WO2007141737A2 - Targeted gene expression for the treatment of primary tumors, and a therapeutic extension to the treatment of metastases. - Google Patents

Abstract

A method for treating a primary cancer and its metastases, said method comprising the steps of: a) Inserting genes into viral vectors, each gene being placed under the expression control of heat- inducible promoters, b) Injecting such vectors into tumors in patients or animals, c) Activating the expression of such genes by applying a targeted source of heat.

Description

Targeted gene expression for the treatment of primary tumors, and a therapeutic extension to the treatment of metastases.

FIELD OF THE INVENTION The treatment of cancer has relied for many years on the use of basically three methodologies, either alone or combined. These are surgical interventions, radiotherapy and chemotherapy. More recent additions to this list include the development of cancer vaccines, thermal ablation and gene therapy. These approaches to the treatment of cancer have met with some limited success in the treatment of certain primary tumors, but less success in the treatment of secondary metastases.

One of the major problems in treating primary tumors is that of removing or otherwise destroying all of the tumor mass, and not leaving cancer cells that may migrate and initiate tumors elsewhere in the body. A second problem is the ability of cancer cells to mask tumor-specific antigens, and thus largely escape immune surveillance.

The problem that needs to be solved is that of efficiently alerting the immune system to tumor antigens exposed during the death of tumor cells through induced apoptosis or other means. This can be achieved by targeted gene expression in primary tumors of genes leading to cell death through apoptosis and/or necrosis, as well as genes alerting the immune system to the presence of tumor antigens. This is the subject of this invention.

BACKGROUND TO THE INVENTION

Metastases

Tumors have the inability to invade normal tissues (replacing healthy cells with the cancerous ones), hence they are called "malignant" and spread (metastasize) to other parts of the body.

Metastases formation takes place in many ways: through the lymphatic system, through the bloodstream, by spreading through body spaces such as the bronchi or abdominal cavity, or through implantation.

The most common way for cancer to spread is through the lymphatic system. This process is called "embolization". The lymph system has its own channels that circulate throughout the body, similar to the veins and arteries of the bloodstream. These channel are very small and carry a tissue fluid called lymph throughout the body.

Often when a solid tumor is removed by surgery, the surgeon will remove not only the tumor but the neighboring lymph glands, even though there is no visible sign of cancer in those glands. This is done as a precautionary measure, because if even one cell has broken away from the tumor and lodged in the lymphatic system, the cancer could continue growing and metastasizing.

Cancer can also metastasize through the bloodstream. Cancer cells, like healthy cells, must have a blood source in order to grow. Malignant cells can break off from the tumor and travel through the bloodstream until they find a suitable place to start forming a new tumor. (Tumors almost always metastasize through the veins rather than through the arteries.) Sarcomas spread through the bloodstream, as do certain types of carcinomas, like carcinoma of the kidneys, testicular carcinoma, and Wilms' tumor, a type of kidney cancer seen in young children. Cancers may spread by more than one route.

Cancers can also spread by local invasion — that is, by intruding on the healthy tissue that surrounds the tumor. Some cancers that spread this way do not venture very far from the original site. An example of this kind of cancer is basal cell carcinoma of the skin. When this kind of cancer is removed by a surgeon, a wide area of healthy tissue surrounding it is also removed and it is usually "cured" immediately. Unless some cells have been left behind, it is very unlikely that it will recur. (However, it is possible that a second cancer of the same kind may start to grow at a later time at a completely different site -- the new growth having nothing to do with the first.)

A very rare type of metastasis is caused by implantation or inoculation. This can happen accidentally when a biopsy is done or when cancer surgery is performed. In this case malignant cells may actually drip from a needle or an instrument (this is also called a "spill"). It is desirable, therefore, if possible and if the cancer is small to remove it completely at the initial surgery — that is at the time of the biopsy. Cancers do not spread in a completely random fashion. Some parts of the body are more vulnerable to becoming metastatic sites than others. For example, cancers rarely metastasize to the skin, but they often metastasize to the liver and lungs. Each type of cancer has its own pattern for metastases.

Metastases formation is a multi-step complex process, which begins with the changes in the genetic material of a cell followed by the uncontrolled multiplication of altered cells and it continues with the development of a new blood supply for the tumor, invading the circulatory system, dispersing of small clumps of tumor cells to other parts of organs or parts of the body and the growth of the secondary tumors in those sites. Some of the specific types of metastases are: Brain metastases, bone metastases, lung metastases and liver metastases.

Metastases treatment

Metastases treatment entirely depends on where the cancer has started and to where it spread to, and knowing the source clearly helps the oncologist in predicting whether it will respond properly to the therapies, such as chemotherapy, radiation therapy or hormonal therapy and selected drugs to treat them.

Chemotherapy

This uses the anticancer drugs, which are usually injected through a vein or given by mouth, and these drugs enter the bloodstream to reach the tumor, which has been metastasized. Chemotherapy is used as a source of primary therapy with the sole aim to curing some metastatic cancers such as lymphomas and germ cell tumors of the ovaries, testicles or placenta. Chemotherapy drugs kill the cancer cells, so appropriate care should be taken while administering them, as serious damage to normal cells can often occur.

Hormonal Therapy

There are some drugs, which can be given to block the action of certain hormones or suppress their growth or production. There are some side effects, which depend upon the type of hormonal treatments used, which might include hot flashes, loss of libido, blood clots and increased risk of other cancers. Radiation Therapy

Radiation may be administered in the form of gamma rays or x-rays. They differ only in their origin, but not in their ultimate biological effects. Radiation therapy is administered to those cancers where there is a selective ability for the radiation to destroy cancer cells while allowing the adjacent normal cells to repair themselves from the injury.

The reason that the treatment course for some cancers is so relatively long is to allow for normal tissue repair and to minimize permanent injury. Relatively small doses given over a long period of time allow for normal tissues to recover at the expense of the cancer cell. (Tissue repair can also be helped by proper nutrition and patients' mental state). The daily dose must also be great enough to destroy the cancer cell while "sparing" the normal tissues. This "balancing act" forms the basis of modern radiation therapy, which has been further complicated in recent years because, in many cases, chemotherapy, which also harms normal tissues, is used in combination with radiation.

Focused Ultrasound (FUS)

Unlike Cryotherapy, Radiation, and Surgery, FUS does not damage surrounding tissue. This cuts down on risk factors, healing time, and the possibility of reduced side effects. Radiation and Chemotherapy can only be performed a certain number of times before they become highly toxic to the body. Surgery creates too much scar tissue to re-operate on a patient multiple times. However, FUS destroys tissue with safe and precise heat energy, and this can be repeated several times. A few patients have been treated up to 4 times with FUS. Because anesthesia is used, a short observation period is required. However this procedure is typically performed as an outpatient procedure. The FUS energy can be precise to the millimeter in its destruction and therefore minimize collateral damage to nearby tissue and structures. Unlike most other procedures, FUS can be done before or after other treatments. All treatments run the risk of having the cancer return. Most of these procedures either can't be repeated or the chances of side effects increase dramatically when mixing treatment options. FUS can be used before or after other treatments without increasing potential side effects.

Current Chemotherapy Standards and Cancer Drug Treatment

Chemotherapy remains the most accepted treatment option for newly diagnosed cancer patients following surgery. The US FDA has approved more than 50 new molecules for cancer use since 1990. The majority of these drugs have received indications for approved claims in breast, lung, ovary, and colon cancers, as well as leukemia and lymphoma. The overwhelming majority of these new and older drugs (about 125) are administered by lengthy multi week or multi day courses of intravenous injection. Other chemotherapeutics are delivered intramuscularly, subcutaneously, and in tablet or capsule forms.

Several new cancer drugs have been approved over the last ten years, and they have mainly been concentrated on killing cancer cells through biological means. The new drugs have failed to adequately improve life expectancy by directly attacking metastases and end stage disease. Indeed, the majority of chemotherapeutics are not highly effective on patients that present with late stage disease on first examination.

Drugs for the treatment of multiple myeloma, lung cancer and head and neck cancer have been approved.

However, none is a cure; median survival benefits are mostly measured in months. Amongst such drugs are Astra Zeneca'a Iressa, Genentech's Tarceva, Avastin and Herceptin, Imclone's Erbitux, and Novartis's Gleevec. In all, around 400 new cancer drugs are under development, but these new molecular targeted molecules have a number of drawbacks; in particular, they only work in a subset of patients. A particular problem in employing chemical drugs is the high risk of drug resistance developing on prolonged use of such drugs. Cancer vaccines are also under development, and these new products aim to stimulate the body's ability to respond to tumors over a prolonged period of time, thus leading to increased life expectancy; little success has been obtained with this approach to date. A particular problem in employing chemical drugs is the high risk of drug resistance developing on prolonged use of such drugs.

Metastases

Metastasis to the brain is the most feared complication of systemic cancer and the most common intracranial tumor in adults. Incidence is rising with improved survival of cancer patients. Currently, cancer patients live longer as a result of important advances in cancer diagnosis and management, and in particular, the widespread use of MRI to detect small metastases. Approximately 40% of intracranial neoplasms are metastatic. Multiple, large autopsy series suggest that, in order of decreasing frequency, lung, breast, melanoma, renal, and colon cancers are the most common primary tumors to metastasize to the brain.

Brain metastases are an increasingly important cause of morbidity and mortality in cancer patients. Thus, brain metastases present a therapeutic challenge for the treating physician and an emotionally and physically debilitating event for the patient. Early diagnosis and aggressive treatment of brain metastases may result in remission of brain symptoms and may enhance the quality of the patient's life and prolong survival. The radiologist plays a primary role in the management of cancer patients by helping detect, localize, and diagnose the lesion.

Since arterial blood must pass through the lungs before entering the brain, and larger aggregates of tumor cells are filtered out in the capillaries, many emboli traveling to the brain via the arterial route originate either from a primary lung tumor or a metastatic site in the lung. However, single tumor cells may pass through the capillaries of the lung and larger tumor emboli also may pass from the venous to the arterial circulation through a persistently patent foramen ovale, between the right and left atrium of the heart.

Metastatic tumor growth in the brain depends on complex organotropic factors as well as passive vascular delivery of tumor cells. Lesions are located in the cerebrum (80-85%), in the cerebellum (10-15%), and in the brain stem (3-5%). Slightly more than 50% of the time, multiple as opposed to solitary metastases occur, but this varies with the type of primary tumor. Melanoma, lung, and breast primaries are more likely to produce multiple metastases.

Intracranial metastases can be categorized by location as skull, dura, leptomeninges, and parenchymal brain metastases. Lesions of the brain and leptomeninges comprise 80% of intracranial metastases. Meningeal carcinomatosis most commonly occurs in patients with breast carcinoma, malignant melanoma, and, less commonly, with lymphoma, leukemia, and other tumors. Patients usually present with headache, vague neurologic complaints, and one or more cranial nerve palsies. Frequency

In the US: Approximately 170,000 cancer patients develop brain metastases annually. Intracranial metastases are seen in approximately 24% of patients that die from cancer.

Brain metastases represent the most common neurologic manifestation of cancer, occurring in 15% of cancer patients. Approximately one third of patients with lung carcinoma develop intracranial metastases eventually, and

50% of brain metastases result from this type of cancer.

Brain metastases are less frequent in children, with an approximate incidence of 6%.

Brain metastases from unknown primary neoplasms are most likely to be from a primary lung cancer (72%), followed in frequency by breast cancer, colon carcinoma, and melanoma. Mortality/Morbidity: Prognosis typically is poor. Therapeutic considerations must be individualized and depend on many factors, which include the patient's neurologic status, extent of systemic tumor, number and location of brain metastases, and sensitivity of the tumor to radiation and chemotherapy. Patients with the best prognostic indicators often die within 18-24 months. Of particular relevance to imaging, patients with a solitary brain metastasis treated by surgical resection show an approximately doubled rate of survival after 1 year.

Sex distribution

Predilection for gender follows that of the primary tumor. Lung cancer is the most common source of metastases in male patients, while breast cancer is the most common source in female patients. As the frequency of lung cancer in women increases, it may become the most common primary tumor to metastasize to the brain in women as well.

Age

Incidence of brain metastases based on age parallels that of primary systemic tumors. Most brain metastases occur in patients aged 35-70 years.

Clinical Details:

Approximately two thirds of brain metastases are symptomatic at some point. Symptoms primarily are caused by (1 ) increased intracranial pressure resulting in headache, nausea, vomiting, confusion, and lethargy and (2) focal irritation or destruction of neurons resulting in hemiparesis, visual field defects, aphasia, focal seizures, ataxia, and other focal neurologic signs or deficits.

Different primary tumors spread to the brain at different points in the disease course. The median latent interval between the initial diagnosis of a primary tumor and diagnosis of brain metastases varies from 6-9 months for lung cancer and 2-3 years for melanoma, breast, and colon cancer. In 20% of patients, metastases are detected during diagnosis of the primary tumor, and in 50% of patients, they are detected within 1 year following diagnosis.

In 5-10% of cancer patients, brain metastasis is the first clinical manifestation of systemic cancer. The primary site can be located in 45% of patients, and in those in whom the primary site is discovered, lung carcinoma is the primary tumor in 45%. Surgical resection is the preferred treatment in patients with one apparent metastasis detected on enhanced CT or MRI.

When screening for intracranial metastases, no consensus has been reached concerning when to use CT or MRI for initial staging evaluation of a patient with cancer. However, brain MRI in patients with primary cancers that frequently metastasize to the brain (e.g. bronchogenic carcinoma) is probably cost-effective. Numerous studies have shown that contrast-enhanced MRI detects 2-3 times as many lesions as contrast-enhanced CT, especially lesions less than 5 mm in diameter. In addition, approximately 20% of patients with solitary metastatic lesions on CT show multiple lesions on MRI. Perform imaging on patients with other cancers based on their clinical evaluation.

Gene therapy is an evolving field of medical treatment that is finding applications in a wide variety of diseases. It is finding more notable success in the treatment of cancer than in that of treating genetically inherited diseases. This is largely due to the fact that treatment of inherited diseases requires controlled levels of transgene expression in vivo, and this throughout the lifetime of a patient. Such long term stability of gene expression has proved very difficult to obtain in practice. On the other hand, when applied to the treatment of cancer, a limited length of expression of transgenes in a tumor should suffice.

Heat shock or Stress Proteins and Promoters

Stress genes encode a small number of heat shock or stress gene families. Major families of stress proteins are distinguished on the basis of molecular weight and amino acid sequence, Nover and Scharf, Cell. MoI. Life. Sci. 53:80, 1997. They include hsp 110, hsp 104, hsp90, hsp70, hsp60, hsp27, hsp10 and ubiquitin.

The promoter regions of stress genes include so-called heat shock element sequences that are essential for the activation of these genes by stress; they contain sequences that bind heat shock transcription factors, Wu, Annu. Rev. Cell Dev. Biol. 11 :441 , Heat Sock Transcription Factors; Structure and Regulation, 1995. These promoters, and in particular the hsp-70B promoter, Bromley P, and Voellmy R. US 5,646,010. Methods and Compositions for Expression of Competent Eukaryotic Gene Products, have been employed to render genes of interest activatable by stress, and in general the stress employed has been a heat shock.

DETAILED DESCRIPTION OF THE INVENTION

Bromley (PCT/IB99/00294 and Japanese Application No. 2000-532150) has demonstrated that combinations of apoptosis genes such as GATA 6 and Fas-ligand can kill tumor cells both in vitro and in vivo, and have proposed that the treatment of primary tumors can be improved by using a combination of genes including apoptosis genes such as, Bad, Bak, Bax alpha, Bcl2 alpha, BcI-XI, Bcl-X5, Bik, CASP3, rev CASP3, CASP6a, rev CASP&a, FAIM, FasL/TNF SF6, GAX, GATA6, Noxa/APR, Puma, SARP2, SURVIVIN/BIRC5, SURVIVIN T34A, TGF betai , TGF beta2, Trail/TNFSH10, cell cycle inhibitor genes, where 800 genes involved in cell cycle control have been identified in yeast (Spellman et al., Molecular Biology of the Cell 9: 3273-3297, 1998), suicide genes such as coda.,codA::upp, upp, fcy1 , furl , tdk:tmk, EHV4 tk, HSV1 tk, HSV1 tk::Sh ble, tumor suppressor genes, such as P53,RB1 , WT1 ,NF1 , NF2, APC,TSC1 ,TSC2, DPC4 (Smad4), DCC, BRCA1 , BRAC2,PTEN,LKB1 (STK11 ),MSH2,MLH1 , CDH1 , VHL,p16 INK4a (CDKN2A), PTCH, and MEN1 , anti-angiogenesis genes such as angiostatin, endostatin, antithrombin 111 , VEGF-D, ANGPT-2/ANG-2, ATF, CALR, ENDO XV, ENDO XV111 , ENDO XV 111 ::ANGIO, ENDO XV111 ::PEX, ENDO XV111 ::Kringle5, sFLK-1/KDR, sFLT-1 , IP-10, KI-5, Kringle 5, Maspin, Mig, Mig::IP10, PEDF, PEX, PF4, PRP, sTie2 (Extek), TIMP1 , TIMP2, TIMP3, TIMP4, Trponin1-2, T2- TrpRS, TSP1 , and Tumstatin and anti-sense sequences such as antisense against the type-1 insulin-like growth factor receptor, or IGF-1 R and the anti clustering gene sequence. Further, Bromley (CIP to the PCT/IB99/00294 and Japanese Application No. 2000-532150), Smith et al., Human Gene Therapy 13: 697-706 (2002) and Moonen (PCT/US97/15270) have demonstrated the necessity of targeting gene expression precisely. Their technique of employing heat inducible promoters to control the expression of transgenes and activating them after injection into tumors using focused ultrasound heating, optimally performed inside of an MRI apparatus for temperature imaging, is proving essential for the application of highly toxic genes such as Fas-ligand to the treatment of cancer.

The invention relates to a gene therapy approach whereby expression of genes causing cell death are combined with the expression of genes capable of alerting the immune system to the presence of tumor specific antigens. This invention provides a unique approach to eliciting antigen-specific cellular immunity against metastatic tumors, following destruction of primary tumors.

Tumor antigens and Tumor vaccines

Various tumor antigens are recognized by specific immune cells in patients with cancer, indicating that the immune system is capable of recognizing tumors, Shiku H, Takahashi T, Oettgen HF. Cell surface antigens of human malignant melanoma. II. Serological typing with immune adherence assays and definition of two new surface antigens. J Exp Med 1976; 144: 873-81 , Ueda R, Shiku H, Pfreundschuh M, Takahashi T, Li LT, Whitmore WF, Oettgen HF, Old LJ. Cell surface antigens of human renal cancer defined by autologous typing. J Exp Med 1979; 150: 564-79, Carey TE, Takahashi T, Resnick LA, Oettgen HF, Old LJ. Cell surface antigens of human malignant melanoma: mixed hemadsorption assays for humoral immunity to cultured autologous melanoma cells. Proc Natl Acad Sci USA 1976; 73: 3278-82, Pfreundschuh M, Shiku H, Takahashi T, Ueda R, Ransohoff J, Oettgen HF, Old LJ. Serological analysis of cell surface antigens of malignant human brain tumors. Proc Natl Acad Sci USA 1978; 75: 5122-6, Tureci O, Sahin U, Pfreundschuh M. Serological analysis of human tumor antigens: molecular definition and implications. MoI Med Today 1997; 3: 342-9, Van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van Den Eynde B, Knuth A, Boon T. A gene encoding antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991 ; 254: 1643-7.

However, the problem with most cancer vaccine approaches to date is that they are performed at sites distant from the primary tumors. It is well known that antigen-based vaccination therapies result in an accumulation of specific immune cells at the vaccination site, Kobayashi K, Kaneda K, Kasama T. lmmunopathogenesis of delayed-type hypersensitivity. Microsc Res Tech 2001 ; 53: 241-5, Reinhardt RL, Bullard DC, Weaver CT, Jenkins MK. Preferential accumulation of antigen-specific effector CD4 T cells at an antigen injection site involves CD62E- dependent migration but not local proliferation. J Exp Med 2003; 197: 751-62, and Schrama D, Pedersen LO, Keikavoussi P, Andersen MH, Straten Pt P, Brocker EB, Kampgen E, Becker JC. Aggregation of antigen-specific T cells at the inoculation site of mature dendritic cells. J Invest Dermatol 2002; 119: 1443-8.

Thus intratumoral approaches are of great interest, since they may direct tumor antigen-specific responses inside the tumors, and may also exploit the presence of multiple undefined tumor antigens present in tumors.

Dendritic cells are of critical importance in priming effective, antigen-specific T-cell activation within secondary lymphoid organs, Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S. Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 2002; 20: 621-67, thus attracting dendritic cells to the tumor is required for the optimal initiation of antigen-specific anti-tumor responses, Lespagnard L, Gancberg D, Rouas G et al. Tumor-infiltrating dendritic cells in adenocarcinomas of the breast: a study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome, lnt J Cancer 1999; 84: 309-14, Iwamoto M, Shinohara H, Miyamoto A et al. Prognostic value of tumor-infiltrating dendritic cells expressing CD83 in human breast carcinomas, lnt J Cancer 2003; 104: 92-7, Reichert TE, Scheuer C, Day R, Wagner W, Whiteside TL. The number of intra-tumoral dendritic cells and zeta-chain expression in T cells as prognostic and survival biomarkers in patients with oral carcinoma. Cancer 2001 ; 91 : 2136—47.

In a preliminary clinical study, patients with metastatic dermal or subcutaneous breast and melanoma tumors received autologous dendritic cells intra-tumorally. Regression of injected tumors was observed in 6 out of 10 patients, and biopsies of the regressing tumors indicated the presence of dendritic cells, Triozzi PL, Khurram R, Aldrich WA, Walker MJ, Kim JA, Jaynes S. lntratumoral injection of dendritic cells derived in vitro in patients with metastatic cancer. Cancer 2000; 89: 2646-54. In attempts to enhance the efficacy of this approach, dendritic cells have been genetically engineered to express pro-inflamatory cytokines, Satoh Y, Esche C, Gambotto A et al. Dendritic cells

Local administration of IL-12-transfected dendritic cells induces anti-tumor immune responses to colon adenocarcinoma in the liver in mice. J Exp Therapeutics Oncol 2002; 2: 337-49, Nishioka Y, Hirao M, Robbins PD, Lotze MT, Tahara H. Induction of systemic and therapeutic anti-tumor immunity using intra-tumoral injection of dendritic cells genetically modified to express interleukin 12. Cancer Res 1999; 59: 4035-41 , Bonham L, Skuk D, Koeberl D et al. Intra-tumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific anti-tumor immunity and achieves tumor eradication. Human Gene Ther 2000; 11 : 1277-88. An alternative approach to injecting dendritic cells into tumors is to attract dendritic cells to a tumor site via the action of chemokines, Sallusto F, Lanzavecchia A. Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression. Immunol Rev 2000; 177: 134-40, Cavanagh LL, Von Andrian UH. Travellers in many guises: the origins and destinations of dendritic cells. Immunol Cell Biol 2002; 80: 448-62. A promising approach to achieving this goal is to provide appropriate chemokines at the tumor site. Based on chemkine receptor expression data, it is possible to identify chemokines that should be chemo-attractive for immature dendritic cells, Sallusto F, Lanzavecchia A. Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression. Immunol Rev 2000; 177: 134-40, Dieu M-C, Vanbervliet B, Vicari A et al. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. Journal of Experimental Medicine 1998; 188: 373-86, Foti M, Granucci F, Aggujaro D et al. Upon dendritic cell (DC) activation chemokines chemokine receptor expression are rapidly regulated for recruitment and maintenance of DC at the inflammatory site. International Immunology 1999; 11 : 979-86, Lin CL, Suri RM, Rahndon RA, Austyn JM, Roake JA. Dendritic cell chemotaxis and trans-endothelial migration are induced by distinct chemokines and are regulated on maturation. European Journal of Immunology 1998; 28: 4114-22, Sallusto F, Palermo B, Lenig D et al. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. European Journal of Immunology 1999; 29: 1617-25, Sallusto F, Schaerli P, Loetscher P, Schaniel C, Lenig D, Mackay CR, Qin S, Lanzavecchia A. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. European Journal of Immunology 1998; 28: 2760-9, Sozzani S, Allavena P, D'Amico G et al. Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties. Journal of Immunology 1998; 161 : 1083-6.

Immature dendritic cells express the chemokine receptors CCR1 , CCR5 and CCR6, which bind the important inflammatory chemokines CCL3, CCL5 and CCL20. Injection of recombinant chemokine has been shown to cause infiltration of cells into the injection site, Didier PJ, Paradis TJ, Gladue RP. The CC chemokine MIP-1alpha induces a selective monocyte infiltration following intra-dermal injection into nonhuman primates. Inflammation 1999; 23: 75-86, Lee SC, Brummet ME, Shahabuddin S, Woodworth TG, Georas SN, Leiferman KM. Cutaneous injection of human subjects with macrophage inflammatory protein-1 alpha induces significant recruitment of neutrophils and monocytes. J Immunol 2000; 164: 3392-401. Crittenden M, Gough M, Harrington K, Olivier K, Thompson J, Vile RG. Expression of inflammatory chemokines combined with local tumor destruction enhances tumor regression and long-term immunity. Cancer Res 2003; 63: 5505-12, have demonstrated that mice challenged with immunogenic colorectal tumors, which had been genetically modified to secrete CCL20, failed to develop tumors compared with non-chemokine expressing control cells, and that expression of CCL20 was associated with a significant increase in dendritic cells at the site of tumor inoculation. Similarly, intra-tumoral injection of adenoviral vectors expressing CCL20 resulted in the accumulation of dendritic cells as well as T cells, Sharma S, Stolina M, Luo J, Strieter RM, Burdick M, Zhu LX, Batra RK, Dubinett SM. Secondary lymphoid tissue chemokine mediates T cell-dependent anti-tumor responses in vivo. J Immunol 2000;164: 4558-63, and also inhibited the growth of lung carcinoma and melanoma in a T-cell-dependent manner, see also Kirk CJ, Hartigan- O'Connor D, Mule JJ. The dynamics of the T-cell antitumor response: chemokine-secreting dendritic cells can prime tumor-reactive T cells extranodally. Cancer Res 2001 ; 61 : 8794-802. However, despite these promising results, problems remain; for instance, a number of human tumors constitutively express chemokines that are chemo-attractive to dendritic cells, Kleeff J, Kusama T, Rossi DL et al. Detection and localization of Mip- 3alpha/LARC/Exodus, a macrophage pro-inflammatory chemokine, and its CCR6 receptor in human pancreatic cancer, lnt J Cancer 1999; 81 : 650-7, Luboshits G, Shina S, Kaplan O et al. Elevated expression of the CC chemokine regulated on activation, normal T cell expressed and secreted (RANTES) in advanced breast carcinoma. Cancer Res 1999; 59: 4681-7, Yoong KF, Afford SC, Jones R, Aujla P, Qin S, Price K, Hubscher SG, Adams DH. Expression and function of CXC and CC chemokines in human malignant liver tumors: a role for human monokine induced by gamma-interferon in lymphocyte recruitment to hepatocellular carcinoma. Hepatology 1999; 30: 100-11. Further, in less immunogenic tumor models, expression of chemokines in tumors is insufficient to cause tumor rejection, Crittenden M, Gough M, Harrington K, Olivier K, Thompson J, Vile RG. Expression of inflammatory chemokines combined with local tumor destruction enhances tumor regression and long-term immunity. Cancer Res 2003; 63: 5505-12.

Antigen loading of dendritic cells in tumors

A wide range of strategies have been applied to trying to provide antigen to dendritic cells, with both in vitro and in vivo approaches having been tried. While the in vitro approach has generally indicated very short term duration of efficacy, ex vivo loading has been improved by conjugating exogenous protein antigen to molecules that assist, such as heat shock proteins, Blachere NE, Li Z, Chandawarkar RY, Suto R, Jaikaha NS, Basu S, Udono H, Shvastava PK. Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J Exp Med 1997; 186: 1315-22, complement and antibody. A promising approach would be to provide endogenous tumor antigens to dendritic cells with systemic cytotoxic therapies.

An even more interesting approach, which is a preferred embodiment of this invention, is to combine each of the above procedures in a single event, by introducing toxic genes into tumors, heating to express both the toxic genes, co-expressing genes such as CCL20 to increase dendritic cell concentration in tumors, and heat shock proteins in situ to optimize dendritic cell loading with tumor antigens. Cytotoxicity in this case is not systemic, but is tumor targeted. Support for this approach has come from the observation that intra-tumoral injection of dendritic cells was more effective in a breast tumor model when combined with agents that enhance levels of cell death in the tumor, Tanaka F, Yamaguchi H, Ohta M, Mashino K, Sonoda H, Sadanaga N, lnoue H, Mori M. Intra-tumoral injection of dendritic cells after treatment of anticancer drugs induces tumor-specific anti-tumor effect in vivo, lnt J Cancer 2002; 101 : 265-9.

Activating dendritic cells once loaded with tumor antigens

Dendritic cells are highly responsive to inflammatory stimuli, such as ligands of the tumor necrosis factor (TNF) family, Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S. Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 2002; 20: 621-67, Schoenberger SP, Toes RE, van der Voort El, Offringa R, Melief CJ. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 1998; 393: 480-3, Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, Heath WR. Help for cytotoxic-T-cell responses is mediated by CD40 signaling. Nature 1998; 393: 478-80, Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 1998; 393: 474-8. In a preferred embodiment of this invention, one or more of the toxic genes employed could be selected from the TNF family. Similarly acting ligands which activate the Toll-like receptors on dendritic cells, Roake JA, Rao AS, Morris PJ, Larsen CP, Hankins DF, Austyn JM. Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1. J Exp Med 1995; 181 : 2237^17, Basu S, Binder RJ, Suto R, Anderson KM, Shvastava PK. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF- kappaB pathway, lnt Immunol 2000; 12: 1539—46. This latter reference shows that necrotic cell death is more effective than apoptotic cell death, in causing dendritic cells containing antigen to mature and up-regulate co- stimulatory factors, Basu et al., 2,000 and Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med 1999; 5: 1249-55.

Attraction of primed effector cells to the tumor site

The degree of lymphocyte infiltration into tumors has been used as an independent prognostic marker for improved survival in specific cases of melanoma patients, Clemente CG, Mihm MC, Jr, Bufalino R, Zurrida S, Co Mini P, Cascinelli N. et al. Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer 1996; 77: 1303-10. However, the presence of large numbers o tumor antigen-specific T-cells does not consistently cause regression of tumors in animals and patients, Hermans IF, Daish A, Yang J, Ritchie DS, Ronchese F. Antigen expressed on tumor cells fails to elicit an immune response, even in the presence of increased numbers of tumor-specific cytotoxic T lymphocyte precursors. Cancer Res 1998; 58: 3909-17, Speiser DE, Miranda R, Zakahan A et al. Self antigens expressed by solid tumors do not efficiently stimulate naive or activated T cells. Implications for immunotherapy. J Exp Med 1997; 186: 645-53. It is possible that using intra-tumoral therapies, it may be possible to incorporate features that directly modify the tumor site to increase the efficacy of effector T-cell trafficking. Chemokines known to attract effector T-cells, are CCL3 and CCL20, Chttenden M, Gough M, Harrington K, Olivier K, Thompson J, Vile RG. Expression of inflammatory chemokines combined with local tumor destruction enhances tumor regression and long-term immunity. Cancer Res 2003; 63: 5505-12. The addition of CCL3 to the set of genes injected into tumors would form a preferred embodiment of this invention. Gough et al (Gough M, Chttenden M, Thanarajasingham U, Sanchez-Perez L, Thompson J, Jevremovic D, and Vile R. Gene Therapy to manipulate effector T cell trafficking to tumors for immunotherapy. J. Immunology 2005; 174: 5766-5773) have used adenoviral vectors to express CCL3 within B16ova tumors in vivo, and have shown increases in the efficacy of adoptive transfer of tumor- specific effector OT1 T cells. They further show that such therapies result in endogenous immune responses to tumor antigens that are capable of protecting animals against subsequent tumor challenge. Such approaches, that form part of a preferred embodiment of this invention, are designed to increase the visibility of tumors to the immune system.

The importance of combining heat shock in tumors with immune approaches to cancer treatment

Heat shock protein expression is closely associated with immunogenic forms of cell death; Gough at al 2004 ( Gough MJ, Melcher AA, Chttenden MR, Sanchez-Perez L, Voellmy R and Vile RG 2004. Induction of cell stress through gene transfer of an engineered heat shock transcription factor enhances tumor immunogenicity. Gene. Ther. 2004; 13: 1099-104, show that the activation of the heat shock transcription factor, leads to an immunogenic death. Cells dying through "stressful death" show decreased phagocytosis by macrophages in vitro. Moreover, cells expressing heat shock proteins during cell death ( which is a preferred embodiment of this invention) are significantly more protective against subsequent tumor challenge.

Indeed, hsp's have been shown to be implicated in signaling to the immune system the presence of stressed or diseased tissue ( Galluci and Matzinger, Curr Opin immunol 13: 114-119, 2001 ). Research on hsp's has demonstrated that they can activate both arms of the immune system, i.e. innate and adaptive immunity. Innate immunity generates a relatively non-specific response which recognizes antigens; Cells of the innate immune system include dendritic cells, macrophages, natural killer cells and gamma T cells. Adaptive immunity is a second line of defense; signals from the innate immune system trigger the adaptive immune system to produce an antigen specific response. The adaptive immune system is composed of B cells and T cells, and is capable of persisting for months and frequently for many years.

The unique ability of hsp's to activate both innate and adaptive immunity has been explained through the identification of hsp receptors on dendritic cells and macrophages (e.g. Binder et al., Nat Immunol 1 : 151-155,

2000). These cell types are referred to as "professional" antigen-presenting cells (APC) since they are perfectly suited for initiating antigen-specific immune responses, especially by the T cells. Three of the hsp receptors so far identified, CD14, Tlr4 and CD40 are involved in stimulating cellular immune responses. CD14 and Tlr4 receptors are involved as sensors for the innate immune system, while CD40 receptors allows communication with T cells, thus acting as a bridge between innate and adoptive immunity. The outcome of cd40 signaling is activation of

APC, which in turn amplifies T cell stimulation.

Indications of the hsp's that can be co-expressed in preferred embodiments of this invention, come from studies in a murine leukemia model; (Sato et al., Blood 98(6) 1852-1857, 2001 ) immunized tumor-bearing animals with hsp70 and gp96 derived from leukemia cells during bone marrow reconstruction. They observed that survival times of hsp-treated animals was significantly increased, and that CD8+ and CD4+ T cells were required for this anti-tumor response. While the majority of murine immunogenicity and tumor rejection studies have employed the major hsp60, hsp70 and hsp90 families, such properties have been shown to extend to the hsp110 and the hsp170 families (Wang et al., J Immunol 165: 490-497, 2001 ). This invention incorporates all members of these hsp families and similar proteins as co-expressed genes.

Genes that can be employed in this invention for attacking primary tumors and killing tumor cells can be derived for those inducing apoptotic (programmed) cell death and those inducing necrotic cell death. Specific forms of tumor cell death may play a crucial role in host-tumor interactions. The uptake of apoptotic cells by dendritic cells has been demonstrated (Albert et al., Nature 392: 86-89, 1998 and Henry et al., Cancer Res 59: 3329-3332, 1999); on the other hand activation and maturation of dendritic cells by exogenous material released from necrotic cells have also been reported (Galluci et al., Nat Med 11: 1249-1255, 1999). A non-exhaustive list of genes that can be employed in this invention, as genes to attack primary tumors, and to incite immune responses against metastases, is given below:

CELL RESCUE, DEFENSE, CELL DEATH AND AGING PRE3, PRE1, PUP2, RPN12, RPT1, MAG1, OGG1, SED1, ATH1, SPE2, GRE3, TPS2, TPS1, ATR1, ATX1, SK13, SK12, SK18, APN1, HPR5, ERG5, CCZ1, SRA1, SNF1, YCK1, YCK2, HRR25, CTA1, CTT1, WSC4, PAM1, TIR2, TIR1, HDF2, TFB4, RAD1, HAM1, LYS7, SOD1, KIN28, DIT2, ERG11, CYC7, CCP1, PHR1, DAK2, DAK1, ALR1, ALR2, HOR2, RAD17, DDC1, DDR2, ALK1, HEL1, SSL2, RAD5, SGS1, PIF1, RAD3, CDC9, REV7, NTG1, RAD18, RAD57, RAD55, XRS2, RAD30, MMS21, RAD51, RADIO, PS02, REV1, DIN7, RAD54, CDC2, PES4, POL2, REV3, RPB7, RPB4, SGE1, UBA1, UBC4, UBC5, RAD6, QR18, RNC1, NTG2, ERC1, RAD4, ETH1, FKB2, YHB1, FLR1, MEC3, ZWF1, GSH1, GRX1, TTR1, HYR1, GLR1, YCF1, FPS1, GPD1, RAS2, RAS1, CUPS, HSP26, HSP30, HSP12, HSP104, DDR48, HSC82, HSP82, MDJ1, MDJ2, HSP60, HSP78, ECM10, SSE1, SSA1, SSA3, SSA4, SSA2, SSE2, HSF1, HIG1, HDF1, HMS2, GRE1, DD11, RTA1, SIMI, LAG2, ZDS1, MET18, SNG1, NCA3, KT112, UTH1, SUN4, SSU81, SSD1, TH14, KAR3, LIF1, SFA1, LAG1, LTV1, MDR1, SSK22, SSK2, HOL1, CIS3, HSP150, PIR3, MAC1, CUP1 A, CUP1 B, YDJ1, SSQ1, SSC1, IMP2, MPT5, ATX2, SN02, MLP1, NHX1, NCP1, NSR1, SNF4, RAD16, RAD7, RAD14, RAD23, ROD1, MGT1, OSM1, SIP18, SAT2, MNR2, MMS2, PNT1, CYP2, PAD1, PDR5, PDR3, PDR6, RTS1, PA13, HOR7, DUN1, IRE1, MKK2, MET22, PPZ2, PTC1, PTP2, MMS4, RAD52, PDR13 SLG1, GRR1 HIT1, RDH54 BR01, PIR1 MSRA, RNR4 RNR3, HAL1, YGP1, CDC55, PPZ1, PKC1, HAL5, MKK1, HOG1, SLT2, BCKi, RAD53, SIR4, SIR3, SIR2, MGA1, FUN30, YR02, DNL4, RRD1, SAT4, RAD27, MSN2, ST11, PAU3, PAU2, PAU5, PAU1, PAU4, PAU6, (MLP1), RAD2, FZ_F1, SSU1, SOD2, CRS5, BCK2, ASM4, TIP1, TFB1, CCL1, SSL1, TFB3, TFB2, TSAI, TRX1, TRX2, ROX3, PDR1, GTS1, MCM1, SKN7, CAD 1, MSN4, YAP1, SLN1, SSK1, PBS2, UB14, RSP5, SVS1,ZRC1

CELL GROWTH, CELL DIVISION AND DNA SYNTHESIS GSC2, PLC1, PRE3, PRE2, PRE1, PUP2, RPN12, RPT6, RPT1, DIS3, RP SOA, AGA1, AGA2, ASG7, ACH1, ACT1, SAC6, ARP100, ABP1, PAN1, ARP 2, AREIARE2, SPE2, CYR1, SRV2, ADK2, GCS1, SOH1, TUB1, TUB3 SAG1, AKR1, YAR1, SK18,

ARG82, ABF1, STE6, BAR1, 8011, TUB2, RBI-2, BIG1, BI M1, BAT1, BEM1, BEM4, SBE2, BN14, BUD6, 8012, BUD9, BUD4, BUD8, RC K2, CMK1, CNA1, CMP2, CNB1, CCH1, CMD1, SRA1, YCK1, YCK2, HRR25, CKA2, CKA1, YCK3, EST2, TFS1, SCM4, GIC2, GIC1, CAK1, BUB2, B UB3, ESR1, RAD24, DBF20, PDS1, HPC2, NUD1, CDC47, CDC10, CDC13, C DC37, CDC1, CDC40, CDC4, CDC20, CDC6, CDC46, CDC3, KAR1, BB P1, CDC50, FUS1, KRE9, EGT2, ARP1, CHS1, CHS2, CHS3, CHS5, MS11, CAC2, R LF2, CHL4, SMC1, SMC2, CIN1, SNF7, CLC1, COF1, PAM1, LAS17, HDF2, SEC3, SNF2, SWH, SNF5, SNF1 1, DOC1, APC2, APC5, TAP42, CDC53, KAR 9, CCE1, CLB6, CLB5, CLN3, PCL2, CLN 1, PCL1, CLN2, CI-133, CI-131, CLB 4, CI-132, FAR1, CKS1, CDC28, PH085, KIN28, SSN3, CLG1, DIT2, SLA1, SLA2, SP020, DPP1, RAD17, DDC1, HEL1, DNA2, RAD5, SGS1, HCS 1, PIF1, CDC9, MSH3, MSH6, MLH1, PMS1, MSH2, MSH1, POL4, REV 7, MRE11, RAD26, RAD9, RAD18, RAD57, RAD55, XRS2, MMS21, RAD51, RADIO, RAD 50, RFA3, RFA2, RFA1, RFC4, RFC5, RFC3, RFC2, RFC1, FOB1, TOP1, TO P2, TOP3, RAP1, RAD54, PR12, PR11, POL1, POI-12, CTF4, HUS2, CDC2, P ES4, POL2, DPB2, DPB3, MIP1, REV3, SSN8, GAL11, RGR1, SRB6, RP041, SEC59, DIP2, CDC14, MSG5, DYN1, UBC4, UBC9, CDC34, UBC5, UBC 1, UBC6, RAD6, QR18, ELC1, RNC1, CTS1, KEX2, APG1, SSP1, SUP35, EXM2, S PR1, EXG1, EXG2, DHS1, CAP1, CAP2, BRN1, GPR1, GIF1, MEC3, TU B4, CIS2, LTE1, SDC25, SRM1, CDC25, ROM2, BUDS, ROM1, SPT16, CDC43, G IP1, SIN4, SNF6, KRE6, GFA1, NGR1, WH12, RSR1, CIN4, RAS2, RAS1, GP A1, STE4, STE18, CDC42, MDG1, SEC4, TEM1, RH03, RH04, RI-102, RHO 1, CDC24, BEM2, BUD2, BEM3, LRG1, GPA2, SIS1, HSP82, HSF1, ABF2, HDF1, HDR1, RPD3, HSL7, HO, SBA1, HPR1, IDS2, NFH, CSE2, MDM 1, MI-1 MIDI, SIMI, HIR3, SIS2, MAKI 1, LAS1, SPA2, WH14, ECM33, SET1, CTF19, CIN2, MCM16, SLK19, CYK2, CNM67, SST2, DPB11, DOS2, D FG16, AFR1, ZDS1, SR07, PEA2, FAR3, SMP2, WH13, CDC5, MET30, SAS2, SCC2, CIS 1, STN1, UTH1, PAC2, SSD1, SRP1, KRE5, KIP1, CIN8, SMY1, KIP2, KAR3, KIP3, CBF1, CBF2, SKP1, CEP3, CTF13, DBR1, LAG1, MIH1, BFR1, DIG2, DIG1, MFA1, MFA2, MFAlphal, MFAIpha2, MID2, SSF1, MATALPHA2, MATA LPHA1, ALPHAI, ALPHA2, A2, A1, SAN1, PGD1, SPO11, MSH5, DMC 1, ISC10, MSH4, SP013, NDT80, REC104, HOPI, RED1, SP07, MUM2,ME15, S AE2, NAM8, REC107, REC102, REC114, MER1, RIM01, NDJ1, CDC54, CP R7, SYG1,MCM2, CIS3, HSP150, ACE2, CDC48, ASE1, YTM1, HSM3, YD J1, ERV1, FUS3, JNM1, MCD1, MMC1, MSB1, MSB2, MPT5, ZDS2, MSN5, KEM1, MLC1, MY02, MY04, MY05, MY03, MY01, DEC1, PMD1, M DS3, ASH1, UME1, UME6, NHP6A, RFT1, TRF5, NNF1, NDC1, BIK1, KAR2, KAR5, NUM1, CDC39, MAK16, NAP1, RAD16, RAD23, NBP35, ORC1, ORC6, ORC5, ORC4, ORC3, RRR1, SIC 1, BUD3, PWP2, STE3, STE2, OPY2, STE50, STE5, PEM, TOR1, TOR2, PIK1, STT4, MSS4, SP014, POL32, IME4, SHP1, PDS5, FEN1, CSE1, FL08, PFY1, PHB2, PHB1, POI-30, AXL1, STE23, RAD28, CDC7, SMP3, MKK2, CDC15, ARD1, CHU, PPH3, PPH21, PPH22, PTC1, SE C9, PPS1, PTP3, YVH1, PTP2, PUS4, PCH2, PCH1, CBF5, SEF1, MMS4, SHR5, RAD59, RAD52, RHC18, RGP1, RVS167, RIM9, BNR11, BN11, SPT3, SOK2, KAR 4, DBF4, SDS22, MCM3, CTF18, SR04, SPH1, FUS2, MOB1, FL08, FIG1, FIG2, END3, DFG5, CTR9, TOM1, POP2, GRR1, SCP160, SUR1, MUM3, ZIP2 CDC45, RDH54, SHE3, SHE2, SHE4, GPH, MIF2, ESP1, HOP2, DNA43, SMC3, PAC11, PAC10, RD11, RGA1, RNR1, RNR2, RNR4, RNR3, PRPS 1, RPL10, RPS1A, MTF1, SN12, CDC12, CDC11, SPR28, CDC55, GLC7, PKC1, Gl N4, SPS1, RCK1, BUB1, IME2, YAK1, YPK2, RIM11, CLA4, MKK1, MEK1, I PL1, SGV1, SLT2, KSS1, BCK1, STE11, STE20, DBF2, HSL1, NRK1, SIT4, T PD3, ELM1, MCK1, RAD53, STE7, SWE1, MPS1, SAS3, HST1, SIR4, SIR3, SIR1, SIR2, CTH1, DOM34, HST4, RVS161, DNL4, IQG1, FUN16, HYM1, RT S2, MNN10, PRK1, MCM6, SAP155, SAP4, SAP190, SAP185, MUD13, M AD1, CIK1, NUF1, SPC97, SPC42, SPC98, CDC31, NUF2, MAD3, MAD2, Dl T1, YSW1, SP012, SP016, MCD4, BDF1, SGA1, GSG1, SHC1, CDA1, CD A2, SMK1, SPS2, SPR6, SLZ1, SPS4, SPR3, SPS100, SPS18, RAD27, SNZ 1, SUR4, ST11, SBE22, CSE4, BMH 1, SVL3, SCH9, (MLP1), SSF2, RAD2, CDH1, CDC27, CDC26, CDC23, CDC16, APC1, APC 11, APC4, APC9, SAP30, RSC6, RSC8, STH1, SFH1, SAS5, JSN1, BMH2, SMT4, BCK2, HOC1, ZIP1, UFE1, EST1, TEM, ANC1, CCM, DST1, TRX1, TRX2, TRF4, PAT1, SPT4, SP T6, CDC36, SWI5, SWI4, PHD1, SWI6, GTS1, MCM1, IME1, SKN7, MBP1, SW13, SIN3, STE12, CIN5, SDS3, SP01, MOT2, RPG1, PRT1, CDC33, TPM1, TPM2, TWF1, TEC1, TTP1, STE13, PRP8, UB14, DSK2, RSP5, D OA4, UNG1, VPS45, VAN1, VRP1, DFG10, YHM2, GL'Q3, SFP1, STE24, RME1, SAE3, ME14, NHP6B, MOB2, EST3, RIM1

HEAT SHOCK PROTEINS CAT5, CPH1, CTT1, CYP2, DDR2, FPR2, HSC82, HSP104, HSP12, HSP150, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, KAR2, MDJ1, SIS1, S OD2, SSA1, SSA2, SSA3, SSA4, SSB1, SSB2, SSC1, SSE1, SSE2, ST11, TIP 1, TPS2, UB14, YDJ1

MITOCHONDRIAL AAC1, AAC3, AAT1, ABC1, ABF2, AC01, ACR1, ADH3, ADK2, AEP2, AFG3, ALD1, ALD2, ARG11, ARG2, ARG5,6, ARG7, ARG8, ARH 1, AT M 1, ATP1, ATP10, ATP11, ATP12, ATP14, ATP15, ATP16, ATP2, ATP3, ATP4, A TP5, ATP6, ATP7, ATP8, ATP9, BAT1, BCS1, CBP1, CBP2, CBP3, CBP4, CB P6, CBR1, CBS1, CBS2, CCA1, CCE1, CCP1, CEM1, CIT1, CIT3, COB, CO Q1, COQ2, COQ3, COQ6, COR1, COT1, COX1, COX10, COX11, COX12, C 0X3, COX14, COX15, COX17, COX2, COX3, COX4, COX5A, COX5B, COX6, COX7, COX8, COX9, CPR3, CTP1, CYB2, CYC1, CYC2, CYC3, CYC7, CYT1, CYT2, DB156, DLD1, DTP, ENS2, ERV1, FLX1, FUM1, GCV1, GCV3, GI-04, GPD2, GSD2, GUT2, HEM1, HEM15, HSP10, HSP60, HSP78, HTS1, IDH1, ID H2, IDP1, IFM1, ILV1, ILV2, ILV3, ILV5, ILV6, IMP1, IMP2, INH1, ISM1, KG D1, KGD2, LAT1, LEU4, LIP5, LPD1, LYS12, LYS4, MAE1, MAM33, MAS1, MAS2, MBA1, MCR1, MDH1, MDJ1, MDJ2, MDM10, MDM12, MEF1, MEF2, MET13, MGE1, MGM101, MIP1, MIR1, MIS1, MMM1, MMTi, M MT2, MOD5, MOL1, MRF1, MRP1, MRP13, MRP17, MRP2, MRP20, MRP21, MRP4, MRP49, MRP51, MRP8, MRPL10, MRPL 11, MRPL13, MRPL15, MRPL16, MRPL 17, MRPL 19, MRPL2, MRPL20, MRPL23, MRPL24, MRPL25, MRPL27, MRPL28, MRP L3, MRPL31, MRPL32, MRPL33, MRPL35, MRPL36, MRPL37, MRPL38, MR PI-39, MRPL4, MRPL40, MRPL44, MRPL49, MRPL6, MRPL7, MRPL8, M RPL9, MRPS28, MRPS5, MRPS9, MRS1, MRS11, MRS2, MRS3, MRS4, MRS5, MSD1, MSE1, MSF1, MSH1, MSK1, MSM1, MSP1, MSR1, MS S1, MSS116, MSS18, MSS51, MST1, MSU1, MSW1, MSY1, MTF1, M T01, NAM1, NAM2, NAM9, ND11, NHX1, NUC1, OM45, ORFA04514, OSM1, OXA1, PDA1, PDB1, PDX1, PEL1, PET111, PET112, PET117, PET122, PET123, PET127, PET130, PET191, PET309, PET494, PET54, PET56, PET9, PETCR46, PI-1131, PHB2, PIF1, PIM 1, POR1, POR2, PPA2, PSD1, PUT1, PUT2, QCR10, QCR2, QCR6, QCR7, QCR8, QCR9, RCAi, RF2, RIM 1, RIM2, RIP1, RML2, RNA12, RPM2, RP 041, SC01, SCO2, SDI-11, SDH2, SDI-13, SDI-14, SECY, SHM1, SHY1, SLS1, SMF2, SOD2, SOM1, SSC1, SS.COPYRGT.1, STF1, STF2, SUN4, SUV3, TIM17, TIM22, TIM23 TIM44, TIM54, TOM20, TOM22, TOM37, TOM40, TOM6, TOM7, TOM 70, TOM72, TRM1, TUF1, UNG1, VAR1, YAH1, YAL011W, YAT1, YBL013 W, YCR024C, YDR041W, YDR115W, YDR116C, YER073W, YFH1, YGLO68W, YGR257C, YHM1, YHR075C, YHR148W, YJL200C, YJR113C, YKLO55C, YKL120W, YKL134C, YKL192C, YLR168C, YMC1, YMC2, YML025C, YMR188C, YMR31, YNL081 C, YNL306W, YNR036C, YNR037C, YOR221 C.YPLOISCETF-BETA

PEROXISOMAL CAT2, CIT2, CTA1, DAL7, EHD1, EHD2, FAA2, FAT2, FOX2, ICU, IDP 3, MDH3, MLS1, PEX11, PEX12, PEX13, PEX14, PEX17, PEX2, PEX3, PE X4, PEX6, PEX7, PEXB, POT1, POX1, PXA1, PXA2, SPS19, YBR204C, YDR 449C, YHR180W DNA-ASSOCIATED A1, A2, ABF1, ABF2, ADA2, ADE12, ADR1, ALPHA1, ALPHA2, ANC1, APN1, ARGR1, ARGR2, ARGR3, ARR1, ASH1, AZF1, BAS 1, BDF1, BR F1, BUR6, CAC2, CAD1, CAF17, CATB, CBF1, CBF2, CCE1, CCR4, CDC13, CDC36, CDC39, CDC46, CDC47, CDC54, CDC6, CDC7, CDC73, CDC9, CEF1, CEP3, CHA4, CHD1, CHU, CHL4, CRZ1, CSE1, CSE2, CSE4, CTF13, CUP2, CUP9, DAL80, DAL81, DAL82, DAT1, DBF4, DMC1, DNA2, DNA43, DNL4, D OS2, DOT6, DP131 1, DPB2, DPB3, DST1, ECM22, ENS2, EST1, EZL1, FCP1, FHL1, FKH1, FKH2, FL08, FZF1, GAL11, GAL4, GAT1, GBP2, GCN4, GCNS, GCR1, GCR2, GLN3, GL03, GTS1, GZF3, HAC1, HAP1, HAP2, HAP3, HAP4, HCM1, HDA1, HDF1, HFM1, HHF1, HHF2, HH01, HHT1, HHT2, HM01, HMS1, HMS2, HO, HOP1, HPR1, HPRS, HSF1, HTA1, HTA2, HTA3, HTB1, HT 62, IFH1, IME1, IME4, IN02, IN04, IXR1, KAR4, LEU3, LYS14, LYS20, LYS21, M AC 1, MAGI, MALI 3, MAL23, MAL33, MATALPHA1, MATALPHA2, MBP1, MCD1, MCM1, MCM2, MCM3, MCM6, MED6, MER2, MET18, MET28, MET30, MET31, MET32, MET4, MGA2, MGT1, MIF2, MIG1, MIG2, MIP1, MLH 1, MOL1, MOT1, MPT4, MRE11, MSH1, MSH2, MSH3, MSH4, MSHS, MS11, MSN1, MSN2, MSN4, MTF1, NBN1, NC132, NDJ1, NGG1, NHP2, NHP6 A, NHP6B, NOT3, NUC2, OAF1, OP11, ORC1, ORC2, ORC3, ORC4, ORCS, ORC6, PAF1, PCH1, PCH2, PDR1, PDR3, PGD1, PHD1, PH02, PH04, PHR1, PIF1, PIP2, PMS1, POB1, POL1, POL12, POL2, POL3, POL30, PO L4, POP2, PPR1, PRH, PR12, PS02, PUT3, RAD1, RADIO, RAD14, RAD 16, RAD18, RAD2, RAD23, RAD26, RAD27, RAD3, RAD4, RADS, RAD50, RAD51, RAD52, RAD54 RAD55, RAD57, RAD6, RAD7, RAP1, RAT-1, RCS1, REB1, REC102, RE C104, REC114, RED1, REG1, RET1, REV3, RFA1, RFA2, RFA3, RFC1, RFC2, RFC3, RFC4, RFCS, RGM1, RGT1, RIF1, RIF2, RIM1, RIM101, RLF2, RLM1, R ME1, RMS1, ROX1, ROX3, RPA12, RPA135, RPA14, RPA190, RPA34, RPA43, RPA49, RP1310, RPB11, RPB2, RP133, RP134, RPBS, RPB6, RPB7, RPBB, RPB9, RPC10, RPC19, RPC25, RPC31, RPC34, RPC40, RPC53, RPC82, RPD3, RP021, RP031, RP041, RRN10, RRN11, RRN3, RRNS, RRN6, RRN7, RRN9, RSC4, RSC 6, RSC8, RTG1, RTG3, SASS, SEF1, SET1, SFH1, SFM, SGS1, SIG1, SIN 3, SIN4, SIP2, SIP4, SIR1, SIR2, SIR3, SIR4, SKN7, SK01, SMC1, SMC 2, SMP1, SNF2, SNFS, SNF6, SOK2, SPKi, SPOL, SPS18, SPT10, SPT 15, SPT16, SPT2, SPT21, SPT23, SPT3, SPT4, SPT5, SPT6, SPTB, SRI 32, SRB4, SRBS, S RB6, SRB7, SR138, SR139, SSL2, SSN3, SSN6, SSNB, SSU72, STB4, STBS, S TE12, STH1, SUA7, SWH, SW13, SW14, SW16, SWP73, TAF19, TAF25, TBF1, TEA1, TEC1, TFAI, TFA2, TF131, TF132, TF133, TFB4, TFC1, TFC2, TF C3, TFC4, TFCS, TFG1, TFG2, TH12, TOA1, TOA2, TOP1, TOP2, TOP3, TRF4, TS P1, TUP1, TYE7, UGA3, UME6, UNGI, USV1, XRS2, YAL019W, YAP1, YA P3, YAPS, YBL054W, YBR026C, YBR150C, YBR239C, YCR106W, YDR026 C, YDR060W, YDR213W, YER045C, YER184C, YFL052W, YIL036W, YIL 130W, YJL103C, YJL206C, YKL005C, YKL222C, YKR064W, YLL054C, YLRO 87C, YLR266C, YNL206C, YOL089C, YOR172W, YOR380W, YOX1, YPL133C, YPR008W, YPR196W, YRR1, ZAP1, ZIP1, ZU01

IMMUNOSUPPRESSANT FEN1, SSH4, SHR3 CYCLINS CCU, CLB1, CLB2, CLB3, CL134, CLBS, CLB6, CLG1, CLN1, CLN2, CLN 3, CTK2, PCL1, PCL10, PCL2, PCLS, PCL6, PCL7, PCLB, PCL9, PH080, S SNB, YBR095C

ATP-BINDING CASSETTE PROTEINS ADP1, ATM 1, CAF16, GCN20, MDL1, MDL2, PDR10, PDR11, PDR12, PDR15, PDRS, PXA1, PXA2, SNQ2, STE6, YBT1, YCF1, YDL223C, YD R091C, YEF3B, YER036C, YHL035C, YKR103W, YKR104W, YLL015W, YNR070W, YOR011W, YOR1, YPL226W

CYTOSKELETAL ABP1, ACF2, ACT1, AFR1, AIP1, AIP2, ARP3, AUT2, AUT7, BEM1, BI M1, BN11, BN14, BUD3, BUD6, CAP1, CAP2, CDC10, CDC11, CDC12, CDC 3, CIN1, CIN2, CIN4, CMD1, COF1, CRN1, END3, GIC1, GIC2, GIN4, J NM1, KAR9, KIP2, KIP3, LAS 17, MDM1, MHP1, MY01, MY02, MY03, MY04, MY05, PFY1, RVS161, RVS167, SAC6, SAC7, SEC 1, SHE3, SHM2, SLA1, SL A2, SMY1, SMY2, SPA2, SPH1, SPR28, SPR3, SRV2, TCP1, TPM1, TPM2, TUB1, TUB2, TUB3, VPS16, VRP1 APOPTOSIS ATP1, ATP14, ATP15, ATP16, ATP2, ATP3, ATP4, ATPS, ATP6, ATP7, ATP8, ATP9, CYC1, SH01, SSK2, SSK22, SW13, SXM1

CELL RESCUE ACC1, ALD6, BCK1, BEM 1, BEM2, BIM1, BMH1, BMH2, CAN1,CBF1, CDC1, CDC14, CDC15, CDC20, CDC25, CDC28, CDC33, CDC37, CDC 42, CDC43, CDC53, CDC6, CHC1, CIN8, CKA1, CKA2, CLA4, CLB1, CLB2, CLB3, CLB4, CLB5, CLN1, CLN2, CLN3, CMP2, CNA1, COF1, CTT1, DBF2, DBF20, DPMI, ERG25, GIC1, GIC2, GPA1, GRR1, HCA4, HIS4, HOC1, HSF1, KAR1, KES1, KRE6, KSS1, MBP1, NMT1, ORC2, ORC5, PDE2, PEP12, PEP7, PKC1, P LC1, PMR1, POL30, PRP18, RAMI, RAS1, RAS2, RBL2, RED1, RFC1, RH01, RH03, RH04, SAC1, SEC13, SEC14, SEC22, SEC4, SET1, SIS2, SKP1, SPC98, SRA1, SR04, SRP1, SSA1, SSA2, SSA4, SSN8, STE20, STN 1, STT4, SUJ 3, SWE1, SW14, SW16, TEL1, TOR1, TUB1, TUB4, VMA1, YCK1, YCK2, YPT1

CELL DAMAGE APN1, BUB1, CDC28, CDC45, CDC46, CDC47, CDC54, CDC7, CLB1, CLB2, CLB3, CLB5, DDC1, DDR2, DDR48, DIN7, DUN1, ECM32, HSM3, IMP2, MEC1, MEC3, MGT1, MOL1, MRE11, MUS81, NTG1, PDS1, PGD1, P HR1, POL2, POL3, POL30, POL4, PR11, PS02, RAD14, RAD16, RAD17, RAD18, RA D24, RAD30, RAD51, RAD52, RAD54, RAD55, RAD57, RAD7, RAD9, RDH54, REV3, RFA1, RFC5, RNR1, RNR2, RNR3, RNR4, RPH1, SIC1, SML1, SP K1, STN1, STS1, TEL1, TFA1, TFA2, TUP1, UBC7, UB14, XBP1, YBR098W, YFH1

OTHER RELEVANT MUTANTS AND GENES Y-1, 9520b, C658-K7, JPD 4, JPM 9, Cy32, E354, JC488, PSY 142, 01-2, Y217, JC787-9A, ML1-21, Y500,86-9C, GL1, GT5-1A, HD565A, PZ1, 127-4D, Y229, JC302-26B, JC482, LB2211-2B, MH41-7B/P21 , erg 81 , SEY6211 , GL4, K335, MK20, MK34, DE4-3A, DE4-3B, DE4-3C, MMY011 , UH 1-GRGZ, 2150-2-3a, Y211 , DP1/517,943,1117, C658, 1252, H79.20.3, LB1-3B, C658-K42, R29B, LB54-3A, XW520-9A, ade7, D225-5A, 309, SDH1 , SDH2, SDH3, SDH4, TCM62, PDE1 , PDE2

Cytokines that mediate and regulate innate immunity : Type I interferons, Tumor necrosis factor-a, lnterleukins 1 , 6, 10, 12, and 15, Chemokines, Cytokines that mediate and regulate specific immunity, lnterleukins 2, 4, 5, 13, 16, and 17, Interferon-g, TGF-β, Lymphotoxin, Cytokines that stimulate hematopoiesis, c-kit ligand, lnterleukins 3, 7, 9, 11 , Colony stimulating factors. An encyclopedic web site of the cytokines may be found at: www.copewithcytokines.de

Immunoglobulin superfamily, Cytokine receptor family - Class I, Cytokine receptor family - Class II, TNF receptor family. Seven transmembrane helix family.

The immunoglobulin super-family is characterized by one or more Ig domains which are regions of 70 to 110 amino acid residues homologous to either Ig V or C domains. Examples include receptors for IL-1 and M-CSF.

Heat shock proteins

Heat shock proteins (HSPs) are a ubiquitous family of proteins that exists in all cells. Inside the cell, they act as 'chaperones,' helping nascent proteins fold into shape and helping existing proteins survive environmental stress. HSPs are also believed to play a role in the presentation of proteins and peptides on the cell surface to help the immune system recognize diseased cells.

Because HSPs are normally found inside cells, their presence outside the cell serves as a powerful signal to the immune system that something is wrong. Very sick cells often undergo necrosis, spilling out their contents. The immune system's dendritic cells then pick up the HSPs, which are still associated with the peptides they had been chaperoning within the cell. Once inside the dendritic cells, the peptides are released from the HSPs and presented on the cell surface as a red flag to the immune system. Any peptide that is recognized as foreign, such as that from an infected or cancerous cell, has the potential to trigger an immune response. The dendritic cells alert the immune system by displaying antigenic peptides on their cell surfaces, which then activates 'killer' T cells to target and destroy the diseased cells.

Specific immunotherapy entails the use of a combination of immunogenic agents to elicit an appropriate anti- tumor immune response. In order to achieve such a response, reestablishment of the proper cell mediated immune cascade must be permitted. There are two main arms of the immune system: humoral immunity and cell mediated immunity. The former is concerned mainly with activation and promotion of the B lymphocyte pathway, ultimately leading to the production and secretion of antibodies against a given target. The latter denotes a T-cell mediated response which eventually leads to the development of a specific clone of T cells, educated and determined to attack and destroy any cell bearing the target peptide with which it was presented. Presentation refers to the process wherein fully matured dendritic cells that have captured tumor-specific antigen [TSA] process TSA into a form presentable to T cells, in- or not in-association with the human leukocytic antigen.

Antibody mediated immunity and cell mediated immunity are both under the control of cytokines. Basically, depending on the endogenous cytokine profile bias, one pathway will be more dominant. Antibody mediated immunity is dominant when the phenotype bias are cytokines from T helper type 2 cells (Th2) (Interleukin [ILJ-5,

IL-10 and IL-13 for example) and the cell mediated system becomes dominant when the bias is towards a T helper type 1 response (Th1 ) (IL-12, IL-2 and interferon [IFN]-* for example). The Th1 and Th2 pathways are mutually inhibitory. Literally, a switch of the phenotypic bias of the cytokine system can be achieved by altering the circulating cytokine levels. One immuno-therapeutic strategy is to create an upsurge in the endogenous production of IL-12. Orally administered muramic acid moieties, in particular muramyl polysaccharide glycan complex, exhibit this activity. Establishment of such bias is important to the success of an anti-tumor immune therapy. Once established, monocytes in the body will become converted into immature dendritic cells, that are then able to scavenge both tumor cells as well as freely circulating TSA. This event is followed by a maturation of the dendritic cell into a professional antigen-presenting cell. Immature dendritic cells do not present antigen well; on the contrary, matured dendritic cells are potent antigen presenters, thus the entitlement of "professional." Antigen presentation is a crucial step in the advancement of the proper anti-tumor immune response.

Targeting of gene expression to primary tumors

An efficient expression of transgenes at a tumor site has been demonstrated by Bromley (PCT/IB99/00294). Both viral and non-viral vectors can be employed in such transgene delivery, although modified adenoviral vectors, which have the capacity to infect most types of human cells, both dividing and non-dividing are a preferred mode of this invention. The potential reaction of the host immune system to such adenoviral vectors should not be an impediment, since for tumor therapy, a single exposure to the vector and concomitant expression of transgenes selected a) for inducing cell death, and b) specifically alerting the immune system, should suffice to treat the primary tumor and to launch an immune attack on metastases.

The activation of transgene expression after injection of the vector intra-tumorally, is performed using focused ultrasound heating. The heat pattern is monitored by performing the ultrasound heating either inside of an MRI machine as described by Moonen C, (PCT/US97/15270) or an ultrasound machine for visualization and control. Real-time temperature mapping of heating part of a rabbit liver has been demonstrated, (Weidensteiner C. et al., Magnetic Resonance in Medicine 50: 322-330, 2003). In a similar fashion, (Smith et al., Human Gene Therapy 13: 697-706, 2002) have demonstrated the spatial and temporal control of transgene expression in rodent liver models.

For an extension to the treatment of metastases, one must optimize the visibility of tumor antigens to the immune system; here it is critical to express increased amounts of stress proteins in tumor cells at the time of their death through necrosis or apoptosis, thus permitting an efficient interaction between such stress proteins expressed in tumor cells with tumor antigens that become available on destruction of the tumor cell. This invention, by inducing stress protein synthesis by focused heating of the primary tumor, provides an optimal approach to the treatment of tumor metastases.

EXAMPLES

Example 1

Vectors containing a hsp promoter operably linked to a gene derived from the gene of interest for de-bulking tumors are used in this example; these include, but are not limited to genes inducing apoptosis or necrosis, blockage of cell cycle, and blocking restenosis and FasL, TNF, II2, 1112, Bacterial toxins such as a diphtheria toxin fragment, ricin.

The human hsp-70B promoter is used, as it is strictly heat regulated and can promote a several thousand fold increase in expression upon induction (M. Dreano et al. (1986), "High level heat-regulated synthesis of proteins in eukaryotic cells," Gene 49:1 ).

The vector is constructed by inserting the human hsp70 promoter in front of the gene to be expressed. An SV40 polyadenylation is optionally used together with a inverse terminal repeat (ITR) as an encapsidation signal and enhancer. Further, more powerful and complex inducible promoter systems may be employed in the invention, for example the hsp70 promoter combined with the HIV-2 promoter, Tsang et al., US 6,709,858, "Hyperthermic inducible expression vectors for gene therapy and methods of use thereof «

Example 2 Combinations of genes designed to initiate an immune reaction against tumor antigens, and thus combat metastases of the primary tumor. Using the technology described in this invention for spatial and temporal control of transgene expression, the following combinations of genes are cited as an example, although it is understood that this list is not exhaustive, and other genes having comparable functions can also be used in this invention: Genes inducing an augmentation of an immune response against tumor antigens include hsp proteins such as the hsp70 and hsp90 families, cytokines, chemokines etc.

Example 3

Combinations of genes selected from Example 1 , tumor de-bulking genes, and from Example 2, immune response genes are employed, injected into tumors either together or sequentially, and induced by a heat shock Localized heating of deep lying tissues can be accomplished by invasive or noninvasive methods (without opening the skin). Among the invasive methods, the introduction of a catheter with a heated tip can be used.

Alternatively, a catheter with an optical guide can be used. A laser beam can then be directed through the catheter to the targeted tissue and heat can be deposited using direct radiation (for example using infrared light).

Although irradiation by laser has been proposed for heating deep tissue, its use in medicine has been limited by optical absorption and thermal diffusion.

In the preferred embodiment, local heating is achieved by noninvasive means. FIG. 1 , taken from Ernst et al., Principles Of Nuclear Radiation In One And Two Dimensions, Oxford University Press, 1987, illustrates the attenuation of electromagnetic and ultrasound radiation. For heating of deep lying tissues, a transition area is used (since both absorption and penetration are needed). The X-ray region of the spectrum uses ionizing radiation which is hazardous. The radio frequency region has a wavelength of more than 10 cm. Acoustical radiation is strongly absorbed for wavelengths below 1 mm. Since the ability to focus is limited to approximately half the wavelength, a focus diameter of 5 cm or more can be attained by radio frequency. This is generally not localized enough to treat small lesions. Ultrasound can be applied with a short enough wavelength to be localized and can penetrate deeply and is to some extent absorbed by body tissues. Therefore, the preferred method for noninvasive local heating is focused ultrasound.

It is known that ultrasound can be aimed at a defined target area, and that prolonged exposure of living tissues to ultrasound can raise the temperature of the exposed tissue. In particular focused ultrasound has been known to be very effective to locally heat tissue so long as there is an acoustic path from the surface to the lesion free of air and bone. LeIe, L. L (1962) "A simple method for production of trackless focal lesions with focused ultrasound: physical factors," J. Physiol 160:494-512; Fry et al., (1978) "Tumor irradiation with intense ultrasound", Ultrasound Med. Biol. 4:337-341. Using an array of ultrasound transducers with high precision of heat deposition, focused ultrasound can be delivered at high intensity to a defined very small area of deep tissue. Focusing of the ultrasound is achieved by the shape of the transducer (spherical, parabolical) and/or by combining several different transducer elements and combining their ultrasound waves with individually adjusted phases in order to provide a focal spot. The principles of ultrasound can be found in, for example, Bushberg J. T. et al., The Essential Physics Of Medical Imaging, Williams and Wilkins, Baltimore, 1994, pp. 367-526.

In general, published studies have either sought to use ultrasound to deliberately burn tissue, or to image tissues without significantly raising their temperature. See, e.g., McAllister et al. (1994) Teratology 51 :191 ; Cline et al. (1992) "MR-guided focused ultrasound surgery" J. Comp. Asst. Tomog. 16:956-965. Angles et al. (1991 ) Teratology 42:285 reported that ultrasound can activate heat shock genes in the absence of any detectable rise in temperature. In U.S. Pat. No. 5,447,858, a soybean hop promoter was recombined with a heterologous gene, introduced into plant cells, and the hap promoter was activated using the "heat of day" (column 11 , lines 15-25) or incubation at, for example, 42.5.degree. C. (column 11 , line 37). High intensity focused ultrasound has been used to ablate tumors in animal models (LeIe (1962), J. Physiol. 160:494-512; Fry et al. (1978), Ultrasound Med. Biol. 4:337341 ) and is a proposed surgical technique for treating liver tumors (ter Harr et al. (1991 ), Phys. Med. Biol. 36:1495-1501 ; ter Harr et al. (1991 ), Min. Invas. Ther. 1 :13-19).

In contrast, the present invention sets out to deliberately heat tissue within a target volume, but in a finely controlled fashion within a defined range of temperatures. In the past, several factors have limited the use of ultrasound to locally heat tissue: 1 ) the inability to precisely pinpoint the exact location of heat deposition due to interference near air/water, water/bone, and fat/water boundaries, 2) the inability to precisely quantify temperature elevation, and 3) the inability to simultaneously visualize the target tissue and surrounding tissues to monitor extent and effects of ultrasound heating. One possibility is to use a combination of focused ultrasound and magnetic resonance imaging (MRI). Cline et al. (1994) Magn. Reson. Med. 31 :628636, Cline et al. (1995) J. Comp. Asst. Tom. 16:956-965, De Poorter (1995) Magn. Reson. Ned. 33:74-81.

It should be noted that the heat shock promoter may be activated by phenomena other than ultrasound that can raise body temperature (e.g., fever, hot shower, stress). Thus, it is appropriate to stringently control these variables (closely monitor a patient's temperature, avoid hot showers, avoid stress-producing environments) during the duration of treatment. Another approach is to limit the duration of the gene therapy. Localized heating of deep lying tissues can be accomplished by invasive or noninvasive methods (without opening the skin). Among the invasive methods, the introduction of a catheter with a heated tip can be used. Alternatively, a catheter with an optical guide can be used. A laser beam can then be directed through the catheter to the targeted tissue and heat can be deposited using direct radiation (for example using infrared light). Although irradiation by laser has been proposed for heating deep tissue, its use in medicine has been limited by optical absorption and thermal diffusion.

Example 4

Genes from either Example 1 or Example 2, or combinations such as in Example 3, after injection into tumors, are activated by FUS Heating applied as follows:

A patient is placed on a special bed (e.g., General Electric Co., Milwaukee, Wis., as described in Cline et al. 1994 and 1995, supra) and moved into the magnet of a magnetic resonance imaging (MRI) instrument (e.g., 1.5T Nat Imaging system by Signa, GE Medical Systems, Milwaukee, Wis.). The MRI instrument is equipped with a focused ultrasound device (e.g. from Specialty Engineering Associates, Milpitas, Calif.) under computer control. Specifically, the FUS device can be incorporated in the bed of the MRI in such a way that the transducer can be freely moved under the patient with motional freedom in the three principal directions to allow the focus to be placed anywhere in the human body. Alternatively, the focus can be adjusted electronically by using a more complicated FUS transducer, a so-called phased array FUS transducer, in fact a combination of multiple transducers that can be controlled individually by electronic means thus allowing to move the focus. Acoustic contact between the focus and the FUS transducer is assured using appropriate water, gel, or other means giving an uninterrupted acoustic path from transducer to focus. A Sparc 10 (Sun Microsystems, Mountain View, Calif.) workstation interfaced to the motor controls, the FUS pulse generator and the MR imaging system is used to program, plan, monitor and control therapy. Cline et al., supra, and Zwart et al., supra.

The area of the target is immobilized by gentle straps to the bed. (Note that the more accelerated the procedure, the less the need for immobilization; with very accelerated procedures immobilization is unnecessary.)

Highly detailed MRI images are obtained with a suitable contrast to determine accurately the computer coordinates of the target (e.g. tumor, or ischemic area) as per standard MRI procedures. Based on i) coordinates of the target, ii) estimates of ultrasound attenuation, iii) acoustic impedance transitions in the ultrasound paths, the focus, power and exposure time of the FUS device are targeted to give an increase in temperature of three degrees Celsius in approximately 10 seconds at the target. The FUS device is switched on for 10 seconds.

Immediately following the FUS exposure, a rapid MRI temperature image is taken as per the procedure outlined in J- de Zwart et al. (1996), J. Magn. Reson. Series B, 112:86-90 and references therein). An evaluation is made as to the following criteria: i) Does the heated spot correspond with the target (comparison of anatomical MRI and temperature Mgtl), and ii) Is the temperature elevation indeed 3 degrees Celsius (quantification of temperature, see J. de Zwart et al. (1996). If not, the FUS target is moved in the first case, and the power is adjusted in the second case. The trial heating is repeated until location and power correspond with the target. Note that, since hsp-70B promoter activity is linearly proportional with the duration, gene expression in this adjustment procedure is limited because of its short duration.

Once power and focus have been adjusted, the therapeutic dose of the ultrasound is delivered. For the hsp-70B promoter, an elevation by 3 degrees for 30 minutes gives rise to very large expression of the gene under hsp-70B control. Therefore, the initial exposure is 30 minutes. It can be increased or decreased at the discretion of the attending physician, taking into consideration the severity of the condition treated, the condition (age, health) of the patient, and the size and location of the target area. Similarly the dosage of therapeutic vectors can also be adjusted on the same basis. The patient is then removed from the MRI. Evaluation of therapy is performed by clinical examination and regular follow-up of detailed anatomical MRI to evaluate primary tumor shrinkage and the reduction and disappearance of metastases.

For purposes of therapy, a therapeutic vector or vectors, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount. This is defined as an amount that is physiologically significant. An agent is physiologically significant is its presence results in a detectable change in the physiology of the patient. Stress promoters are only induced at very high levels of stress, i.e. 41-42 degree.C and above in human cells. Such temperatures correspond to extreme fever, and are only reached rarely in humans. A treating physician will do everything possible to prevent a patient from developing such a fever.

Claims

1. A method for treating a primary cancer and its metastases, said method comprising the steps of: a) Inserting genes into viral vectors, each gene being placed under the expression control of heat- inducible promoters, b) Injecting such vectors into tumors in patients or animals, c) Activating the expression of such genes by applying a targeted source of heat.

2. The method of Claim 1 where the de-bulking of primary tumors is achieved by expressing genes inducing necosis and/or apoptosis, such genes being selected from, but not limited to FasL, TNF, II2, 1112, Bacterial toxins such as a diphtheria toxin fragment, ricin.

3. The method of Claim 1 where the immune response gene or genes are selected from, but not limited to hsp proteins such as the hsp70 and hsp90 families, chemokines such as, but not limited to GM-CSF, gamma interferon, chemokines attractive to dendritic cells, CCI3, CCL20, TNF, ligands which activate Toll-like receptors, IM .

4. The method of Claim 1 where the genes from sets of genes noted in Claims 2 and 3, are combined, and injected at the same time.

5. The method of Claim 4 where the subject is a human.

6 The method of Claim 4 where genes from the debulking list and from the immune response list are injected at different times.

7. Where the genes of claim 4 are delivered to tumors in adenoviral vectors.

8. The method of claim 4 where the heating induction step is performed by a Focused Ultrasound Device.

9. The method of Claim 8 where other forms of local or targeted heating than FUS is employed.

10. Where the method of claim 5 is monitored by being placed inside of an MRI machine.

11. The method of claim 5 where the therapeutic target is a solid tumor.

12. The method of claim 5 where the tumor has metastasized.

13. The method of Claim 1 where genes are selected for the treatment of diseases other than cancer, but where cell destruction is a required treatment.