's, fascial , fibroblastic, fowl, giant cell , granulocytic,

hemangioendothelial , Hodgkin ' s , idiopathic multiple pigmented hemorrhagic , immunoblastic sarcoma of B cells, immunoblastic sarcoma of T cells, Jensen ' s , Kaposi ' s , kupffer cell ,

leukocytic, lymphatic, melanotic , mixed cell , multiple ,

lymphangio, idiopathic hemorrhagic , multipotential primary sarcoma of bone , osteoblastic, osteogenic, parosteal ,

polymorphous , pseudo-kaposi , reticulum cell , reticulum cell sarcoma of the brain, rhabdomyosarcoma, rous , soft tissue, spindle cell, synovial, telangiectatic, sarcoma

(osteosarcoma) /malignant fibrous histiocytoma of bone, and soft tissue sarcomas .

Lymphomas that may be treated include AIDS-related, non- Hodgkin ' s , Hodgkin ' s , T-cell , T-cell leukemia/ lymphoma, African, B-cell , B-cell monocytoid, bovine malignant, Burkitt ' s ,

centrocytic , lymphoma cutis, diffuse , diffuse, large cell , diffuse , mixed small and large cell , diffuse , small cleaved cell , follicular, follicular center cell , follicular, mixed small cleaved and large cell , follicular, predominantly large cell , follicular, predominantly small cleaved cell , giant follicle, giant follicular, granulomatous , histiocytic , large cell , immunoblastic, large cleaved cell , large nocleaved cell , Lennert ' s , lymphoblastic , lymphocytic, intermediate;

lymphocytic , intermediately differentiated, plasmacytoid; poorly differentiated lymphocytic , small lymphocytic, well

differentiated lymphocytic , lymphoma of cattle ; MALT, mantle cell , mantle zone , marginal zone , Mediterranean lymphoma mixed lymphocytic-histiocytic, nodular, plasmacytoid, pleomorphic, primary central nervous system, primary effusion, small b-cell , small cleaved cell , small concleaved cell , T-cell lymphomas ; convoluted T-cell , cutaneous t-cell , small lymphocytic T-cell , undefined lymphoma, u-cell , undifferentiated, aids-related, central nervous system, cutaneous T-cell , effusion (body cavity based) , thymic lymphoma, and cutaneous T cell lymphomas .

Leukemias and other blood cell malignancies that may be targeted include acute lymphoblastic, acute myeloid, acute lymphocytic, acute myelogenous leukemia, chronic myelogenous , hairy cell , erythroleukemia, lymphoblastic , myeloid, lymphocytic ,

myelogenous , leukemia, hairy cell , T-cell , monocytic ,

myeloblastic, granulocytic , gross, hand mirror-cell , basophilic , hemoblastic , histiocytic, leukopenic , lymphatic, Schilling ' s , stem cell , myelomonocytic, monocytic , prolymphocytic , promyelocytic , micromyeloblastic , megakaryoblastic ,

megakaryoctyic, rieder cell , bovine, aleukemic , mast cell , myelocytic, plamsa cell , subleukemic , multiple myeloma,

nonlymphocytic, chronic myelogenous leukemia, chronic

lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin ' s disease , non-Hodgkin ' s lymphoma (indolent and high grade forms ) , multiple myeloma, Waldenstrom' s macroglobulinemia, heavy chain disease , myelodysplastic syndrome, and myelodysplasia and chronic myelocytic leukemias .

Brain and central nervous system (CNS) cancers and tumors that may be treated include astrocytomas (including cerebellar and cerebral) , brain stem glioma, brain tumors , malignant gliomas , ependymoma, glioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumors , visual pathway and

hypothalamic gliomas , primary central nervous system lymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamic glioma, extracranial germ cell tumor, medulloblastoma,

myelodysplastic syndromes , oligodendroglioma,

myelodysplastic/myeloproliferative diseases , myelogenous leukemia, myeloid leukemia , multiple myeloma , myeloproli ferative disorders , neuroblastoma, plasma cell neoplasm/multiple myeloma, central nervous system lymphoma, intrinsic brain tumors , astrocytic brain tumors , gliomas , and metastatic tumor cell invasion in the central nervous system .

Gastrointestimal cancers that may be treated include

extrahepatic bile duct cancer, colon cancer, colon and rectum cancer, colorectal cancer, gallbladder cancer, gastric ( stomach) cancer, gastrointestinal carcinoid tumor, gastronintestinal carcinoid tumors , gastrointestinal stromal tumors , bladder cancers , islet cell carcinoma (endocrine pancreas) , pancreatic cancer, islet cell pancreatic cancer, prostate cancer rectal cancer, salivary gland cancer, small intestine cancer, colon cancer, and polyps associated with colorectal neoplasia . Lung and respiratory cancers that may be treated include bronchial adenomas/carcinoids , esophagus cancer esophageal cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, lung carcinoid tumor, non-small cell lung cancer, small cell lung cancer , small cell carcinoma of the lungs , mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, oral cancer, oral cavity and lip cancer, oropharyngeal cancer;

paranasal sinus and nasal cavity cancer, and pleuropulmonary blastoma .

Urinary tract and reproductive cancers that may be treated include cervical cancer, endometrial cancer, ovarian epithelial cancer, extragonadal germ cell tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, spleen, kidney cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor , ovarian low malignant potential tumor, penile cancer, renal cell cancer (including carcinomas ) , renal cell cancer, renal pelvis and ureter (transitional cell cancer) , transitional cell cancer of the renal pelvis and ureter, gestational trophoblastic tumor, testicular cancer, ureter and renal pelvis , transitional cell cancer, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, ovarian carcinoma, primary peritoneal epithelial neoplasms , cervical carcinoma, uterine cancer and solid tumors in the ovarian follicle) , superficial bladder tumors , invasive transitional cell carcinoma of the bladder, and muscle-invasive bladder cancer .

Skin cancers and melanomas (as well as non-melanomas ) that may be treated include cutaneous t-cell lymphoma, intraocular melanoma, tumor progression of human skin keratinocytes , basal cell carcinoma, and squamous cell cancer . Liver cancers that may be targeted include extrahepatic bile duct cancer, and hepatocellular cancers . Eye cancers that may be targeted include intraocular melanoma, retinoblastoma, and intraocular melanoma .

Hormonal cancers that may be treated include: parathyroid cancer, pineal and supratentorial primitive neuroectodermal tumors , pituitary tumor, thymoma and thymic carcinoma, thymoma, thymus cancer, thyroid cancer, cancer of the adrenal cortex, an ACTH-producing tumors .

Miscellaneous other cancers that may be targeted include advanced cancers , AIDS-related, anal cancer adrenal cortical , aplastic anemia, aniline , betel , buyo cheek, cerebriform, chimney-sweeps , clay pipe, colloid, contact, cystic, dendritic, cancer a deux, duct, dye workers, encephaloid, cancer en cuirasse, endometrial , endothelial , epithelial , glandular, cancer in situ, kang, kangri , latent , medullary, melanotic , mule-spinners ' , non-small cell lung, occult cancer , paraffin, pitch workers ' , scar, schistosomal bladder, scirrhous , lymph node, small cell lung, soft, soot, spindle cell , swamp, tar, an tubular cancers .

Miscellaneous other cancers that may be targeted also include carcinoid (gastrointestinal and bronchal ) Castleman ' s disease chronic myeloproliferative disorders , clear cell sarcoma of tendon sheaths , Ewing ' s family of tumors , head and neck cancer, lip and oral cavity cancer, Waldenstrom's macroglobulinemia, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome , multiple myeloma/plasma cell neoplasm, Wilms ' tumor, mycosis fungoides , pheochromocytoma, sezary syndrome , supratentorial primitive neuroectodermal tumors , unknown primary site , peritoneal effusion, malignant pleural effusion, trophoblastic neoplasms , and

hemangiopericytoma .

Miscellaneous other cancers that may be treated include carcinoid (gastrointestinal and bronchal ) Castleman ' s disease chronic myeloproliferative disorders, clear cell sarcoma of tendon sheaths , Ewing ' s family of tumors , head and neck cancer, lip and oral cavity cancer, Waldenstrom's macroglobulinemia, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome , multiple myeloma/plasma cell neoplasm, Wilms ' tumor, mycosis fungoides , pheochromocytoma, sezary syndrome , supratentorial primitive neuroectodermal tumors , unknown primary site , peritoneal effusion, malignant pleural effusion, trophoblastic neo-plasms , and

hemangiopericytoma .

Further described herein are methods of stimulating an immune response in a mammalian subj ect compri sing administering to the subj ect an HO-1 inhibitor and an immunotherapeutic agent described herein . The HO-1 inhibitor and the immunotherapeutic agent may be administered directly to the subj ect in the same manner as a vaccine . In some embodiments , HO-1 inhibitors and immunotherapeutic agents described herein may be useful for the induction of an immune response to a tumor antigen, one or more pathogenic organisms , or other antigen as described herein .

The term "treating" a disorder, disease or deleterious

physiological response refers to an approach for obtaining beneficial or desired results , including clinical results . As used herein, "treating" or "treatment" includes preventing, delaying, abating or arresting the clinical symptoms and/or signs of a disorder, disease or deleterious physiological response . Treatment also alleviation or amelioration of one or more symptoms or conditions , diminishment of extent of disease, stabilization of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset ,

amelioration or palliation of the disease state, and remission (whether partial or total ) . "Treating" can also mean prolonging survival of a subj ect or patient beyond that expected in the absence of treatment . As used herein, a "subject" or "individual" may refer to a patient and frequently a human patient . However this term is not limited to humans and encompasses a variety of mammals such as a human, non-human primate , cow, horse , pig, sheep, goat , dog, cat , or rodent . In preferred embodiments , the subj ect is a human .

As used herein "administration" or "administering" refers to any suitable method of providing a composition of the present invention to a subj ect . It is not intended that the present invention be limited to any particular mode of administration . In certain embodiments , the compounds and pharmaceutical compositions of the present invention are administered by a parenteral route, e.g. via intramuscular, intraperitoneal, intravenous , intracisternal or subcutaneous inj ection or infusion . The pharmaceutical compositions may be formulated in suitable dosage unit formulations appropriate for each route of administration .

As used herein, the term "effective amount" or "therapeutically effective amount" of a compound refers to a nontoxic but sufficient amount of the compound to provide the desired therapeutic or prophylactic effect to most patients or

individuals . In the context of treating a haematological malignancy, a nontoxic amount does not necessarily mean that an agent is not used, but rather means the administration of a tolerable and sufficient amount to provide the desired

therapeutic or prophylactic effect to a patient or individual . The effective amount of a pharmacologically active compound may vary depending on the route of administration, as well as the age , weight, and sex of the individual to which the drug or pharmacologically active agent is administered. Those of skill in the art given the benefit of the present disclosure can easily determine appropriate effective amounts by taking into account metabolism, bioavailability, and other factors that affect plasma levels of a compound following administration within the unit dose ranges disclosed further herein for different routes of administration. As used herein, a subj ect having a disorder, disease , such as cancer, or deleterious physiological response is a subj ect with at least one identifiable sign, symptom, or laboratory finding sufficient to make a diagnosis of the disorder, disease or deleterious physiological response in accordance with clinical standards known in the art for identifying such disorders .

Examples of such clinical standards can be found in textbooks of medicine such as Harrison' s Principles of Internal Medicine, 15th Ed . , Fauci AS et al . , eds . , McGraw-Hill , New York, 2001. In some instances , a diagnosis of a disorder, disease or deleterious physiological response will include identification of a particular cell type present in a sample of a body fluid or tissue obtained from the subj ect .

In some embodiments , a method of treating a subj ect using a HO-1 inhibitor and an immunotherapeutic agent as disclosed herein, may further comprise administering one or more tumour therapies to treat the tumour . Such therapies include , for example, tumour medicaments , radiation and surgical procedures . As used herein, a "tumour medicament" refers to an agent which is administered to a subj ect for the purpose of treating a cancer . Various types of medicaments for the treatment of tumours are described herein . Tumour medicaments function in a variety of ways . Some cancer medicaments work by targeting physiological mechanisms that are specific to tumour cells . Examples include the targeting of specific genes and their gene products (i.e. proteins primarily) which are mutated in tumours . Such genes include but are not limited to oncogenes (e.g. , Ras , Her2, bcl-2 ) , tumour suppressor genes (e.g. , EGF, p53 , Rb) , and cell cycle targets (e.g. CDK4 , p21 , telomerase) .

Tumour medicaments can alternately target signal transduction pathways and other molecular mechanisms which are altered in tumour cells . Chemotherapeutics for use in combination

therapies as disclosed herein may include aspirin, sulindac, curcumin, alkylating agents including : nitrogen mustards , such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil ; nitrosoureas , such as carmustine (BCNU) , lomustine

(CCNU) , and semustine (methyl-CCNU) ; thylenimines/methylmelamine such as thriethylenemelamine (TEM) , triethylene,

thiophosphoramide (thiotepa) , hexamethylmelamine (HMM,

altretamine ) ; alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC) ; antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine,

gemcitabine , cytosine arabinoside (AraC, cytarabine) , 5- azacytidine , 2 , 2 ^' -difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2 ' - deoxycoformycin (pentostatin) , erythrohydroxynonyladenine

(EH A) , fludarabine phosphate, and 2-chlorodeoxyadenosine

( cladribine , 2-CdA) ; natural products including antimitotic drugs such as paclitaxel , vinca alkaloids including vinblastine

(VLB) , vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate ; epipodophylotoxins such as etoposide and tenyposide; antibiotics such as actimomycin D, daunomycin

( rubidomycin) , doxorubicin, mitoxantrone , idarubicin,

bleomycins , plicamycin (mithramycin) , mitomycinC , and

actinomycin; enzymes such as L-asparaginase .

Immunotherapeutic agents for use in combination with HO-1 inhibitors , as described herein may include biological response modifiers . Biological response modifiers for use in combination therapies as disclosed herein may include interferon-alpha, IL-2 , G-CSF and GM-CSF; miscellaneous agents including platinum coordination complexes such splatin and carboplatin, anthracenediones such as mitoxantrone , substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane ( o , p ^' -DDD) and aminoglutethimide ; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents , dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate , medroxyprogesterone acetate and megestrol acetate; estrogen such as

diethyl stilbestrol and ethinyl estradiol equivalents ;

antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents ; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide;

kinase inhibitors , histone deacetylase inhibitors , methylation inhibitors , proteasome inhibitors , oxidants , anti-oxidants , telomerase inhibitors , BH3 mimetics , ubiquitin ligase

inhibitors , stat inhibitors ; receptor tyrosine kinase inhibitors such as Imatinib (originally STI571, now marketed as Gleevec or Glivec) , and Erlotinib (an EGF receptor inhibitor) now marketed as Tarceva .

Additional biological response modifiers may include inhibitors of the immunomodulatory enzymes indoleamine 2, 3-dioxygenase (IDO or (IDO or INDO EC 1.13.11.52) tryptophan 2 , 3-dioxygenase (TDO, (EC 1.13.11.11) .

Tryptophan 2, 3-dioxygenase (TDO) is a homotetrameric heme- containing cytosolic enzyme encoded by gene TD02 and expressed at high levels in the liver . It catalyses the first and rate- limiting step of tryptophan degradation along the kynurenine pathway and thereby regulates systemic tryptophan levels . The role of TDO in cancer was shown in studies by Pilotte et al (Proc Natl Acad Sci U S A. 2012 Feb 14; 109 (7) : 2497-502. Epub 2012 Jan 30) . These studies detected TDO expression in a significant proportion of human tumors . In a preclinical model , TDO expression by tumors prevented their rej ection by immunized mice . The studies used a TDO inhibitor, which, upon systemic treatment , restored the ability of mice to rej ect TDO-expressing tumors thus providing evidence that TDO inhibitors can be effective in cancer therapy . TDO inhibitors include LM10. Indoleamine 2 , 3 dioxygenase (IDO) is an enzyme that in humans is encoded by the ID01 gene . This enzyme also catalyzes the first and rate-limiting step in the degradation of the essential amino acid L-tryptophan to N-formylkynurenine , and is normally expressed in tumor cells and in activated immune cells . IDO dampens the immune response by degrading the indole moiety of tryptophan, locally depleting tryptophan levels, and increasing proapoptotic kynurenines . Consequently, IDO blocks the

proliferation and activation of T cells, which are extremely sensitive to Trp shortage . This creates an environment in which tumor-specific cytotoxic T lymphocytes are rendered functionally inactive or are no longer able to attack a patient ' s cancer cells . The observation that many human tumors constitutively express IDO introduced the hypothesis that its inhibition could enhance the effectiveness of cancer immunotherapy . Results from in vitro and in vivo studies show that the efficacy of

therapeutic vaccination of cancer patients may indeed be improved by concomitant administration of an IDO inhibitor .

Small molecule inhibitors of IDO are available in the art to treat IDO-related diseases such as cancer . For example,

W099/ 29310 reports methods for altering T-cell mediated immunity comprising altering local extracellular concentrations of tryptophan and tryptophan metabolites , among others . Additional compounds having IDO inhibitory activity are reported in

WO2004/094409, US8088803 (which reports the INCB024360

compound) , US20110165188 and US 20110159017. Immunotherapeutic agents for use m combination with HO-1 inhibitors , as described herein may include cytokines .

Cytokines that are effective in inhibiting tumor

growth/metastasis may be used in combination with HO-1

inhibitors . Such cytokines , lymphokines , or other hematopoietic factors include , but are not limited to, M-CSF, GM-CSF, TNF, IL- 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, I FN, TNFcx, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin , stem cell factor, and erythropoietin .

As used herein, the term "immunotherapy" , "immunotherapeutic agent" or "immunotherapeutic" generally refers to any

therapeutic approach aimed at mobilizing or manipulating a patient ' s immune system to treat or cure disease , particularly diseases such as cancer . The term "immunotherapy" ,

"immunotherapeutic agent" or "immunotherapeutic" includes the targeting of tumour cells via the recognition of immunogenic proteins or antigens expressed by said tumour cells,

accomplished by utilizing either pas sively trans ferred immune molecules such as antibodies , or cancer vaccine preparations designed to induce antibodies or T lymphocytes (T cells) recognizing a localized region of an antigen or epitope specific to the tumour cell .

Immunotherapeutic agents for use in combination with HO-1 inhibitors , as described herein may be antibodies .

Suitable antibodies include anti-Her2/neu receptor antibody for example trastuzumab (marketed as Herceptin ) ; Alemtuzumab , a CD52 antibody marketed as Campath, MabCampath or Campath-1H currently under further development as Lemtrada; Gemtuzumab, an anti-CD33 monoclonal antibody linked to a calicheamicin marketed by Wyeth as Mylotarg; an anti-CD20 antibody, such as Rituximab (marketed as Rituxan and MabThera) or Ibritumomab tiuxetan sold under the trade name Zevalin; anti-TNF-alpha antibodies such as Infliximab (marketed as Remicade) , or Adalimumab (marketed as Humira) , or a soluble TNFR2 molecule such as etanercept (also known as Enbrel ) ; an antibody to the CD25 chain of the IL-2 receptor such as basiliximab (trade name Simulect) ; an anti CD40/CD40L antibody such as a humanized IgGl anti-human CD40 antibody (SGN-40) ; an anti-CTLA-4 blocking antibody such as Ipilimumab (also known as MDX-101 or MDX-010, and marketed as Yervoy) ; anti-PD-1 antibody (programmed cell death protein 1 , also designated as CD279); an anti-PDL-1 (programmed cell death ligand) ; an antibody against glucocorticoid-induced TNFR family-relate gene , or anti-GITR antibody) ; or an anti-OX-40 (CD134 ) antibody;

Other suitable immunotherapeutic agents may include soluble Lymphocyte-activation gene 3 (also known as LAG3 or CD223) -based immune modulators such as LAG3-Ig ( IMP321 ) ; Toll-like receptor agonists like MPL, CpG, single-stranded R A, nucleotides , nucleotide analogue, CL087 (a TLR7-specific ligand) , loxoribine , polyinosine-polycytidylic acid, flagellin, resiquimod,

immiquimod, gardiquimodNOD ligands like muramyl dipeptide, murabutide, peptidoglycan, muramyldipeptide and anti-virals such as oseltamivir phosphate , Amphotericin B, and palivizumab .

In some embodiments , a therapy as disclosed herein may comprise administration of a HO-1 inhibitor, such as SnMP, and one or more antibodies selected from the group consisting of an anti- PD1 antibody, an anti-PDL-1 antibody, an anti-CTLA-4 antibody an anti-GITR antibody and an anti-OX40 antibody . An anti-PDl antibody may be a monoclonal antibody directed against the negative immunoregulatory human cell surface receptor PD-1 with immunopotentration activity . An exemplary anti-PDl antibody is human monoclonal antibody MDX-1106 which binds and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2 , resulting in the activation of T-cells and cell-mediated immune responses against tumor cells . In some embodiments , the anti-PD-Ll antibody is a monoclonal antibody directed against the protein ligand PD-L1 with immunomodulating and

antineoplastic activities . An exemplary anti-PD-Ll antibody is human monoclonal antibody MDX-1105 which binds PD-L1 and blocks its binding to and activation of its receptor PD-1 , which may enhance the T-cell-mediated immune response to neoplasms and reverse T-cell inactivation in chronic infections disease . PD- Ll is expressed broadly on hematopoietic and parenchymal tissues .

An anti-CTLA-4 antibody may be a monoclonal antibody directed against the T-cell receptor protein cytotoxic T-lymphocyte associated protein 4 (CTLA-4 ) . An exemplary anti-CTLA-4 antibody is human IgG2 monoclonal antibody tremelimumab which binds to CTLA4 and blocks binding of the antigen presenting cell ligands B7-1 and B7-2 to CTLA-4 , resulting in inhibition of B7- CTLA4 -mediated down-regulation of T-cell activation . Another exemplary anti-CTLA-4 antibody is human IgGl monoclonal antibody ipilimumab which binds to CTLA4 and blocks binding of the antigen presenting cell ligands B7-1 and B7-2 to CTLA-4 , resulting in inhibition of B7-CTLA4-mediated down-regulation of T-cell activation . Ipilimumab is undergoing clinical trials for the treatment of non-small cell lung carcinoma, small cell lung cancer and metastatic hormone-refractory prostate cancer .

An anti-GITR antibody may be a monoclonal antibody directed against glucocorticoid-induced tumor necrosis factor receptor (GITR) which blocks the interaction of GITR with its ligand, enhances cytotoxicity of natural human killer cells and/or down- modulates GITR expression on peripheral blood lymphocytes .

An anti-OX40 antibody may be an agonistic monoclonal antibody that mimicks the natural 0X40 ligand and selectively binds to and activates the OX40 receptor . Receptor activation induces proliferation of memory and effector T cells In some embodiments, an immunotherapeutic agent for

administration with an HO-1 inhibitor as described herein may ^'. a cancer vaccine . A cancer vaccine is an agent , a cell-based agent , molecule , or immunogen which stimulates or elicits an endogenous immune response in an individual or subj ect against one or more tumour antigens .

As used herein, a tumour antigen is broadly defined as an antigen specifically expressed by a tumour or cancer cell . The antigen may be expressed at the cell surface of the tumour cell where it is recognized by components of the humoral immune system such as B lymphocytes (B cells) . Intracellular tumour antigens are processed into shorter peptide fragments which form complexes with maj or histocompatibility complex (MHC ) molecules and are presented on the cell surface of cancer cells, where they are recognized by the T cell receptors (TCF s) of T lymphocytes (T cells ) . Preferably, the tumour antigen is one which is not expressed by normal cells , or at least not

expressed to the same level as in tumour cells . Currently produced cancer vaccines being developed for the treatment of humans activate the humoral immune system (i.e., the antibody dependent immune response) or the cell-mediated immune system including T lymphocytes (T cells ) which are capable of

specifically recogni zing and killing tumour cells.

A cancer vaccine may enhance the presentation of one or more tumour antigens to both antigen presenting cells (e.g. ,

macrophages and dendritic cells) and/or to other immune cells such as T cells , B cells , and NK cells . In some examples , preparations and/or formulations of cancer vaccines may be used together with one or more adj uvants that are well known in the art , to induce an immune response or to increase an immune response .

An adj uvant is a substance incorporated into or administered with antigen which potentiates the immune response . Adj uvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages) , activating macrophages and stimulating specific sets of lymphocytes .

Adj uvants of many kinds are well known in the art . Specific examples of adj uvants include monophosphoryl lipid A (MPL,

SmithKline Beecham) , a congener obtained after purification and acid hydrolysis of Salmonella Minnesota Re 595

lipopolysaccharide; saponins , including QS21 ( SmithKlineBeecham) a pure QA-21 saponin purified from Quillj a saponaria extract ; DQS21 , described in PCT application WO96/33739 ( SmithKline

Beecham) ; QS-7, QS-17, QS-18, and QS-L1 (So et al . , MoI Cells (1997) 7 : 178-186) ; ISCOMATRIX adjuvant, a cage-like structure composed of saponin, phospholipid, and cholesterol ( see, e.g., Maraskovsky et al . , Clin. Cancer Res . (2004) 10:2879-2890) ;

incomplete Freund ' s adj uvant ; complete Freund ' s adj uvant ;

montanide ; alum; CpG oligonucleotides (see e.g. Kreig et al . , Nature 374:546-9, 1995) and other immunostimulatory

oligonucleotides including poly-IC and poly-ICLC (Hiltonol©) ; and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol .

In some embodiments , cancer cells or a tumour in the individual may be resistant to immunotherapy . In some embodiments , the individual may have minimal residual disease (MRD) after an initial cancer treatment .

Currently, vaccines comprising CT-X antigens including MAGE-A3 ,

FRAME and NY-ESO-1 are in clinical trials in patients with melanoma and lung cancer where such antigens are frequently expressed (Bender et al . , Cancer Immunol (2007 ) 7 , 16;

Atanackovic et al . , PNAS (2008 ) 105, 1650-1655; Jager et al . ,

PNAS (2006) 103, 14453-14458; van Baren et al . , J Cln. Oncol .

(2005) 23, 9008-9021 ; Valmori et al . , PNAS (2007) 104, 8947- 8952; Odunsi et al . , PNAS (2007) 104, 12837-12842; Davis et al . ,

PNAS (2004) 101, 10697-10702; Theurillat et al . , Intern J Cancer (2007 ) 120, 2411-2417) . The reported results of clinical trials of immunotherapeutic agents such as cancer vaccines in patients or individuals with advance stage cancer have been disappointing and, in such patients , tumour cells may be non-responsive or resistant to immune responses to tumour antigens which are displayed by the tumour cells . In such patients , cancer cells or tumours in the individual may, for example , be associated with an immunosuppressive tumour microenvironment .

The presence of immunosuppression within the microenvironment of cancer cells or tumours in an individual may be determined . For example , a method as described herein may comprise the step of identifying one or more FAP+ stromal cells in a tumour sample obtained from the individual .

Immunosuppressive FAP+ stromal cells may be further

characterized by their expression of the CD45 marker and may be referred to as CD45+FAP+ stromal cells . CD45+, FAP+ stromal cells may be identified using any suitable technique . In particular, immunological techniques such as

immunocytochemistry, flow cytometry, fluorescent confocal microscopy or immunohistochemistry may be employed .

Specifically, immunosuppressive CD45+FAP+ cells may be

identified further by flow cytometry by characterizing the expression of various cell surface proteins including but not limited to CDllb, MHC class II , Sca-1 , but not Gr-1/.

Selectively depleting or functionally inhibiting

immunosuppressive CD45+, FAP+ stromal cells through inhibition of heme oxygenase-1 (HO-1) expression or activity may alter the immunological status of tumour microenvironment and improve the efficacy of immunotherapeutic agents such as cancer vaccines , as described herein . Heme oxygenase 1 (HO-1 or H OX1 : GenelD: 3162) is the first rate-limiting enzyme in the degradation of heme into biliverdin, iron and carbon monoxide (ECl .14.99.3) and free iron . HO-1 plays a role as a sensor and regulator of oxidative stress . HO-1 expression is often increased in tumour tissues suggesting that HO-1 may have a role in tumour induction, growth and metastasis . Constitutively elevated levels of HO-1 have been observed in a number of human tumours including glioma, melanoma, prostate , pancreatic and renal cell carcinoma, lymphosarcomas , Kaposi sarcoma (Antiox. Redox Signal 2007, 9 (12) : 2009-2117) .

Some observations indicate that HO-1 exerts anti-inflammatory effects via the degradation products of heme , to help modulate the growth and proliferation of tumour cells , possibly via inhibiting adaptive immune responses (Andersen et al, JCI , Vol . 119 No.8, p2245 , 2009) . For example, carbon monoxide has been reported to induce strong anti-inflammatory responses including the suppression of TNF-a production, interleukin-ΐβ and

macrophage inflammatory protein-ΐβ (Annu . Rev . Pharmcol . Toxicol 46:411, 2006) . In mice , HO-1 deficiency results in increased generation of pro-inflammatory cytokines , including IL-lb, interferon-g, TNF, IL- 6 and in general , pro-inflammatory immune activity (Kapturczak et al , 2004 Am J Pathol 165 (3) 1045-1053; Poss et al, 1997 PNAS USA 94 (20) 10919-10924) .

HO-1 is thought to be predominantly immune-suppressive and has anti-inflammatory effects . Regulatory T cells (CD4+ and CD8+ Tregs ) accumulate in the microenvironment of tumours and are associated with prevention of antitumor immunity . The isolation of naturally occurring Tregs specific for HO-1 from the

peripheral blood of cancer patients has been reported (Andersen et al . J. Clin . Invest . 119: 2245-2256) . These cells are reported as being able to inhibit the activity of effector immune cells, which is suggested to be associated with

prevention of antitumor immunity and anticancer immunotherapy . HO-1 activity might provide a braking mechanism allowing homeostatic proliferation of T cells , but preventing

uncontrolled activation and proliferation of naive T cells.

Pharmacologic inhibition of HO-1 using the inhibitor SnMP as a single agent can lead to activation and proliferation of naive CD4+ and CD8+ T cells and reduces the suppressive capacity of Treg cells (Burt et al . 2010. J. Immunol. 185 : 5279-5288) .

However, the identity of the cells expressing HO-1 is not clear from these studies .

Pharmacologic inhibition of HO-1 has previously been proposed as a therapeutic option and potential sensitizer to chemotherapy for a wide range of cancers (Was et al . 2010. Current Drug Targets, 11 : 1551-1570) .

Another aspect of the invention provides a method comprising identifying CD45+FAP+ stromal cells expressing high levels of HO- 1 in an individual with cancer and admini stering a

therapeutically effective amount of an HO-1 inhibitor or modulator in combination with a immunotherapeutic agent, such as a cancer vaccine, to the individual .

HO-1 inhibitors inhibit, reduce or abolish or otherwise modulate the activity of HO-1 in a cell . The inhibition, modulation or reduction, can be due to a reduced amount of HO-1 , a reduced amount of active HO-1 or due to reduced efficiency of HO-1.

Preferably, the HO-1 inhibitor inhibits an inducible form of HO- 1. Suitable HO-1 inhibitors are well-known in the art (see for example WO97/ 35569) .

Synthetic derivatives of heme, called metalloporphyrins are known to be competitive inhibitors of HO-1 ( Drummond and Kappas , 1981 PNAS USA 78(10) 6466-70; Frydman et al, 1981 Biochemistry 20(18) 5177-5182; Maines 1981 Biochem Biophys Acta 673 (3) 339- 350 ) . In one embodiment , suitable metalloporphyrins include analogues of ferriprotoporphyrin including metal mesoporphyrins , such as tin mesoporphyrin (SnMP) , inc mesoporphyrin ( ZnMP) , iron mesoporphyrin (FeMP) , mangane e mesoporphyrin (MnMP) , and chromium mesoporphyrin (CrMP) , and non-ferrous protoporphyrins , such as tin protoporphyrin (SnPP) , zinc protoporphyrin ( ZnPP) , chromium protoporphyrin (CrPP) and manganese protoporphyrin (MnPP) .

In other embodiments , an HO-1 inhibitor may be an imidazole dioxalane-small molecule inhibitor, such as OB-24 having the structure of formula 1 ;

Formula 1 as disclosed in US20090176831; an triterpenoid compound, such a RTA-403 which has the structure of formula 2 ;

Formula 2

as disclosed in WO09965478; or a pegylated ZnPP Syb-0702 having the structure of formula 3.

Formula 3

In some preferred embodiments, the HO-1 inhibitor is tin mesoporphyrin ( SnMP; CASIO 6344-20-1 ; formula 4) .

Formula 4

The preparation of SnMP and its formulation into therapeutic compositions is well-known in the art (see for example US4657902 and US5010073) . Preferred HO-1 inhibitors may display HO-1 inhibitory activity which is the same as or greater than the HO-1 inhibitory activity of SnMP under the same conditions .

In some preferred embodiments the HO-1 inhibitor is administered in combination with an immunotherapeutic agent , such as a cancer vaccine .

In a further embodiment such combination is administered to patient , such as a patient with cancer, or a patient in need treatment with an immunotherapeutic agent, such as a cancer vaccine .

In a further embodiment, treatment of a patient involves the scheduled administration of an HO-1 inhibitor and an

immunotherapeutic agent, such as a cancer vaccine, enabling the coordination and resulting enhancement of the beneficial activities of the individual agents . For example , the temporal modulation of the tumour microenvironment by the HO-1 inhibitor may be scheduled to complement and enhance stimulation of anti- tumour immunity, e.g. cellular immunity, by the

immunotherapeutic agent or cancer vaccine .

A tumour or cancer antigen is an immunogenic molecule, such as a peptide or a polypeptide , which is capable of eliciting an immune response in an individual . A cancer antigen which is present on the surface of cancer cells in an individual but which is not present on the surface of normal somatic cells of the individual i.e. the antigen is exposed to the immune system in cancer cells but not in normal somatic cells . The antigen may be expressed at the cell surface of the tumour cell where it is recognized by components of the humoral immune system such as B lymphocytes (B cells ) . Intracellular tumour antigens are processed into shorter peptide fragments which form complexes with maj or histocompatibility complex (MHC ) molecules and are presented on the cell surface of cancer cells, where they are recognized by the T cell receptors (TCR's) of T lymphocytes (T cells) . Preferably, the cancer antigen is one which is not expressed by normal cells, or at least not expressed to the same level as in tumour cells . An immunotherapeutic agent , such as a cancer vaccine or immunogen, may comprise one or more epitopes or antigenic determinants , e.g. peptide epitopes or antigenic determinants , from a tumour or cancer antigen, such that the immune response generated by the cancer vaccine is reactive against the tumour antigen .

A cancer vaccine may enhance the presentation of one or more cancer antigens to both antigen presenting cells (e.g. ,

macrophages and dendritic cells) and/or to other immune cells such as T cells , B cells , and NK cells . In some examples , preparations and/or formulations of cancer vaccines may be used together with one or more adj uvants that are well known in the art , to induce an immune response or to increase an immune response ,

Tumour antigens may include, for example , cancer-testis antigens encoded by cancer-germ line genes . Cancer-testis (CT) antigens constitute a unique group of genes which are predominantly expressed in human germ line cells such as placenta and testis but become reactivated in various malignancies (Simpson et al . , Nature Rev (2005) 5 , 615-625 ) . Most of these genes are located as multigene families on the X-chromosome and are also referred to as CT-X antigens (Simpson et al . , Nature Rev (2005) 5 , 615- 625 ) . Analogies have been drawn between their expression pattern during germ maturation and neoplastic transformation, thus suggesting their involvement in several steps of tumorigenesis (Simpson et al . , Nature Rev (2005) 5, 615-625) . The CT-X antigens are broadly expressed in a wide variety of cancer types including for example bladder cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, Brain cancer, glioma, glioblastoma, hepatocellular carcinoma and melanoma . Moreover, their expression pattern is closely associated with advanced disease and poor outcome and might thus be of diagnostic and/or prognostic relevance (Gure et al . , Clin Cancer Res (2005) 11, 8055-8062; Velazquez et al . , Cancer Immun (2007) 7, 1 1 ;

Andrade et al . , Cancer Immun (2008 ) 8 , 2 ; Tinguely et al . ,

Cancer Science (2008) ; Napoletano et al . , Am J of Obstet Gyn (2008 ) 198, 99 e91-97. Due to their highly restricted expression in malignant tissues , their tumour associated peptide epitopes provide promising targets for anticancer immunotherapy ( Scanlan et al . , Immunol Rev (2002) 188 , 22-32 ) . Indeed, clinical trials evaluating the role of CT antigens , namely MAGE- A3 , Frame and NY-ESO-I , as targets for specific immunotherapy have already been initiated in a number of different malignancies (Bender et al . , Cancer Immunol (2007) 7 , 16 ; Atanackovic et al . , PNAS (2008) 105, 1650-1655; Jager et al . , PNAS (2006) 103, 14453- 14458; van Baren et al . , J Clin Oncol (2005) 23, 9008-9021 ; Valmori et al . , PNAS (2007) 104, 8947-8952; Odunsi et al . , PNAS (2007) 104, 12837-12842; Davis et al . , PNAS (2004) 101, 10697- 10702 ( 9-15 ) . Tumour antigens which may be comprised of the full-length polypeptide sequence of the tumour antigen or an immunogenic fragment , or epitope derived from the full-length polypeptide sequence of the tumour antigen . Tumour antigens include the corresponding nucleotide sequence encoding for the full-length polypeptide , immunogenic fragment , or epitope derived from the full-length polypeptide sequence of the tumour antigen .

A fragment of a tumour antigen is a contiguous stretch of amino acid residues from the sequence of the antigen which is shorter than the full length antigen (i.e. it consists of fewer amino acid residues ) . For example, a fragment may comprise less than 500 , less than 400, less than 300, less than 200 , less than 100 amino acids , or less than 50 amino acids . A fragment will generally consist of at least 5 amino acids , for example , at least 10 amino acids , at least 15 amino acids , at least 20 amino acids , at least 25 amino acids , at least 30 amino acids or at least 35 amino acids. Fragments of tumour antigens may include immunogenic regions or epitopes that bind to MHC class I or class II molecules and are recognized by TCR' s of T lymphocytes . Many such epitopes of tumour antigens are known in the art and available from public databases (e.g.

www . cancerimmunity . org/peptidedatabase/Tcellepitopes ) .

Suitable tumour antigens for use in a immunotherapeutic agent such as a cancer vaccine include : MAGE-A1 , MAGE-A2 , MAGE- A3 , MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8 , MAGE-A9 , MAGE-A10 , MAGE-A11 , MAGE-A12 , GAGE-I, GAGE-2 , GAGE-3, GAGE-4, GAGE-5, GAGE- 6, GAGE- 7, GAGE-8, BAGE-I, RAGE- 1, LB33/MUM-1, FRAME , NAG, MAGE-Xp2 (MAGE-B2) , MAGE-Xp3 (MAGE-B3) , MAGE-Xp (MAGE-B4) , MAGE- C1/CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX-2 (HOM-MEL- 40) , SSX-3, SSX-4, SSX-5, SCP-I and XAGE and immunogenic fragments thereof . Other types of tumour antigens include overexpressed or mutated proteins and differentiation antigens particularly melanocyte differentiation antigens such as p53 , ras, CEA, MUC1, PMSA, PSA, tyrosinase, Melan-A, MART-1 , gplOO, gp75, alpha-actinin-4 , Bcr-Abl fusion protein, Casp-8 , beta- catenin, cdc27 , cdk4 , cdkn2a, coa-1 , dek-can fusion protein, EF2 , ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2 , HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3 , neo-PAP, myosin class I , OS-9 , pml-RAR. alpha . fusion protein, PTPRK, K-ras , N-ras , Triosephosphate isomeras, GnTV, Herv-K-mel , NA-88, SP17, and TRP2-Int2, (MART- I) , E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens , EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE- 4, MAGE-5, MAGE- 6, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, alpha . -fetoprotein, 13HCG,

BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA) , CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM) , HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA- 90 (Mac-2 binding protein\cyclophilin C-associated protein) , TAAL6 , TAG72 , TLP, and TPSand tyrosinase related proteins such as TRP-1 , TRP-2. Other suitable antigens include cancer antigens in the following classes : cancer testis antigens (e.g. , HOM-MEL-40) , differentiation antigens (e.g. , HOM-MEL-55) , overexpressed gene products (HOM-MD-21 ) , mutated gene products (NY-COL-2 ) , splice variants (HOM-MD-397 ) , gene amplification products (HOM-NSCLC-11 ) and cancer related autoantigens (HOM-

MEL-2.4 ) as reviewed in Cancer Vaccines and Immunotherapy (2000 ) Eds Stern, Beverley and Carroll , Cambridge University Press, Cambridge . In some exemplary embodiments , the antigen is a tumor antigen selected from the group consisting of MUCl , MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, FRAME, PSMA, tyrosinase, melan-A, and mixtures thereof . In some variations , the cancer antigen is a mammalian protein . In some variations , the cancer antigen is a human protein . In some variations , the full-length protein may be employed as the antigen . In some variations , peptides comprising an antigenic fragment of these proteins may be used as the tumor antigen .

Other suitable tumour antigens are well known in the art ( see for example O00/ 20581 ) The sequences of these tumour antigens are readily available from public databases but are also found in WO 1992/020356 Al , WO 1994/005304 Al, WO 1994/023031 Al , WO 1995/020974 Al, WO 1995/023874 Al, WO 1996/026214 Al which are incorporated by reference . Suitable cancer vaccines are known in the art and may be produced by any convenient technique .

For example , a cancer vaccine may be generated wholly or partly by chemical synthesis . For example, a peptide-based vaccine or immunogen may be synthesised using liquid or solid-phase synthesis methods ; in solution; or by any combination of solid- phase , liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate , after removal of any protecting groups being present , by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof . Chemical synthesis of peptides is well-known in the art (J.M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984) ; M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984) ; J. H . Jones , The Chemical

Synthesis of Peptides . Oxford University Press , Oxford 1991; in Applied Biosystems 430A Users Manual , ABI Inc . , Foster City, California; G . A. Grant, (Ed . ) Synthetic Peptides , A User' s Guide . W. H . Freeman & Co . , New York 1992, E. Atherton and R.C. Sheppard, Solid Phase Peptide Synthesis, A Practical Approach . IRL Press 1989 and in G.B. Fields , (Ed . ) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol . 289) . Academic Press , New York and London 1997) . Cancer vaccines may comprise or may be produced by recombinant vectors . For example a cancer vaccine may comprise a

recombinant virus encoding the tumor antigen of interest . Any suitable vector may be used to introduce a polynucleotide that encodes a polypeptide of the invention encoding one of the tumor antigen proteins into the host . Exemplary vectors that have been described in the literature include replication deficient retroviral vectors , including but not limited to lentivirus vectors [Kim et al . , J . Virol . , 72(1) : 811-816 (1998) ; Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43 46.]; adeno- associated viral (AAV) vectors [ U.S. Patent No . 5,474,935; U.S. Patent No . 5, 139, 941; U.S. Patent No . 5, 622, 856; U.S. Patent No . 5, 658, 776; U.S. Patent No. 5, 773, 289; U.S. Patent No. 5, 789, 390; U.S. Patent No. 5, 834, 441; U.S. Patent No. 5, 863, 541; U.S.

Patent No . 5,851,521; U.S. Patent No . 5,252,479; Gnatenko et al. , J. Invest. Med. , 45 : 87 98 (1997)]; adenoviral (AV) vectors [See, e.g. , U.S. Patent No . 5,792, 453; U.S. Patent No.

5,824,544; U.S. Patent No. 5,707, 618; U.S. Patent No. 5, 693, 509; U.S. Patent No. 5, 670, 488; U.S. Patent No. 5, 585, 362; Quantin et al. , Proc. Natl . Acad. Sci . USA, 89: 2581 2584 (1992) ; Stratford Perricadet et al . , J . Clin . Invest . , 90 : 626 630 (1992) ; and Rosenfeld et al . , Cell , 68 : 143 155 (1992) ] ; an adenoviral adenoassociated viral chimeric (see for example, U.S. Patent No. 5,856,152) or a vaccinia viral or a herpesviral (see for example, U.S. Patent No. 5,879, 934; U.S. Patent No . 5,849,571; U.S. Patent No. 5, 830, 727; U.S. Patent No. 5, 661, 033; U.S.

Patent No . 5,328,688; Lipofectin mediated gene transfer (BRL) ; liposomal vectors [See , e.g., U.S. Patent No . 5, 631, 237

(Liposomes comprising Sendai virus proteins ) ] ; and combinations thereof .

Alternatively, peptide-based cancer vaccines may be generated wholly or partly by recombinant techniques . For example, a nucleic acid encoding a cancer antigen may be expressed in a host cell and the expressed antigen isolated and/or purified from the cell culture . For example , antigen may be expressed in E . coli either in soluble form or in inclusion bodies , which may be solubilized and refolded. After expression, the antigen may be isolated and/or purified. Cancer antigen may be analysed by standard techniques , such as mass spectrometry and western blot analysis .

The use of tumour antigens to generate immune responses is well- established in the art ( see for example; Kakimi K, et al . Int J Cancer . 2011 Feb 3 ; Kawada J, Int J Cancer . 2011 Mar 16; Gnj atic S, et al . Clin Cancer Res . 2009 Mar 15 ; 15 ( 6 ) : 2130- 9 ; Yuan J, et al. Proc Natl Acad Sci U S A. 2008 Dec 23; 105 (51) : 20410-5;

Sharma P, et al . J Immunother . 2008 Nov-Dec; 31(9) :849-57; Wada H, et al . Int J Cancer . 2008 Nov 15; 123(10) :2362-9; Diefenbach CS, et al . Clin Cancer Res . 2008 May 1 ; 14 ( 9 ) : 2740-8 ; Bender A, et al . Cancer Immun . 2007 Oct 19 ; 7 : 16 ; Odunsi K, et al . Proc

Natl Acad Sci U S A. 2007 Jul 31; 104 (31) : 12837-42; Valmori D, et al. Proc Natl Acad Sci U S A. 2007 May 22; 104 (21) : 8947-52;

Uenaka A, et al . Cancer Immun . 2007 Apr 19 ; 7 : 9 ; Kawabata R, et al. Int J Cancer. 2007 May 15 ; 120 (10) : 2178-84; Jager E, et al . Proc Natl Acad Sci U S A. 2006 Sep 26; 103 (39) : 14453-8; Davis ID Proc Natl Acad Sci U S A. 2005 Jul 5; 102 (27) : 9734; Chen Q, Proc Natl Acad Sci U S A. 2004 Jun 22; 101 (25) : 9363-8; Jager E, Proc Natl Acad Sci U S A. 2000 Oct 24; 97 (22) : 12198-203; Carrasco J, et al . J Immunol. 2008 Mar 1 ; 180 (5 ) : 3585-93 ; van Baren N, et al . J Clin Oncol. 2005 Dec 10; 23 (35) : 9008-21; Kruit WH, et al . Int J Cancer. 2005 Nov 20; 117 (4) :596-604; Marchand M, et al . Eur J Cancer . 2003 Jan; 39 (1) : 70- 7 ; Marchand M et al . Int J Cancer . 1999 Jan 18 ; 80 ( 2 ) : 219-30 ; Atanackovic D, et al . Proc Natl Acad Sci U S A. 2008 Feb 5; 105 (5) : 1650-5) . Typically, an immunotherapeutic agent, such as a cancer vaccine , is administered to the individual whose tumour expresses the said antigen . Cancer cells from the individual may be analysed to identify the tumour antigen and patients are then identified for administration of the appropriate immunotherapeutic agent or cancer vaccine . For example, a method as described herein may comprise the step of identifying a tumour antigen which is displayed by one or more cancer cells in a sample obtained from the individual . A biological sample may be obtained from the subj ect such as a biopsy, blood or bone marrow sample and tested for the presents of cancer cells which may be identified as displaying the tumour antigen using any standard techniques including but not limited to immunological techniques , such as immunocytochemistry and immunohistochemistry may be employed . Additional techniques include immunological analysis such as serologically determining an autologous immune response to said cancer antigen, see

WO2001/ 007917. Analysis of gene expression can be performed using methods known in the art such as polymerase chain reaction or microarray analysis .

HO-1 inhibitors and immunotherapeutic agents , such as cancer vaccines , as described herein, will usually be administered in the form of pharmaceutical compositions , which may comprise at least one additional component . The HO-1 inhibitor and the immunotherapeutic agent may be formulated in the same or separate pharmaceutical compositions .

A pharmaceutical composition may comprise, in addition to the HO-1 inhibitor and/or an immunotherapeutic agent such as a cancer vaccine, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art . Suitable materials will be sterile an pyrogen free, with a suitable isotonicity and stability .

Examples include sterile saline (e.g. 0.9% NaCl) , water, dextrose, glycerol , ethanol or the like or combinations thereof Such materials should be non-toxic and should not interfere wit the efficacy of the active compound. The precise nature of the carrier or other material will depend on the route of

administration, which may be by bolus , infusion, inj ection or any other suitable route , as discussed below . Suitable material will be sterile and pyrogen free, with a suitable isotonicity and stability . Examples include sterile saline (e.g. 0.9% NaCl) , water, dextrose , glycerol , ethanol or the like or combinations thereof . The composition may further contain auxiliary substances such as wetting agents , emulsifying agents pH buffering agents or the like .

Usually administration will be by the intravenous route, although other routes such as intraperitoneal, subcutaneous , transdermal , oral , nasal , intramuscular or other convenient routes are not excluded.

The cancer vaccine may be administered in conj unction with an adj uvant . Suitable adj uvants are well known in the art and include aluminum salts , such as alum (aluminium potassium sulphate dodecahydrate ) , aluminum hydroxide and aluminum phosphate and organic compounds , such as squalene .

In addition to a cancer antigen, an immunotherapeutic,

immunogenic or vaccine formulation may comprise an adj uvant . For example , a formulation may comprise 1-500 ]ig, preferably 1- 50 μg, of cancer antigen and 0.5 to 20 mg, preferably 1-10 mg, of adjuvant in a pharmaceutically acceptable carrier or diluent as mentioned above. A vaccine formulation may comprise a Toll-like Receptor (TLR) ligand. Suitable TLR ligands include polyinosinicpolycytidylic acid (poly I : C) , lipopolysaccharide (LPS ) , CpG

oligodeoxynucleotide , poly LC, poly ICLC , MPL (Corixa Corp) and imidazoquinolines, such as imiquimod and R848. The use of TLR ligands to modulate immune responses is well-known in the art

(see for example, Weiner et al (1997) PNAS USA 94 10833-10837; Vabulas et al J . Immunol . (2000) 164 2372-2378; Gunzer et al

(2005) Blood 106 2424-2432) . Formulations of immunotherapeutic agents , such as cancer vaccines , are well-known in the art and include MAGE-A3 ASCI, NY-ESO-1 ASCI and PRAME ASCI (GSK Bio) ; Provenge (Dendreon) , Abogovomab (Meranini ) , , M-Vax (Avax) , Allovectin-7 (Vial ) for metastatic melanoma, GSK1572932A (GSK Bio) Belagenpumatucel-L (Novarex) BMP-25 (Merck Serono ) , BiovaxID (Biovest/Accentia) , MDX-1379 (Medarex/BMS) , Ipilimumab (BMS) Trovax (Oxford

Biomedical ) Oncophage (Antigenics ) and PR1 leukemia peptide (The Vaccine company) . The HO-1 inhibitor and the immunotherapeutic agent may be administered in same or separate pharmaceutical compositions .

The HO-1 inhibitor may be comprised in a pharmaceutical

formulation . For example, suitable formulations of SnMP are well-known in the art for the treatment of neonatal j aundice , porphyria and inherited hyperbilirubinemia syndromes (Kappas et al . 2004. Pediatrics 113:119-123; Drummond G et al . 1987

Arch . Biochem. Biophys .255 : 64-74; Delaney, J 1988 : Pediatrics . 81 : 498-5041; Galbraith et al . 1989 Hepatology 9, No. 6: 882- 8438, Kappas A et al . 2001 Pediatrics . 108(6) : 1374-7 & Kappas A et al . 1993 Pediatrics 91 ; 537-539 ) . Generally the preparation of pharmaceutical compositions comprising a heme oxygenase inhibitor for oral use is described in US Patent Nos . 4657902 and 5010073. In some embodiments , immunotherapeutic agents and/or HO-1 inhibitors may be provided in a lyophilised form for

reconstitution prior to administration . For example, lyophilised reagents may be re-constituted in sterile water and mixed with saline prior to admini stration to an individual .

Doses of any individual immunotherapeutic agent and/or HO-1 inhibitor will be in accordance with conventional practice in the art , and at the discretion of the physician . SnMP is a potent inhibitor of HO-1 mediated heme catabolism which has been provided to many patients for the treatment of inherited hyperbilirubinemia and neonatal j aundice where it has been shown to be safe and effective (Kappas et al . 2004. Pediatrics , 113 : 119-123) . The HO-1 inhibitor SnMP, has been given either by intravenous (i.v.) infusion and intramuscular (i.m.) inj ection which has been previously described as safe in and effective in normal individuals (Galbraith et al . Hepatology Vol . 9 , No. 6, pp. 882-8438. 1989) . SnMP is cleared from the plasma of normal subj ects with dose-dependent pharmacokinetics (T, = 3.8 hr following i.v. administration of 1 μττιοΐβ per kg body

weight) . Plasma concentrations two hours following intramuscular administration of SnMP were identical to those obtained

following i.v. administration of SnMP . This route of

administration is consistent with existing reports of

administration of SnMP in registered clinical trials .

Pharmacokinetic (PK) data from the preclinical studies would support more or less frequent administration; allowing protocols to be adj usted accordingly .

For safety, clinical doses of SnMP are based on existing data, which has proved to be effective and safe in humans .

Investigational clinical studies using multiple doses of SnMP in porphyria patients (4 doses of 0.5 μτηοΐβ per kg body weight either i.v or i.m) and in normal adults (single dose i.v. 0.1- 1. Ομτηοΐβ per kg body weight) have been demonstrated as being safe and effective . Blood counts and serum biochemistries remained normal following SnMP treatment , throughout all studies . Coagulation tests and platelet counts were monitored during the study but no abnormalities were noted . The only dose- limiting side effect was transient cutaneous photosensitivity (Galbraith et al . Hepatology Vol. 9, No. 6, pp. 882-8438. 1989) . In additional studies a total of 68 porphyria patients received a single dose i.v. and 20 porphyria patients received multiple doses i.v. weekly for 6 months for a total of 24 doses

(Clinicaltrial.gov study No's: NCT00004398, NCT00004397,

NCT00004396, NCT00004789) . Compassionate studies in two 17 year old patients with a lethal hereditary liver disease

characterized by severe, sustained j aundice received 110 doses over 400 day treatment period (40 x 0.5 mole/Kg every 2 days , 70 x 1. ΟμΜοΙθ/Kg every 2 days , (Kappas A, et al . Pediatrics 1993 91 ; 537-539. ) . Prolonged administration of 110 doses of SnMP over a 400-day study period was well tolerated in both patients .

Compositions comprising cancer immunotherapeutic agents and/or HO-1 inhibitors may be prepared in the form of a concentrate for subsequent dilution, or may be in the form of divided doses ready for administration . Alternatively, the reagents may be provided separately within a kit , for mixing prior to

administration to a human or animal subj ect .

Administration is normally in a therapeutically effective amount, this being sufficient to show benefit to a patient .

Such benefit may be at least amelioration of at least one symptom .

It will be appreciated that appropriate dosages of the active compounds can vary from patient to patient . Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the administration. The selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the active compound, other drugs ,

compounds , and/or materials used in combination, and the age , sex, weight , condition, general health, and prior medical history of the patient . The amount of active compounds and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve concentrations of the active compound at a site of therapy without causing substantial harmful or deleterious side-effects ,

Routes of administration of an HO-1 inhibitor and an

immunotherapeutic agent is optionally via any conventional pharmaceutical route , including but not limited to oral , rectal , subcutaneous , parenteral , intravenous , intraperitoneal , and topical .

Administration in vivo can be effected in one dose , continuously or intermittently (e.g. , in divided doses at appropriate intervals ) . Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subj ect being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician .

Administration of the immunotherapeutic agent such as a cancer vaccine and the HO-1 inhibitor may be simultaneous , separate or sequential . By "simultaneous" administration, it is meant that the immunotherapeutic agent such as a cancer vaccine and the HO- 1 inhibitor are administered to the individual in a single dose by the same route of administration . By "separate" administration" , it is meant that the

immunotherapeutic agent and the HO-1 inhibitor are administered to the individual by two different routes of administration which occur at the same time . This may occur for example where one agent is administered by infusion or parenterally and the other is given orally during the course of the infusion or parenteral administration .

By "sequential" it is meant that the immunotherapeutic agent and the HO-1 inhibitor are administered at different points in time , provided that the activity of the first administered agent is present and ongoing in the subj ect at the time the second agent is administered. For example, the immunotherapeutic agent may be administered first, such that an immune response against a tumour antigen is generated, followed by administration of the HO-1 inhibitor compound of the invention such that

immunosuppression at the site of the tumour is reduced .

Preferably, a sequential dose will occur such that the second of the two agents is administered within 48 hours , preferably within 24 hours , such as within 12 , 6, 4 , 2 or 1 hour ( s ) of the first agent .

Multiple doses of the HO-1 inhibitor may be administered, for example 2 , 3 , 4 , 5 or more than 5 doses may be administered after administration of the immunotherapeutic agent . The administration of the HO-1 inhibitor may continue for sustained periods of time after admini stration of the immunotherapeutic agent . For example treatment with the HO-1 inhibitor may be continued for at least 1 week, at least 2 weeks , at least 3 weeks , at least 1 month or at least 2 months . Treatment with the HO-1 inhibitor may be continued for as long as is necessary to achieve complete tumour rej ection .

Multiple doses of the immunotherapeutic agent may be

administered, for example 2 , 3 , 4 , 5 or more than 5 doses may be administered after administration of the HO-1 inhibitor . The administration of the immunotherapeutic agent may continue for sustained periods of time after administration of the HO-1 inhibitor . For example treatment with the immunotherapeutic agent may be continued for at least 1 week, at least 2 weeks , at least 3 weeks , at least 1 month or at least 2 months . Treatment with the immunotherapeutic agent may be continued for as long as is necessary to achieve complete tumour rej ection .

The immunotherapeutic agent and HO-1 inhibitor may be

administered alone or in combination with other treatments , either simultaneously or sequentially dependent upon the individual circumstances .

For example , the immunotherapeutic agent and the HO-1 inhibitor may be admini stered in conj unction with other anti-cancer agents . Other anti-cancer agents may include any known agent with desirable anti-cancer properties , including taxoids such as Taxol®, Taxotere© or other chemotherapeutics , such as cis-platin

(and other platin intercalating compounds ) , etoposide and etoposide phosphate, bleomycin, mitomycin C, CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, and the like . Other anticancer agents may be Contemplated chemotherapeutics for use in combination therapies as disclosed herein include aspirin, sulindac, curcumin, alkylating agents including : nitrogen mustards , such as mechlor-ethamine , cyclophosphamide ,

ifosfamide, melphalan and chlorambucil ; nitrosoureas , such as carmustine (BCNU) , lomustine (CCNU) , and semustine (methyl- CCNU) ; ethy1enimines /methylmelamine such as thriethylenemelamine

(TEM) , triethylene, thiophosphoramide (thiotepa) ,

hexamethylmelamine (HMM, altretamine ) ; alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC) ; antimetabolites including folic acid analogs such as methotrexate and

trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine) , 5-azacytidine , 2,2^' -difluorodeoxycytidine , purine analogs such as 6-mercaptopurine, 6-thioguanine , azathioprine, 2 ' -deoxycoformycin (pentostatin) , erythrohydroxynonyladenine (EHNA) , fludarabine phosphate, and 2-chlorodeoxyadenosine

( rubidomycin) , doxorubicin, mitoxantrone , idarubicin,

bleomycins , plicamycin (mithramycin) , mitomycinC , and

actinomycin; and enzymes such as L-asparaginase . The anticancer agent may also be a biological agent such as a protein that inhibits tumour growth, such as interferon (I N) -gamma, tumour necrosis factor (TNF) -alpha, TNF-beta, GM-CSF, and similar cytokines , or an anti-angiogenic factor such as

angiostatin and endostatin or inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors , including soluble VGF/VEGF receptor . Further anti-cancer agents can be platinum coordination complexes such 3.3 CI splatin and

carboplatin, anthracenediones such as mitoxantrone , substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH ) and procarbazine , adrenocortical suppressants such as mitotane (o , p ^' -DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid

antagonists such as prednisone and equivalents , dexamethasone and aminoglutethimide ; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethyl stilbestrol and ethinyl estradiol equivalents ; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents ;

antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide ; non-steroidal antiandrogens such as flutamide ; kinase inhibitors , histone deacetylase inhibitors , methylation inhibitors , proteasome inhibitors , monoclonal antibodies , oxidants , anti-oxidants , telomerase inhibitors , BH3 mimetics , ubiquitin ligase inhibitors , stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate

(marketed as Gleevac or Glivac ) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; and anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab .

Various embodiments are disclosed above for combinations of HO-1 inhibitors , such as SnMP, and different immunotherapeutic agents . Aspects and embodiments of the invention relating to combinations of two or more compounds or agents (i.e. an HO-1 inhibitor and an immunotherapeutic agent and optionally one or more other agents ) disclosed above include disclosure of the administration of the compounds or agents separately

(sequentially or simultaneously) or in combination ( co- formulated or mixed) . For each aspect or embodiment , the specification further discloses a composition comprising the two or more compounds or agents (i.e. the HO-1 inhibitor and the immunotherapeutic agent and optionally one or more other agents ) co- formulated or in admixture with each other and further discloses a kit or unit dose containing the two or more

compounds/agents packaged together, but not in admixture .

Optionally, such compositions , kits or doses further comprise one or more carriers in admixture with one or both agents or co- packaged for formulation prior to administration to a subj ect . The reverse also is true : some aspects of the invention are disclosed herein as compositions useful for therapy and

containing two or more therapeutic agents . Equivalent methods and uses are specifically contemplated .

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure .

All documents mentioned in this specification are incorporated herein by reference in their entirety .

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other . For example "A and/or B" is to be taken as specific disclosure of each of ( i ) A, (ii) B and ( iii ) A and B, j ust as if each is set out individually herein .

Unless context dictates otherwise, the descriptions and

definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described .

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above .

Experiments

A CD45+ subset of FAP+ stromal cells was found to express heme oxygenase-1 (HO-1) . EGFP/BAC transgenic mice were inj ected subcutaneously with LL2 /OVA tumours . At day 10 the palpable tumour was harvested . Four color fluorescent confocal microscopy of the LL2/OVA tumour was carried out in which FAP+ cells

(Green) , CD45+ (Red) and HO-1+ (White) were visualized,

represented in Figure 1.

Nuclei were stained with DAPI (Blue) . HO-1 expression co- localized with CD45+/FAP+ cell s (merge panel : triple positive cells are yellow) . HO-1 expres sion was found to co-localize with

CD45+/ FAP+ cells (Merge panel : triple positive cells are yellow ) . Many CD45+ cells were single positive , as are two FAP+ cells . No double positive cell s were observed.

Groups of non-transgenie mice were inj ected subcutaneously with either the immunogenic tumour LL2/OVA or the non-immunogenic LL2 tumours . At day 10 the tumours became palpable and the mice were inj ected intraperitoneally with the HO-1 inhibitor SnMP

(25 Mol/kg) or PBS control , every other day for the duration of the experiment . The tumour growth was measured every 2-3 days and the growth curves for each group of mice are represented in Figure 2. Inhibiting HO-1 with SnMP caused acute hypoxic tumour necrosis and reduced growth of the established LL2/OVA tumours compared to treatment with PBS. The effect was not observed in the non-immunogenic LL2 tumours where similar growth kinetics was observed following treatment with either SnMP or PBS, indicating that the necrotic response is immunologically mediated and dependent upon stimulation of a robust endogenous stimulation of anti-tumour immunity, specifically to the Ova antigen . Outgrowth of LL2/OVA tumours was noted following long- term treatment of with SnMP . Analysis of these outgrowing tumours indicated that the OVA expression had been lost . SnMP induced selection of OVA loss variants further indicates that the response was immunologically mediated and that the anti- tumour effect was dependent upon both the pharmacologic

inhibition of HO- 1 and specific anti-tumour immunity . The non- immunogenic LL2 tumour were unable to, resulting in limited response to SnMP treatment induce a robust endogenous anti- tumour immunity .

Further groups of non-transgenie mice were inj ected

subcutaneously with LL2 /OVA tumours . At day 10 , the tumours became palpable and the mice were inj ected intraperitoneally with the HO-1 inhibitor SnMP ( 25pMol/kg) . Tumours were harvested at 0, 12 and 25 hours following SnMP administration . The number of viable cell per gram tissue was measured as represented in Figure 3. As expected there was reduced proportion of viable cells over time following SnMP administration suggesting that inhibition of HO-1 activity by SnMP caused rapid cell death in the immunogenic tumours .

Further groups of non-trangenic mice were inj ected subcutaneously with LL2 /OVA tumours . At day 10 , the tumours became palpable and the mice were inj ected with pimonidazole one hour before being inj ected intraperitoneally with the HO-1 inhibitor SnMP (25pMol/kg) . In vivo pimonidazole conj ugates to proteins in hypoxic regions of low p0₂. Tumours were harvested after 0, 12 and 24 hours following SnMP administration and conj ugated pimonidazole was detected by immunoperoxidase staining. As represented in Figure 4 , SnMP treatment induced increased levels of acute hypoxia within the immunogenic tumour over time .

Further groups of non-trangenic mice were inj ected

subcutaneously with LL2/OVA tumours . At day 10 the tumours became palpable and the mice were inj ected intraperitoneally with the HO-1 inhibitor SnMP ( 25 Mol/kg) . Tumours were harvested after 0, 12 and 24 hours following SnMP administration and single cell preparations were stained with antibodies to CD31 and Tissue factor and analyzed by FACS . Figure 5 represents the mean fluorescence index (MFI ) of Tissue Factor staining of CD31+ cells . SnMP induced inhibition of HO-1 results in rapid

expression of Tissue Factor by endothelial cells within the immunogenic tumour . Activated endothelial cells would lead to clotting in intratumour vessels causing hypoxia and likely tumour cell death .

Frozen sections of a murine LL2/OVA tumor were stained with antibodies to CD4 , Foxp3 , and HO-1 and analysed by confocal microscopy (Figures 6 and 7 ) . Foxp3+ CD4+ Treg cells were found to be distinct from HO-1+ cells .

Additional immunogenic tumour models and transgenic models are used to demonstrate that pharmacologic inhibition of HO-1 by SnMP would modulate the immune suppression of the FAP+ stromal cells in the tumour microenvironment . Pharmacologic inhibition of HO-1 by an inhibitor such as SnMP would complement many of the current strategies and immunotherapeutic agents being used or evaluated as immunotherapies for the treatment of cancer patients .

P815 is a mastocytoma tumour line derived from a methyl- cholanthrene induced tumour in DBA/2 mice . It can be

successfully transplanted in naive syngeneic mice but is fully rej ected in mice previously immunized against P1A, the maj or antigen naturally expressed by the P815 tumour cells (Brandle, 1994, Van den Eynde, 1991; Lethe, 1992, Van den Eynde, 1994) . PIA is expressed in various tumour cells but not in normal somatic tissues . The only normal cells expressing such genes are male germ cells , which do not express MHC molecules at their surface and therefore cannot present the peptide antigen . The PlA-encoded antigen is representative tumour model for the important group of tumour-specific antigens encoded by cancer- germline genes . The corresponding human cancer-germline genes or cancer testis antigens , such as MAGE-A3 , NY-ESO-1 , LAGE and PRAME are currently being tested in clinical trials of

experimental cancer vaccines . A number of preclinical models of cancer vaccine rely on the use of this antigen (Uyttenhove , 1997, Naslund, 2007, Bilsborough, 1999, Uyttenhove , 2003) .

Groups of DBA/2 mice are inj ected subcutaneously with P815 tumours following the standard protocols in Uyttenhove , 1997, Naslund, 2007 , Bilsborough, 1999, Uyttenhove , 2003 which are incorporated herein in their entirety . Once tumours are palpable the mice are inj ected intraperitoneally with the HO-1 inhibitor SnMP (25 Mol/kg) or PBS control , every other day for the duration of the experiment . The tumour growth was measured every 2-3 days to establish the growth curves for each treatment group of mice . Reduced growth of established P815 tumours following treatment with SnMP compared to with treatment confirms in an additional tumour model that Inhibition of HO-1 with SnMP can result in acute hypoxic tumour necrosis . Analysis of PIA expression in any outgrowing P815 tumours following long term treatment of with SnMP as a result of SnMP induced selection of PIA loss variants will indicate that the anti- tumour response was immunologically mediated . In addition, the DBA/2 mice can be immunized against PIA using various vaccination protocols and scheduling protocols for combination with SnMP treatment , to elucidate the relationship between : pre-existing immunity on the induction of tumour necrosis following inhibition of HO-1 with SnMP treatment, inhibition of HO-1 with SnMP treatment and therapeutic immunization with a PIA vaccine. Additionally, combinations of SnMP treatment and other types of

immunotherapeutic agents such as immune checkpoint blockers (anti-CTLA-4 antibody, anti-PDl , anti-PDLl antibodies ) will be tested in this preclinical model . Further combinations of SnMP treatment with biological modifiers such as IDO and TDO

inhibitors , including but not limited to INCB24360 and LM10 will be tested in this model . DBA/2 mice were inj ected with 10⁶ living P815 s.c. tumor cells on day 0. On day 4, they received the anti-tumor vaccine in the form of subcutaneous inj ections of living L1210 leukemia cells engineered to express the tumor antigen encoded by PIA as well as the costimulatory molecule B7-1 (Brandle et al Eur . J.

Immunol . 28:4010-4019,⁾ (Figure 8 ) . These immunizing

L1210. P1A.B7-1 cells (21 x 10⁶ cells in total ) were inj ected in three s.c. sites that were distinct from the site of P815 tumor inj ection . As expected, the immunizing cells were completely rej ected through the induction of a strong anti-PlA immune response . This however did not prevent the growth of the P815 tumor inoculated before the vaccination : on day 15 , all

vaccinated mice bore tumors of 200-300 mm3 in size . On day 15 , a group of vaccinated mice started receiving i.p. inj ections of SnMP to inhibit HO-1. SnMP was administered every other day at a dose of 18.8 mg/kg in 100 μΐ . Tumor sizes were measured every 4 days with a caliper (Figure 9 ) . On day 25, the mean tumor volume in the [vaccine + SnMP] group was 722 ± 203 mm^J, while it was 1684 ± 324 mm^:i in the group treated with the vaccine alone ( see figure 9 ) . Data were analyzed using the Student T-test . The P value was 0.0257 ( [vaccine + SnMP] versus [vaccine alone ] ) .

The results showed that SnMP synergizes with the tumor vaccine to induce a therapeutic effect allowing the rej ection of preexisting P815 tumors (Figure 9 ) . TIRP is an inducible murine melanoma model expressing the PIA cancer-germline antigen (Huijbers, I . J, Cancer Res 2006; 66 : 3278-86) . The transgenic mouse strain develops autologous melanomas expressing a defined PIA tumour antigen. Melanocytes are transformed by simultaneously activating the Ras pathway and inactivating tumour suppressor Ink4a/Arf , reproducing two genetic events frequently observed in human melanoma . Melanomas are induced with 4-OH-tamoxifen (OHT ) have an activated Ras pathway, deletion of gene Ink4a/Arf and express PIA and are recognized by PlA-specific T cells . This model enables optimal characterization of the interactions between the immune response and naturally occurring tumours and tumour microenvironment . Melanoma are induced with OHT in groups of TIRP mice following the standard protocols in Huij bers , I . J, Cancer Res 2006; 66 : 3278-86 which are incorporated herein in their entirety . Once tumours are palpable , the mice are inj ected intraperitoneally with the HO-1 inhibitor SnMP ( 25pMol/kg) or PBS control , every other day for the duration of the experiment . The tumour growth was measured every 2-3 days to establish the growth curves for each treatment group of mice . Reduced growth of established tumours following treatment with SnMP compared to with treatment confirms in an additional tumour model that Inhibition of HO-1 with SnMP can result in acute hypoxic tumour necrosis . Analysis of PIA expres sion in any outgrowing tumours following long term treatment of with SnMP as a result of SnMP induced selection of PIA loss variants will indicate that the anti- tumour response was immunologically mediated . In addition, the mice can be immunized against PIA using various vaccination protocols and scheduling protocols for combination with SnMP treatment , to elucidate the relationship between : pre-existing immunity on the induction of tumour necrosis following inhibition of HO- 1 with SnMP treatment, inhibition of HO-1 with SnMP treatment and therapeutic immunization with a PIA vaccine . Additionally, combinations of SnMP treatment and other types of

inhibitors , including but not limited to INCB2 360 and LM10 will be tested in this model .

The CT26 is a tumour cell line derived from a chemically induced murine colon carcinoma (Brattain et al . , 1980) . The BALB/c transplantable tumour was transfected with human NY-ESO-1 antigen, an important member of cancer germline gene family (cancer-testis antigen ) widely expressed among many cancer types which has been the focus of a large number of human cancer vaccine clinical trials (Muraoka . D et al . 2010 : J . Immunol , 185 : 3768-3776) . Groups of BALB/c mice are inj ected subcutaneously with CT26-NY- ESO-l tumour following the standard protocols in Muraoka . D et al . 2010 : J . Immunol , 185 : 3768-3776 this is incorporated herein in its entirety . Once tumours are palpable the mice are inj ected intraperitoneally with the HO-1 inhibitor SnMP ( 25 Mol/kg) or PBS control , every other day for the duration of the experiment . The tumour growth was measured every 2-3 days to establish the growth curves for each treatment group of mice . Reduced growth of established CT26-NY-ESO-l tumours following treatment with SnMP compared to with treatment confirms in an additional tumour model that Inhibition of HO-1 with SnMP can result in acute hypoxic tumour necrosis . Analysis of NY-ESO-1 expression in any outgrowing CT26-NY-ESO-l tumours following long term treatment of with SnMP as a result of SnMP induced selection of NY-ESO-1 loss variants will indicate that the anti- tumour response was immunologically mediated . In addition, the BALB/ c mice can be immunized against NY-ESO-1 using various vaccination protocols and scheduling protocols for combination with SnMP treatment , to elucidate the relationship between : pre-existing immunity on the induction of tumour necrosis following inhibition of HO- 1 with SnMP treatment, inhibition of HO-1 with SnMP treatment and therapeutic immunization with a NY-ESO-1 vaccine . Additionally, combinations of SnMP treatment and other types of immunotherapeutic agents such as immune checkpoint blockers (anti-CTLA-4 antibody, anti-PDl , anti-PDLl antibodies ) will be tested in this preclinical model . Further combinations of SnMP treatment with biological modifiers such as IDO and TDO

inhibitors , including but not limited to INCB24360 and LM10 will be tested in this model .

We cloned the CT26 line by limited dilution and used CT26 clone 1. Female BALB/c mice (9 to 10 weeks of age) were inoculated s.c. with 5 x 10^Λ5 CT26 tumor cells on day 0. Tumor sizes were measured after becoming visible two times weekly in two

dimensions using a caliper, and the volume presented in mm³ using the formula : V = 0.5 (A x Β^Λ2 ) , where A and B are the long and short diameters of the tumor, respectively . On day 8 , tumor- bearing animals were sorted into groups ( 10 mice each) with similar mean tumor volumes prior to treatment, usually 32 to 64 mm3. One group received the vehicle control (100 μΐ i . p . ) , the other group received SnMP i.p. at a dose of 18 mg/ kg in 100 μΐ ( see Figure 10 ) . Inj ections were repeated every other day . Data were analyzed using the Student T-test .

The progression of CT26 tumors in mice treated or not with SnMP is shown in Figures 11 and 12. SnMP was found to slow tumor growth in this model .

CMS-5 is a methylcholanthrene-induced fibrosarcoma of BALB/c origin, shown to be devoid of viral antigens ( DeLeo et al . , 1977) and weakly immunogenic when compared with CMS-13 or Meth A, two nonerossreacting methylcholanthrene-induced tumours of the same genetic background (Srivastava et al . , 1986) . The BALB/ c transplantable tumour was transfected with human NY-ESO-1 antigen, an important member of cancer germline gene family (cancer-testis antigen) widely expressed among many cancer types which has been the focus of a large number of human cancer vaccine clinical trials (Muraoka . D et al . 2010 : J . Immunol, 185 : 3768-3776) .

Groups of BALB/c mice are inj ected subcutaneously with CMS-5 - NY-ESO-1 tumour following the standard protocols in Muraoka . D et al . 2010 : J. Immunol , 185: 3768-3776. Once tumours are palpable the mice are inj ected Intraperitoneally with the HO-1 inhibitor SnMP (25 Mol/kg) or PBS control , every other day for the duration of the experiment . The tumour growth was measured every 2-3 days to establish the growth curves for each treatment group of mice . Reduced growth of established CMS-5 -NY-ESO-1 tumours following treatment with SnMP compared to with treatment confirms in an additional tumour model that Inhibition of HO-1 with SnMP can result in acute hypoxic tumour necrosis . Analysis of NY-ESO-1 expression in any outgrowing CMS-5 -NY-ESO-1 tumours following long term treatment of with SnMP as a result of SnMP induced selection of NY-ESO-1 loss variants will indicate that the anti- tumour response was immunologically mediated . In addition, the BALB/c mice can be immunized against NY-ESO-1 using various vaccination protocols and scheduling protocols for combination with SnMP treatment, to elucidate the relationship between : pre-existing immunity on the induction of tumour necrosis following inhibition of HO-1 with SnMP treatment, inhibition of HO-1 with SnMP treatment and therapeutic

immunization with a NY-ESO-1 vaccine . Additionally, combinations of SnMP treatment and other types of immunotherapeutic agents such as immune checkpoint blockers (anti-CTLA-4 antibody, anti- PD1, anti-PDLl antibodies ) will be tested in this preclinical model . Further combinations of SnMP treatment with biological modifiers such as IDO and TDO inhibitors , including but not limited to INCB24360 and LM10 will be tested in this model .

Bl 6 melanoma is a murine melanoma of C57B1/ 6 origin, which rapidly develops as a tumour when inoculated into syngeneic immunocompetent hosts (Fidler, 1973) . Nevertheless , Bl 6 tumours are considered to be immunogenic since tumour regression can be induced by means of immunotherapeutic intervention . Furthermore , B16 melanoma cells express several melanoma-associated antigens that may serve as targets for autologous T cells . The C57B1/ 6 transplantable tumour was transfected with human NY-ESO-1 antigen, an important member of cancer germline gene family (cancer-testis antigen) widely expressed among many cancer types which has been the focus of a large number of human cancer vaccine clinical trials (Maraskovsky E . , 2004, Clin Cancer Res .10 (8) :2879-90) .

Groups of C57B1/ 6 mice are inj ected subcutaneously with B16-NY- ESO-1 tumour following the standard protocols in Maraskovsky E . , 2004, Clin Cancer Res .10 (8) : 2879-90 which is incorporated herein in its entirety . Once tumours are palpable the mice are inj ected intraperitoneally with the HO-1 inhibitor SnMP

(25pMol/kg) or PBS control , every other day for the duration of the experiment . The tumour growth was measured every 2-3 days to establish the growth curves for each treatment group of mice . Reduced growth of established Bl 6 -NY-ESO-1 tumours following treatment with SnMP compared to with treatment confirms in an additional tumour model that inhibition of HO-1 with SnMP can result in acute hypoxic tumour necrosis . Analysis of NY-ESO-1 expression in any outgrowing Bl 6 -NY-ESO-1 tumours following long term treatment of with SnMP as a result of SnMP induced selection of NY-ESO-1 loss variants will indicate that the anti- tumour response was immunologically mediated . In addition, the C57B1/ 6 mice can be immunized against NY-ESO-1 using various vaccination protocols and scheduling protocols for combination with SnMP treatment, to elucidate the relationship between : preexisting immunity on the induction of tumour necrosis following inhibition of HO-1 with SnMP treatment , inhibition of HO-1 with SnMP treatment and therapeutic immunization with a NY-ESO-1 vaccine . Additionally, combinations of SnMP treatment and other types of immunotherapeutic agents such as immune checkpoint blockers (anti-CTLA-4 antibody, anti-PDl , anti-PDLl antibodies ) will be tested in this preclinical model . Further combinations of SnMP treatment with biological modifiers such as IDO and TDO inhibitors , including but not limited to INCB2 360 and LM10 will be tested in this model.

The transgenic and non-transgenie animal models described herein demonstrate that pharmacologic inhibition of HO-1, for example by SnMP, modulates the immune suppression of the FAP+ stromal cells in the tumour microenvironment .

In summary, HO-1 inhibitor, SnMP, was found to slow the growth of immunogenic LL2/OVA tumours . This growth control involved acute hypoxic necrosis . SnMP increased expression of Tissue Factor by endothelial cells and this may be involved in

mediating microvascular thrombosis and hypoxic necrosis . (Tissue Factor expression is a characteristic of TNF- β- stimulated endothelial cells ) . SnMP-induced hypoxic necrosis was found to resemble the necrosis of LL2 /OVA tumours in which FAP+ stromal cells are ablated . HO-1 was found to be expressed the CD45+ subset of FAP+ cells . As only the CD45-/FAP+ subset may be involved in hematopoiesis and metabolism, inhibition of HO-1 in CD45+/FAP+ cells may not have the adverse effects seen with ablating all FAP+ cells .

HO-1 may therefore be a therapeutic target for enhancing the efficacy of anti-cancer vaccines and pharmacologic inhibition of HO-1 by an inhibitor such as SnMP, may complement many of the current strategies being used or evaluated as immunotherapies for the treatment of cancer patients .

Claims

Claims :

1. A method of treatment of cancer comprising administering a HO-1 inhibitor and an immunotherapeutic agent to an individual in need thereof.

2. A HO-1 inhibitor and an immunotherapeutic agent for use in the treatment of cancer .

3. An HO-1 inhibitor for use in combination with an

immunotherapeutic agent in the treatment of cancer .

4. An immunotherapeutic agent for use in combination with an HO-1 inhibitor in the treatment of cancer .

5. Use of an HO-1 inhibitor and an immunotherapeutic agent in the manufacture of a medicament for use in the treatment of cancer .

6. Use of an immunotherapeutic agent in the manufacture of a medicament for use for use in combination with an HO-1 inhibitor in the treatment of cancer .

7. Use of an HO-1 inhibitor in the manufacture of a

medicament for use in combination with an immunotherapeutic agent for the treatment of cancer .

8. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 1 to 7 wherein the HO-1 inhibitor is a metalloporphyrin taken from the group SnMP, ZnMP, FeMP, MnMP, CrMP, SnPP, CrPP, MnPP .

9. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 8 wherein the HO-1 inhibitor is Tin

Mesoporphyrin ( SnMP) .

10. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 1 to 9 wherein the HO-1 inhibitor is comprised in a pharmaceutical formulation .

11. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 1 to 10 wherein the

immunotherapeutic agent is a cancer vaccine .

12. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 11 wherein cancer cells in the individual express a tumour antigen which is immunologically cross reactive with the cancer vaccine .

13. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 12 wherein the tumour antigen is expressed in the cancer cells but not normal somatic cells of the individual .

14. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 12 or claim 13 wherein the tumour antigen is a cancer-testis antigen encoded by a cancer-germ line gene .

15. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 12 to 14 wherein the tumour antigen is selected from the group consisting of : P1A, MAGE-A1 , MAGE-A2 , MAGE- A3, MAGE-A4, MAGE-A5, MAGE-A6 , MAGE-A7, MAGE-A8, MAGE-A9 , MAGE-A10 , MAGE-A11 , MAGE-A12, GAGE-I , GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6 , GAGE-7, GAGE-8, BAGE-I, RAGE-1,

LB33/MUM-1, FRAME, NAG, MAGE-Xp2 (MAGE-B2) , MAGE-Xp3 (MAGE-B3) , MAGE-Xp4 (MAGE-B4 ) , tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE- C1/CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX- 2 (HOM-MEL-40) , SSX-3, SSX-4, SSX-5, SCP-I and XAGE .

16. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 12 to 15 wherein the treatment comprises identifying the presence or amount of the tumour antigen in one or more cancer cells in a sample obtained from the individual .

17. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 1 to 10 wherein the

immunotherapeutic agent is a therapeutic antibody .

18. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 17 wherein the therapeutic antibody is Ipilimumab or Tremelimumab .

19. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 17 wherein the therapeutic antibody is an anti-PDl antibody .

20. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 17 wherein the therapeutic antibody is an anti-PDLl antibody .

21. A method, immunotherapeutic agent, HO-l inhibitor or use according to claim 17 wherein the therapeutic antibody is an anti-OX40 antibody .

22. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 17 wherein the therapeutic antibody is an anti-GITR antibody .

23. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 1 to 10 wherein the

immunotherapeutic agent is a biological response modifier .

24. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 23 wherein the biological response modifier is an indoleamine 2 , 3 dioxygenase ( IDO) inhibitor .

25. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 24 wherein the indoleamine 2, 3 dioxygenase ( IDO) inhibitor is 1-methyl- [D] -tryptophan .

26. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 23 wherein the biological response modifier is a tryptophan 2 , 3-dioxygenase (TDO) inhibitor .

27. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 24 wherein the indoleamine 2 , 3 dioxygenase (IDO) inhibitor is INCB024360.

28. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 26 wherein the tryptophan 2 , 3-dioxygenase (TDO) inhibitor is LM10.

29. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of the preceding claims wherein the immunotherapeutic agent is comprised in an immunotherapeutic formulation .

30. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 29 wherein the immunotherapeutic formulation comprises a TLR ligand .

31. A method, immunotherapeutic agent, HO-1 inhibitor or use according to claim 30 wherein the TLR ligand is poly LC, poly ICLC (TLR3) , MPL (TLR4), imiquimod (TLR7) , R848 (TLR8) or CpG (TLR9 )

32. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 29 to 31 wherein the

immunotherapeutic formulation comprises an adjuvant .

33. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 29 to 32 wherein the immunotherapeutic formulation is selected from MAGE-A3 ASCI ; Provenge ; Abogovomab; M-Vax; Allovectin-7; GSK1572932A;

Belagenpumatucel-L ; BMP™25 ; BiovaxID; MDX-1379 ; Trovax ;

Oncophage and PR1 leukaemia peptide .

34. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 1 to 33 wherein the cancer is bladder cancer, ovarian cancer, breast cancer, prostate cancer, melanoma, metastatic melanoma, lung cancer, non-small cell lung cancer, ovarian cancer, non-Hodgkin ' s lymphoma, acute myeloid leukaemia, hepatocellular carcinoma, brain cancer, glioma, or glioblastoma .

35. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 1 to 35 wherein the cancer is resistant to treatment with the immunotherapeutic agent in the absence of the HO-1 inhibitor .

36. A method, immunotherapeutic agent, HO-1 inhibitor or use according to any one of claims 1 to 35 wherein the individual has one or more tumours which comprise CD45+, HO-1+ stromal cells .

37. A method, use, immunotherapeutic agent or HO-1 inhibitor according to claim 36 wherein the treatment comprises

identifying one or more CD45+ HO-1+ stromal cells in a tumour biopsy from the individual .

38. A composition comprising an HO-1 inhibitor and an immunotherapeutic agent .

A composition according to claim 38 for use in a method ording to any one of claims 1 and 8 to 38.