Oncolytic vaccinia virus cancer therapy

(54) Title: CANCER THERAPY WITH VIRUSES FROM (57) Abstract: ONCOLYTIC VACCINE.

(30) Unionist Priority: 3/15/2007 us 60 / 894,932 (73) Owner (s): Jennerex, Inc (72) Inventor (s): David Kirn (74) Attorney (s): Dannemann .Siemsen, Bigler & Ipanema Moreira (86) International Order: pct US2008057257 of 17/03/2008 (87) International Publication: wo 2008 / 113078de 18/09/2008

<td>Coon.</td><td>Mode Patient</td><td>Demographics of Patients</td><td>Tumor Type (Live Target LD)</td><td>No. of Cicloe (Oats of the 1st Treatment)</td><td>state</td>

<td> 1</td><td> 101</td><td>M 55</td><td>(8.5 cm)</td><td>1 (09-Jan-2006)</td><td>(roneu «n W / marchCOM)</td>

<td> 1</td><td> 102</td><td>F 56</td><td>(4.1 cm)</td><td>3 (06-Feb-2006)</td><td>(died in llSúíuisnvaOM)</td>

<td> 1</td><td> 103</td><td>M 53</td><td>(9.8 cm)</td><td>6 + 2HAI (16-Mor-2006)</td><td>Treatment completed</td>

<td>Λ »</td><td></td><td colspan="2"></td><td></td><td></td>

<td> 2</td><td> 201</td><td>M 66</td><td>HCC (6.2 cm)</td><td>8 (30-Μαι-2006)</td><td></td>

<td> 2</td><td> 202</td><td>M 50</td><td>(9.7 cm)</td><td>4 (12-Jun-2006)</td><td>Treatment completed</td>

<td> 2</td><td> 203</td><td>M 56</td><td>HCC (6.1 cm)</td><td>5 (12-JuJ-2006)</td><td>Treatment completed</td>

Descriptive Report of the Invention Patent for CANCER THERAPY WITH VACCINE VIRUS ONCOLYTIC.

Background of the Invention

Technical Field of the Invention

The present invention generally relates to the fields of oncology and virology. More particularly, it refers to poxviruses, specifically including oncolytic vaccinia viruses suitable for the treatment of cancer.

Background of the Technique

Normal tissue homeostasis is a highly regulated process of cell proliferation and cell death. An imbalance of cell proliferation or cell death can develop in a cancerous state (Solyanik et al., 1995; Stokke et al., 1997; Mumby and Walter, 1991; Natoli et al., 1998; Magi-Galluzzi et al. , 1998). For example, cervical, kidney, lung, pancreatic, colorectal, and brain cancer are just a few examples of the many cancers that can result (Erlandsson, 1998; Kolmel, 1998; Mangray and King, 1998; Mougin et al., 1998). In fact, the occurrence of cancer is so high that more than 500,000 deaths per year are attributed to cancer in the United States alone.

The maintenance of cell proliferation and cell death is regulated at least partially by proto-oncogenes and tumor suppressors. A proto-oncogene or tumor suppressor can encode proteins that induce cell proliferation (for example, sis, erbB, src, ras and myc), proteins that inhibit cell proliferation (for example, Rb, p16, p19, p21, p53, NF1 and WT1) or proteins that regulate programmed cell death (for example, bc1-2) (Ochi etal., 1998; Johnson and Hamdy, 1998; Liebermann etal., 1998). However, genetic rearrangements or mutations of these proto-oncogenes and tumor suppressors result in the conversion of a proto-oncogene into a potent cancer-causing oncogene or a tumor suppressor in an inactive polypeptide. Often, a single point mutation is sufficient to achieve the transformation. For example, a point mutation in the tumor suppressor protein p53 results in complete loss of the function of wild type p53 (Vogelstein and Kinzler, 1992).

Currently, there are few effective options for treating many types of common cancers. The course of treatment for a given individual depends on the diagnosis, the stage at which the disease has developed and factors such as age, sex, general health of the patient. The most conventional cancer treatment options are surgery, radiation therapy and chemotherapy. Surgery plays a key role in the diagnosis and treatment of cancer. Typically, a surgical approach is required for biopsy and to remove cancerous growth. However, if the cancer has metastasized and is widespread, surgery is unlikely to result in a cure and an alternative approach should be taken.

Radiation therapy and chemotherapy are the most common alternatives to surgical treatment of cancer (Mayer, 1998; Ohara, 1998; Ho et at., 1998). Radiation therapy involves the precise assertion of high-energy radiation to destroy cancer cells, and very similar to surgery, it is especially effective in treating localized cancer cells that have not metastasized. Side effects of radiation therapy include skin irritation, difficulty swallowing, dry mouth, nausea, diarrhea, hair loss, and loss of energy (Curran, 1998; Brizel, 1998). Chemotherapy, the treatment of cancer with anticancer drugs, is another mode of cancer therapy, and most chemotherapy approaches include the combination of more than one anticancer drug, which is known to increase the response rate of a wide range of cancers ( US patent number 5,824,348; US patent number 5,633,016 and US patent number 5,798,339, incorporated herein by reference). However, an important side effect of chemotherapy drugs is that they also affect cells in normal tissues, and the cells that are most likely to be affected are those that divide rapidly in some cases (for example, bone marrow, gastrointestinal tract, reproductive system and hair follicles). Other side effects of chemotherapy drugs may include mouth sores, difficulty swallowing, dry mouth, nausea, diarrhea, vomiting, fatigue, bleeding, hair loss, and infection.

Selective replicating oncolytic viruses are promising for the treatment of cancer (Kirn et al., 2001). These viruses can cause the death of tumor cells through oncolytic effects dependent on direct replication and / or dependent on gene expression (Kirn et al., 2001). In addition, viruses are able to enhance the induction of cell-mediated antitumor immunity within the host (Todo et al., 2001; Sinkovics et al., 2000). These viruses can also be created by genetic engineering to express therapeutic transgenes within the tumor, to increase antitumor effectiveness (Hermiston, 2000). However, there are also important limitations to this therapeutic approach.

Therefore, more additional therapies for the treatment of cancer are needed. The use of oncolytic viruses presents a potential area for development.

Summary of the Invention

The embodiments of the invention relate to methods that include the administration of a thymidine kinase deficient vaccinia virus. Methods include administering the vaccinia virus in increasing viral concentrations. In certain aspects, the methods include inducing oncolysis or collapse of the tumor vasculature in an individual who has a tumor, comprising administering to said individual at least 1x10⁸ viral particles of a replicating competent vaccinia virus vector, which expresses GM-CSF and is TK deficient, sufficient to induce cell oncolysis in the tumor. In another aspect of the invention, the methods may exclude pretreatment of an individual with a vaccine vaccine, for example, an individual who does not need to be vaccinated 1, 2, 3, 4, 5, or more days, weeks, months or years before to administer the therapy described here. In some respects, tumors or uninjected cancer will be infected with the therapeutic virus, thereby treating a patient by local administration and systemic spread.

In certain aspects, the individual is given at least 2 x 10®, 5 x 10®, 1 x 10⁹, 2 x 10⁹, 5 x 10⁹, 1 x 10¹°, 5 x 10¹°, 1 x 10¹¹, χ 10¹¹, 1 χ 10¹², 5 χ 10¹² or more viral particles or plaque-forming units (pfu), including the various values ​​and ranges between them. The viral dose can be administered in 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more milliliters, including all values ​​and ranges between them. In one aspect, the dose is sufficient to generate a detectable level of GM-CSF in the patient's serum, for example, at least about, at most about, or about 5, 10, 40, 50, 100, 200, 500 , 1,000, 5,000, 10,000, 15,000 to 20,000 pg / mL, including all values ​​and ranges between them. It is contemplated that a single dose of the virus refers to the amount administered to an individual or within a tumor over a period of 1, 2, 5, 10, 15, 20, or 24 hours. The dose can be spread over time or by separate injection. Typically, multiple doses are administered to the same generic target region, such as in the vicinity of a tumor or, in the case of intravenous administration, a specific point of entry into an individual's bloodstream or lymphatic system. In certain respects, the viral dose is delivered by an injection device that comprises a needle that produces multiple orifices on a single needle or multiple nozzles (prongs) coupled to a syringe, or a combination of them. In another aspect, the vaccinia virus vector is administered 2, 3, 4, 5, or more times. In yet another aspect, the vaccinia virus is administered for 1, 2, 3, 4, 5, 6, 7 or more days or weeks.

In certain modalities, the individual is a human being. The individual may be afflicted with cancer and / or a tumor. In certain embodiments, the tumor may be non-resectable before treatment and resectable after treatment. In certain aspects, the tumor is located on the liver or inside the liver. In other respects, the brain cancer tumor, a head and neck cancer tumor, an esophageal cancer tumor, a skin cancer tumor, a lung cancer tumor, a thymus cancer tumor, a tumor stomach cancer, colon cancer tumor, liver cancer tumor, ovarian cancer tumor, uterine cancer tumor, bladder cancer tumor, testicular cancer tumor, cancer tumor rectal cancer, a breast cancer tumor, or a pancreatic cancer tumor. In other embodiments, the tumor is a bladder tumor. In still other modalities, the tumor is melanoma. The tumor can be a recurrent, primary, metastatic tumor, and / or a tumor resistant to multiple drugs. In certain embodiments, the tumor is a hepatocellular tumor or a metastatic tumor that originated from another tissue or location. In some ways, the tumor is in the liver.

In certain aspects, the method further comprises administering to the individual a second cancer therapy. The second cancer therapy can be chemotherapy, biological therapy, radiation therapy, immunotherapy, hormonal therapy, antivascular therapy, cryotherapy, toxin therapy or surgery, including combinations of them. In another aspect, chemotherapy can be taxed) or sorafenib. In yet another aspect, surgery includes transarterial chemoembolization (TACE procedure, see Vogl et al., European Radiology 16 (6): 1393, 2005). The method may further comprise a second administration of the vaccinia virus vector. The methods of the invention may further comprise assessing the viability of tumor cells before, during, or after treatment, or a combination of them. In certain modalities, the virus is administered intravascularly, intratumorally, or combinations of them. In another aspect, administration is by injection into a tumor mass. In yet another embodiment, administration is by injection into the region of the tumor vasculature. In yet another embodiment, administration is by injection into the lymphatic system or the vasculature of the site of said tumor. In some ways, the method includes reproducing images of the tumor before or during administration. In some ways, a patient is or is not pre-immunized with a vaccine virus vaccine. In another aspect, the individual may be immunocompromised, naturally or clinically.

In some respects, the virus is administered in an amount sufficient to induce oncolysis in at least 20% of the cells in an injected tumor, in at least 30% of the cells in an injected tumor, in at least 30% of the cells in a tumor that received injection, in at least 40% of the cells in a tumor that received injection, in at least 50% of the cells in a tumor that received injection, in at least 60% of the cells in a tumor that received injection, in at least 70% of the cells in an injected tumor, in at least 80% of the cells in an injected tumor, or in at least 90% of the cells in an injected tumor.

In certain embodiments, the vaccinia virus comprises one or more modified viral genes. The one or more modified genes can comprise one or more among (a) an interferon modulating polypeptide; (b) a complementary control polypeptide; (c) a TNF-modulating polypeptide or chemokines; (d) a serine protease inhibitor; (e) a ΙΙ_-1β modulator polypeptide; (f) a polypeptide in the form of non-infectious EEV; or (g) a viral polypeptide that acts to inhibit the release of infectious virus from cells (polypeptide in the form of anti-infectious virus), which act to inhibit the release of infectious virus from cells (polypeptide in the form of anti-virus) infectious) or combinations of them.

The modalities of the invention target common critical cancer pathways. Targeting these pathways involves the modulation of various cellular mechanisms (for example, cell levels of thymidine kinase: responsive to E2F; activation of the EGF-R pathway; immune sanctuary: response to antiviral IFN (ras, p53); vascular pore size induced by VEGF: deposition IV) leading to multiple mechanisms of efficacy, such as oncolysis: necrosis, vascular arrest, induction of CTL attacks (cytotoxic T lymphocytes), systemic: IT, IV; Tumor-specific CTLs.

The modalities of the invention are based on phase I clinical trials, which demonstrate the safety and efficacy of the vaccinia virus as a cancer treatment. A clinical trial on metastatic melanoma with seven patients with an average life expectancy &lt;6 months enrolled was conducted using intratumoral injections in a study with dose rise twice a week. The trial indicated that the vaccinia virus was safe, well tolerated and resulted in tumor responses in 5 patients (71%) with two long-term disease-free survivors.

The initial results of phase 1 / ll tests also demonstrated the continued safety of JX-594. Influenza-like symptoms have been observed for 5-8 days. A transient decrease in platelets (plt), lymph, absolute neutrophil count (ANC) (typically Gr1-2) was also observed. There was one death on Day 8 of the study, but it was not attributed to treatment. In total, JX-594 viremia was well tolerated with an immediate post-injection (15-30 min.): Maximum 3 x 10⁸ total blood genomes and peak replication (Day 5-8): maximum 10¹⁰ total genomes in the blood.

Other embodiments of the invention are discussed in this entire patent application. Any modality discussed with respect to one aspect of the invention also applies to other aspects of the invention, and vice versa. The modalities in the example section are to be understood as being modalities of the invention that are applicable to all aspects of the invention.

The terms inhibit, reduce or prevent, or any variation of these terms, when used in the claims and / or in the specification, include any measurable decrease or complete inhibition to achieve a desired result.

The use of the word one or one when used in conjunction with the term understand in the claims and / or in the specification can mean one, but it is also consistent with the meaning of one or more, at least one, and one or more than one.

It is contemplated that any modality discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. In addition, the compositions and kits of the invention can be used to carry out the methods of the invention.

In this entire patent application, the term about is used to indicate that a value includes the standard deviation of the error for the device or method being employed to determine the value.

The use of the term or in the claims is intended to mean and / or, unless explicitly stated to refer to alternatives only or the alternatives are mutually exclusive, although the description substantially defines only alternatives and and / or .

As used in this specification and in the claims, the words understand (and any form of understanding, as they understand and understand), have (and any form of have, as they have and have), include (and any form of include, as as it includes and includes) or contain (and any form of contain, as it contains and contain) are inclusive or unlimited and do not exclude elements or steps of the additional methods not listed here.

Other objects, characteristics and advantages of the present invention will be evident from the detailed description that follows. It should be understood, however, that the detailed description and specific examples, while indicating specific modalities of the invention, are provided for illustrative purposes only, as various changes and modifications within the spirit and scope of the invention will become evident to versed in the technique from the detailed description.

Brief Description of Drawings

The following drawings are part of this specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1 is the design of the clinical trial study for liver tumors using JX-594 by intratumor injection.

Figure 2 is the design of the clinical trial study for melanoma using JX-594 by intratumor injection.

Figure 3 represents the oncolytic viral therapy that has multiple unusual mechanisms for the eradication of cancer.

Figure 4 represents disease-free long-term survivors after the phase I clinical trial with JX-594 for metastatic melanoma. Patient 1, at the top, is a 32-year-old woman: Refractory: DTIC, IL-2; tumors that received an injection: CR; metastases that did not receive an injection: - dermal: CR; breast: CR with surgery. Alive, disease free 1.5+ year. Patient 2, below, a 75-year-old man: multiple metastatic sites (n = 24); tumors that received an injection: CR; metastasis that did not receive an injection: -CR; Alive, disease free 3+ years.

Figure 5 represents responses from the phase I clinical trial with JX594 in tumors that received an injection and that did not receive an injection.

Figure 6 represents JX594-IT-hep001 - patient demographics and treatment status - cohorts 1 and 2.

Figure 7 represents JX594-IT-hep001 - patient demographics and treatment status - cohort 3.

Figure 8 represents the intravenous spread of JX-594 in the bloodstream: early leakage from the tumor corresponds to the dose, mostly cleared in 6 h.

Figure 9 represents the JX-594 replication viremia evident in 80% of patients: secondary wave of JX-594 in the blood, demonstrated in cycles 1-7. (+) after the purified intake dose, (-) Level below the detection limit, squares = patient out of the study, and (p) = pending data. Detection limit = 700 genomes / mL.

Figure 10 represents the JX-594 replication viremia evident in 80% of patients: secondary wave of JX-594 in blood cycles 1-7, days 3 - 22.

Figure 11 depicts avascular necrosis induced by acute treatment of vascular arrest associated with JX-594 replication (pt. 1, gastric cancer).

Figure 12 represents long-term stable disease with control of squamous cell carcinoma with JX-594 (lung - cohort 2).

Figure 13 represents the metabolic response (PET) of tumor melanoma that received an injection of JX-594 response after 2 cycles of JX-594 (cohort 3).

Figure 14 represents the metabolic response (PET) of liver tumor carcinoma that received an injection of JX-594 with long-term control (cohort 2) for 9+ months.

Figure 15 represents Response to Tumor Marker: 99.9% decrease in rapid destruction of AFP liver cancer by the blood marker.

Figure 16 represents the Body Weight Gain with JX-594 10% increase (6 kg; 14 Ib) demonstrates tolerability, effectiveness.

Figure 17 represents the systemic viremia and the tumor response: the viremia associated with JX-594, the resulting systemic efficacy (HCC-cohort 2) - decrease of AFP 40%.

Figure 18 represents the systemic distribution of JX-594 for tumors and the answer: Efficacy in distant tumors that did not receive an injection after the injection found the liver. Metabolic PET response in two tumors that did not receive an injection after 2 cycles (Pt. 304, cohort 3).

Figure 19 represents the Treatment Effectiveness and Survival Data: Tumor responses by CT and PET, long-term survivors.

Figure 20 represents the profile of the assay.

Figures 21A-21B represent important hematological tests and liver function tests (error bars, standard error of the mean). (A) Increase in ANC correlates with increased dose of JX-594 and expression of hGM-CSF. Full bars: ANC; hollow bars: GM-CSF. X-axis: Patient identification number. (B) ALT levels of patients in cohorts 3 and 4 in the first cycle. Most patients did not experience any significant change in ALT levels over time; mild transient transaminitis was also observed.

Figures 22A-22C represent changes in hematological tests. (A) Dose-dependent thrombocytopenia. (B) Magnitude of thrombocytopenia is independent of cycles. (C) Magnitude of changes in ANC, eosinophils and monocytes were more significant in cycle 1 compared to subsequent cycles. White bars: ANC; gray bars: eosinophils; black bars: monocytes. The error bars represent standard error of the mean.

Figures 23A-23F represent the pharmacokinetics, blood-borne spread and distant tumor infection by JX-594. (A) Acute concentrations of the circulating genome. The genomes of JX-594 were detected as early as 15 minutes after injection. In the case of cohorts 1 to 3, acute clearance rates were consistent across cohorts. (B) concentrations of the JX-594 genomes from cohorts 1 and 3 in cycle 1 are illustrated. Concentrations of JX-594 genomes, including secondary peak levels of viremia, were dose related. LOQ: limit of quantification. The error bars represent standard error of the mean. (C) Representative concentrations of the JX-594 genomes in cycle 1. (D) Recovery of JX-594 and expression of hGM-CSF from a melanoma patient (cohort 3). High levels of JX-594 and GM-CSF genomes were detected in circulation, as well as malignant body fluids (cohort 3, melanoma patient). Asterisk: not detectable; PE: pleural effusion. (E) The presence of infectious JX-594 was demonstrated by expression of lac-Z (blue) from the cells in a malignant pleural effusion. (F) Sample for liver cancer metastasis biopsy that did not receive an injection (neck), illustrating the staining of the vaccinia virus with B5R (arrows; brown).

Figures 24A-24B represent anti-tumor efficacy. (A) Representative CT scans and tumor measurements of a non-small cell lung cancer tumor. Circles: tumors. Arrow: time when the administration of JX-594 started. Note the changes in the cross-sectional area of ​​the tumor over time. (B) Results of physical scans representative of CT and PET-CT, demonstrating the objective response of tumors (after 4 cycles) of metastatic tumor in the neck that received an injection, after inducing neutralizing antibodies with high titre for JX-594.

Detailed Description of the Invention

The present invention relates to the use of oncolytic poxviruses for the treatment of cancer. In particular, the use of a vaccinia virus that expresses GM-CSF to achieve a specific degree of oncolysis is described. In another embodiment, a poxvirus that expresses GM-CSF can be engineered to be more effective or more efficient in killing cancer cells and / or to be less toxic or damaging to non-cancer cells, by mutating or modifying gene products, in such a way that the changes make viruses more capable of infecting the host, less toxic to host cells, and / or more capable of infecting cancer cells. A specific modification is to make the virus deficient in thymidine kinase (TK) function.

I. Poxvirus

Poxviruses have been known for centuries, and the characteristic pustule marks produced by the smallpox virus give this family its name. It appears that smallpox first appeared in China and the Far East more than 2,000 years ago. Fortunately, this often fatal virus has now been eradicated, with the last outbreak in 1977 in Somalia.

The viral particle of the poxvirus is oval or brick-shaped, measuring approximately 200-400 nm in length. The outer surface is curled in parallel rows, sometimes arranged helically. The particles are extremely complex, containing more than 100 distinct proteins. Extracellular forms contain two membranes (EEV - extracellular enveloped virions), whereas intracellular particles have only one inner membrane (IMV - mature intracellular virions). The outer surface is composed of lipid and protein and surrounds the nucleus, which is made up of strongly compressed nucleoprotein. Antigenically, poxviruses are also very complex, inducing specific antibodies and also cross-reacting. There are at least ten enzymes present in the particle, mostly concerned with nucleic acid metabolism / genome replication.

The poxvirus genome is linear double-stranded DNA with 130-300 kbp. The ends of the genome have a terminal loop in the form of a clamp (hairpirí) with several repeated tandem sequences. Several poxvirus genomes have been sequenced, with most essential genes located in the central part of the genome, while non-essential genes are located at the ends. There are about 250 genes in the poxvirus genome.

Replication occurs in the cytoplasm, as the virus is complex enough to have acquired all the functions necessary for genome replication. There is some contribution by the cell, but the nature of this contribution is not clear. However, even though poxvirus gene expression and genome replication occur in anucleated cells, maturation is blocked, indicating some of the cell's role.

Receptors for poxviruses are not generally known, but there are likely to be multiple numbers and in different cell types. In the case of vaccinia, one of the likely receptors is the epidermal growth factor (EGF) receptor (McFadden, 2005). Penetration can also involve more than one mechanism. The stripping occurs in two stages: (a) removal of the outer membrane as the particle enters the cell and cytoplasm, and (b) the particle is further stripped and the nucleus passes into the cytoplasm.

Once inside the cell's cytoplasm, gene expression is driven by viral enzymes associated with the nucleus. Expression is divided into 2 phases: early genes: which represent about 50% of the genome, and are expressed before genome replication, and late genes, which are expressed after genome replication. Temporal expression control is provided by late promoters, which are dependent on DNA replication for activity. Genome replication is believed to involve self-conditioning, leading to the formation of high molecular weight concatamers, which are subsequently cleaved and repaired to produce viral genomes. Viral assembly occurs in the cytoskeleton and probably involves interactions with cytoskeletal proteins (for example, actin-binding proteins). Inclusions are formed in the cytoplasm, which mature to give viral particles. Cell-to-cell spread can provide an alternative mechanism for spreading the infection. In total, the replication of this large complex virus is somewhat rapid, taking only 12 hours on average.

At least nine different poxviruses cause disease in humans, but smallpox and vaccinia viruses are the best known. Smallpox strains are divided into major smallpox (25-30% fatalities) and minor smallpox (the same symptoms, but less than a 1% mortality rate). Infection with both viruses occurs naturally in the respiratory route and is systemic, producing a series of symptoms, but more noticeably pustules characteristic of smallpox and excoriation of the skin.

A. Vaccinia virus

The vaccinia virus is a large complex enveloped virus that has a linear double-stranded DNA genome of about 190 kbp and that encodes approximately 250 genes. Vaccinia is well known for its role as a vaccine that eradicated smallpox. After smallpox eradication, scientists have been exploring the use of vaccinia as a tool to distribute genes into biological tissues (gene therapy and gene engineering). The vaccinia virus is unique among DNA viruses because it replicates only in the cytoplasm of the host cell. Therefore, the large genome is necessary to encode various enzymes and proteins necessary for viral DNA replication. During replication, vaccinia produces several infectious forms that differ in its outer membranes: the mature intracellular virion (IMV), the intracellular enveloped virion (IEV), the enveloped virus associated with cells (CEV) and the extracellular enveloped virion (EEV). IMV is the most abundant infectious form and is believed to be responsible for spread between hosts. On the other hand, CEV is believed to play a role in cell-to-cell spread and EEV is believed to be important for far-reaching spread within the host organism.

Vaccinia encodes several proteins that give virus resistance to interferons. K3L is a protein that has homology to elF-2a. The K3L protein inhibits the action of PKR, an activator for interferons. E3L is another vaccinia protein that also inhibits PKR activation and is also capable of binding to double-stranded RNA.

The vaccinia virus is closely related to the virus that causes cow pox (cowpox). The precise origin of vaccinia is unknown, but the most common view is that the vaccinia virus, cowpox virus, and human smallpox virus (the causative agent of smallpox) were all derived from a common ancestral virus. It is also speculated that the vaccinia virus was originally isolated from horses. A vaccinia virus infection is mild and typically asymptomatic in healthy individuals, it can cause a mild skin rash and fever, with an extremely low fatality rate. An immune response generated against a vaccinia virus infection protects this person from a lethal smallpox infection. For this reason, the vaccinia virus was used as a live virus vaccine against smallpox. The vaccinia virus vaccine is safe because it does not contain smallpox virus, but occasionally complications and / or adverse effects of the vaccine may arise, especially if the vaccine is immunocompromised.

As discussed above, vaccinia viruses were engineered to express numerous foreign proteins. One of these proteins is the granulocyte and macrophage colony stimulating factor, or GM-CSF. GM-CSF is a protein secreted by macrophages, which stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and macrophages. Human GM-CSF is glycosylated at amino acid residues 23 (leucine), 27 (asparagine), and 39 (glutamic acid) (see US patent number 5,073,627, incorporated by reference). GM-CSF is also known as molgramostim or, when the protein is expressed in yeast cells, sargramostim (trademark: Leukine®), which is used as a medication to stimulate the production of leukocytes, especially granulocytes and macrophages, after chemotherapy. A vaccinia virus that expresses GM-CSF has been reported previously. However, it was not distributed as an oncolytic agent, but merely as a distribution vector for GM-CSF. Therefore, it was administered to patients at a dosage below that which can achieve significant oncolysis. In this invention, the use of a vaccinia virus that expresses GM-CSF is described, which, in some modalities, is administered in concentrations greater than 1 x 10⁸ plaque forming units (pfu) or particles.

B. Modified Poxviruses

Viruses are often inactivated, inhibited or cleared by immunomodulatory molecules such as interferons (-α, -β, -γ) and tumor necrosis factor-α (TNFa) (Moss, 1996). Immune tissues and cells infl16 host killers often secrete these molecules in response to viral infection. These molecules can have direct antiviral effects and / or indirect effects through the recruitment and / or activation of inflammatory cells and lymphocytes. Given the importance of these immunological clearance mechanisms, viruses have evolved to express gene products that inhibit the induction and / or function of these cytokines / chemokines and interferons. For example, the vaccinia virus (W, and some other poxviruses) encodes the secreted protein vCKBP (B29R) that binds and inhibits CC chemokines (for example, RANTES, eotaxin, ΜΙΡ-1-alpha) (Alcami et al, 1998). Some strains of W also express a secreted viral protein that binds and inactivates TNF (for example, Lister A53R) (Alcami et al, 1999). Most poxvirus strains have genes that encode secreted proteins that bind to and inhibit the function of interferons-α / β (eg, B18R) or interferon (B8R). vC12L is a protein that binds to IL-18, which prevents IL-18 from inducing IFNγ and activating NK cells / cytotoxic T cells.

Most research on poxvirus virulence has been carried out in mice. Many, but not all, of these proteins are active in mice (B18R, for example, is not). In situations where these proteins are active against murine versions of the target cytokine, deletion of these genes leads to reduced virulence and increased safety with W mutants with deletions or functional mutations in these genes. In addition, the inflammatory immune response to these mutants and the viral clearance of these mutants are often increased compared to the strain of the parental virus that expresses the inhibitory protein. For example, deletion of the T1 / 35kDa family of proteins secreted by the poxvirus (chemokine-binding / binding proteins) can lead to a marked increase in leukocyte infiltration into tissues infected by the virus (Graham et al., 1997). Deletion of the vC12L gene in W leads to reduced viral titers / toxicity after nasal administration in mice; in addition, the activity of NK cells and cytotoxic T lymphocytes is increased along with IFN-γ induction (Smith et al, 2000). Deletion of the myxoma virus T7 gene (capable of binding to IFN-γ and a wide range of chemokines) results in reduced virulence and significantly increased inflammation / tissue infiltration in a toxicity model (Upton et al., 1992 ; Mossman et al., 1996). Deletion of the myxoma virus M-T2 gene also resulted in reduced virulence in a rabbit model (Upton et al. 1991). The deletion of the anti-interferon-a / -p gene product, B18R, also leads to increased viral sensitivity to IFN-mediated clearance, reduced titers in normal tissues and reduced virulence (Symons et al., 1995; Colamonici et al., 1995; Alcami et al., 2000). In short, these viral gene products work to decrease the antiviral immune response and the infiltration of inflammatory cells into tissues infected by the virus. The loss of protein function through deletion / mutation leads to decreased virulence and / or increased pro-inflammatory properties of the virus within host tissues.

Cytokines and chemokines can have potent anti-tumor effects (Vicari et al., 2002; Homey et al., 2002). These effects can be on the tumor cells themselves directly (for example, TNF) or they can be indirect through effects on non-cancer cells. An example of the latter is TNF, which can have antitumor effects by causing toxicity to blood vessels associated with tumors; this leads to a loss of blood flow to the tumor after tumor necrosis. In addition, chemokines can act to recruit (and in some cases, activate) immune effector cells such as neutrophils, eosinophils, macrophages and / or lymphocytes. These immune effector cells can cause the destruction of tumors by countless mechanisms. These mechanisms include the expression of antitumor cytokines (eg TNF), expression of fas ligand, expression of perforin and granzyme, recruitment of natural killer cells, etc. The inflammatory response may eventually lead to the induction of tumor-specific systemic immunity. Finally, many of these cytokines (eg, TNF) or chemokines can act synergistically with chemotherapy or radiation therapy to destroy tumors.

The clinically effective systemic administration of recombinant versions of these immunostimulating proteins is not feasible due to (1) severe toxicity induction with systemic administration, and (2) local expression within the tumor tissue is necessary to stimulate local infiltration and antitumor effects. Approaches are needed to achieve high local concentrations of these molecules within tumor masses, while minimizing levels in the systemic circulation. Viruses can be engineered to express genes for cytokines or chemokines in an attempt to enhance their effectiveness. Expression of these genes from selective replication vectors has potential advantages over expression from non-replicating vectors. Expression from replicating viruses can result in higher local concentrations within tumor masses; in addition, replicator viruses help induce anti-tumor immunity through cell destruction / oncolysis and the release of tumor antigens in a pro-inflammatory environment. However, there are several limitations to this approach. Serious safety concerns arise from the potential for release within the environment of a competent replicating virus (albeit tumor-selective) with a gene that can be toxic if expressed in high local concentrations. Viruses that express potent pro-inflammatory genes from their genome may therefore present safety risks for the treated patient and the general public. Even with selective replication viruses that target the tumor, which express these genes, gene expression can occur in normal tissues, resulting in toxicity. In addition, size limitations prevent the expression of multiple genes and / or large genes from viruses such as adenovirus; these molecules will definitely work more effectively in combination. Finally, many of the oncolytic viruses in use express anti-inflammatory proteins and, therefore, these viruses will counteract the induction of a pro-inflammatory medium within the infected tumor mass. The result will be the inhibition of the induction of antitumor immunity, antivascular effects and sensitization to chemotherapy / radiotherapy.

C. Modified Vaccinia Virus

1. Interferon Modulating Polypeptides Interferon-a / -p blocks viral replication through various mechanisms. Interferon-γ has weaker direct viral inhibitory effects, but it is a potent inducer of cell-mediated immunity through various mechanisms. Viruses have evolved to express secreted gene products that are able to counter the antiviral effects of interferons. For example, the vaccinia virus (and other poxviruses) encodes the secreted proteins B8R and B18R that bind to interferon-γ and -α / -β, respectively (Smith et al., 1997; Symons et al., 1995; Alcami et al., 2000). An example of a vaccinia gene product that reduces interferon induction is the caspase-1 inhibitor, B13R, which inhibits the activation of the interferon-γ-inducing factor, IL-18. The interferon-modulating polypeptides include, but are not limited to, B18R, which can be called B19R in other viral strains, such as the Copenhagen strain of the vaccinia virus; B8R; B13R; vC12L; A53R; E3L and other viral polypeptides with similar activities or properties. IFN modulating polypeptides can be divided into non-exclusive categories of those that preferentially modulate IFNa and / or β pathways (such as B18R, B8R, B13R, or vC12L) and those that modulate IFNy pathways (for example B8R.B13R , or vC12L).

Cancer cells are often resistant to the effects of interferons. Numerous mechanisms are involved. They include the fact that activation of signal transduction pathways (for example, by ras mutation, upstream of growth factor receptor overexpression / mutation, etc.), a common feature of cancer cells, leads to inhibition of PKR. In addition, lymphocytes are often inhibited in tumor masses by a number of mechanisms, including production of IL-10 and expression of fas-L by tumor cells. Since lymphocytes are an important source of interferon-γ production, inhibition of lymphocytes leads to a decrease in the production of interferon-γ in tumors. Therefore, tumor masses tend to be sanctuaries due to the effects of interferons. In addition, interferons themselves can have anti-tumor effects. For example, IFN-γ can increase the presentation of MHC class I-associated antigens; this will allow for more efficient CTL-mediated extermination of tumor cells. IFN-α / β, for example, can block angiogenesis within tumor masses and thus block the growth of tumors.

2. Complement Control Polypeptides

An important mechanism for the clearance of viral pathogens is the extermination of infected cells within the host or of virions within an organism by complement-dependent mechanisms. As the infected cell dies, it is unable to continue producing infectious viruses. In addition, during apoptosis, intracellular enzymes are released that degrade DNA. These enzymes can lead to the degradation of viral DNA and inactivation of the virus. Apoptosis can be induced by numerous mechanisms, including binding of activated complement and complement membrane attack complex. Poxviruses, such as vaccinia, have evolved to express gene products that are able to counter complement-mediated virus clearance and / or virus-infected cells. These genes thus prevent apoptosis and inhibit viral clearance by complement-dependent mechanisms, thus allowing viral infection to continue and viral virulence to be increased. For example, vaccine virus complement control proteins (VCP; for example, C21L) play roles in preventing complement-mediated cell extermination and / or inactivating the virus (Isaacs et a!., 1992). VCP also has anti-inflammatory effects, as its expression decreases the infiltration of leukocytes into tissues infected with viruses. Complement control polypeptides include, but are not limited to, VCP, also known as C3L or C21L.

Cancer cells often overexpress cellular anti-complement proteins; this allows cancer cells to survive the onslaught of supplements. Therefore, agents that preferentially target tumor cells due to their inherent resistance to complement-mediated extermination would have selectivity and potential efficacy in a wide range of human cancers (Durrant et al., 2001). In addition, one of the characteristics of cancer cells is a loss of normal apoptotic mechanisms (Gross et al., 1999). Resistance to apoptosis promotes carcinogenesis as well as resistance to antitumor agents, including immunological, chemotherapeutic and radiotherapy agents (Eliopoulos et al, 1995). Inhibition of apoptosis can be mediated by a loss of function of pro-apoptotic molecules (eg, bax), an increase in the levels / function of anti-apoptotic molecules (eg, bc1-2) and finally a loss of sensitivity to add-ons.

3. TNF Modulating Polypeptides

One of several mechanisms for the clearance of viral pathogens is the extermination of infected cells within the host by inducing apoptosis, as described above. Apoptosis can be induced by a number of mechanisms, including the binding of TNF and lymphotoxin-alpha (LTa) to TNF cell receptors, which triggers intracellular signaling cascades. Activation of TNF receptors works in regulating immune and inflammatory responses, as well as inducing apoptotic cell death (Wallach et al, 1999).

Several strains of poxvirus, including some strains of the vaccinia virus, have evolved to express gene products that are able to counteract TNF-mediated virus clearance and / or cells infected with the virus. The proteins encoded by these genes prevent pro-inflammatory and apoptosis-inducing TNF activities by extracellular TNF binding and sequestration, resulting in inhibition of viral clearance. Due to the fact that the viruses are not cleared, the viral infection is allowed to proceed, and therefore, the viral virulence is increased. Several members of the poxvirus family express secreted viral TNF receptors (vTNFR). For example, several poxviruses encode vTNFRs, such as myxoma (T2 protein), cow pox strains, and vaccinia viruses, such as Lister, can encode one or more of the CrmB, CrmC (A53R), CrmD, CrmE, B28R proteins and / or its equivalents. These vTNFRs have roles in preventing TNF-mediated cell extermination and / or virus inactivation (Saraiva and Alcami, 2001). TNF-modulating polypeptides include, but are not limited to, A53R, B28R (this protein is present, but may be inactive in the Copenhagen strain of the vaccinia virus) and other polypeptides with similar activities or properties.

One of the characteristics of cancer cells is the aberrant expression of genes, which can lead to a loss of sensitivity to numerous molecular mechanisms for modulating growth, such as sensitivity to anti-cancer activities of TNF. Therefore, viral immunomodulatory mechanisms may not be necessary for the spread of a virus within the tumor microenvironment.

4, Serina Protease Inhibitors

An important mechanism for the clearance of viral pathogens is the induction of apoptosis in infected cells within the host. As the cell dies, it is unable to continue producing infectious viruses. In addition, during apoptosis, intracellular enzymes are released that degrade DNA. These enzymes can lead to the degradation of viral DNA and inactivation of the virus. Apoptosis can be induced by a number of mechanisms, including cytokine binding (eg, tumor necrosis factor), granzyme production by cytotoxic T lymphocytes or fas ligand binding; caspase activation is a critical part of the final common apoptosis pathway. Viruses have evolved to express gene products that are able to counter the intracellular signaling cascade induced by these molecules, including tumor ligand or molecules related to tumor necrosis factor (TNF) (eg, E3 10.4 / 14.5, adenovirus 14.7 genes (Wold et al., 1994); adenovirus E1B-19kD (Boyd et al., 1994); cowpox virus crmA; vaccinia virus B13R) (Dobbelstein et al., 1996; Kettle et al., 1997) ). These gene products prevent apoptosis by apoptosis-inducing molecules, and thus, allow viral replication to proceed despite the presence of antiviral apoptosis-inducing cytokines, fas, granzyme or other apoptosis stimulants.

W SPI-2 / B13R is highly homologous to cow's smallpox CrmA; SPI-1 (W) is weakly homologous to CrmA (Dobbelstein et al., 1996). These proteins are serpins (inhibitors of serine proteases) and CrmA and also SPI-2 have roles in the prevention of various forms of apoptosis. Inhibition of the interleukin-ΐβ-converting enzyme (ICE) and granzyme, for example, can prevent apoptosis of the infected cell. These gene products also have anti-inflammatory effects. They are able to inhibit IL-18 activation, which in turn would decrease IL-18-mediated IFN-γ induction. The immunostimulatory effects of IFN-γ on cell-mediated immunity are thus inhibited (Kettle et al., 1997). SPis include, but are not limited to, B13R, B22R, and other polypeptides with similar activities or properties.

One of the characteristics of cancer cells is a loss of normal apoptotic mechanisms (Gross et al., 1999). Resistance to apoptosis promotes carcinogenesis as well as resistance to antitumor agents, including immunological, chemotherapeutic and radiotherapy agents (Eliopoulos et al., 1995). Inhibition of apoptosis can be mediated by a loss of pro-apoptotic molecule function (eg, bax) or an increase in the levels / function of anti-apoptotic molecules (eg, bcl-2).

5. IL-18 Modulating Polypeptides

IL-Ιβ is a biologically active factor that acts locally and also systemically. Only a few functional differences between IL-1 β and IL-1a have been described. The numerous biological activities of IL-Ιβ are exemplified by the many different acronyms under which IL-1 has been described. IL-1 has no species specificity with the exception of human IL-Ιβ, which is inactive in porcine cells. Some of the biological activities of IL-1 are mediated indirectly by inducing the synthesis of other mediators, including ACTH (corticotropin), PGE2 (prostaglandin E2), PF4 (platelet factor 4), CSF (colony stimulating factors), IL-6, and IL8. The synthesis of IL-1 can be induced by other cytokines including TNF-a, IFN-a, IFN-β and IFN-γ and also bacterial endotoxins, viruses, mitogens, and antigens. The main biological activity of IL-1 is the stimulation of helper T cells, which are induced to secrete IL-2 and to express IL-2 receptors. Macrophages infected with the virus produce large amounts of an IL-1 inhibitor that can support opportunistic infections and cell transformation in patients with defects in T cell maturation. IL-1 acts directly on B cells, promoting their proliferation and synthesis immunoglobulins. IL-1 also functions as one of the factors of osmotic conditioning that makes B cells responsive to IL-5. IL-1 stimulates the proliferation and activation of NK cells and fibroblasts, thymocytes, glioblastoma cells.

Blocking the synthesis of IL-Ιβ by the viral protein is seen as a viral strategy that allows systemic antiviral reactions elicited by IL-1 to be suppressed or decreased. Binding proteins that effectively block IL-1 functions with B1 5R-like activity have also been shown to be encoded by cowpox virus genes. The vaccinia virus also encodes another protein, called B8R, that behaves as a receptor for cytokines (Alcami and Smith, 1992; Spriggs et al., 1992). The IL-1 modulating polypeptides, however without limitations, B13R, B15R, and other polypeptides with similar activities or properties.

One of the characteristics of cancer cells is the aberrant expression of genes, which can lead to a loss of sensitivity to numerous molecular mechanisms for growth modulation, such as a sensitivity to IL-1 anticancer activities. Therefore, viral immunomodulatory mechanisms may not be necessary for the spread of a virus within the tumor microenvironment.

6. Form EEV

Viral spread to metastatic tumor sites, and even spread within an infected solid tumor mass, is generally inefficient (Heise et al, 1999). Intravenous administration typically results in viral clearance or inactivation by antibodies (eg, adenovirus) (Kay et al, 1997) and / or the complement system (eg, HSV) (Ikeda et al, 1999). In addition to these immunomediated mechanisms, the biodistribution of these viruses results in the vast majority of intravenous viruses that deposit within normal tissues rather than tumor masses. Intravenous adenovirus, for example, ends mainly within the liver and spleen; less than 0.1% of the incoming virus deposits within tumors, even in immunodeficient mice (Heise et al, 1999). Therefore, although moderate efficacy can be demonstrated with extremely high relative doses in models of immunodeficient mouse tumors, intravenous delivery is extremely inefficient and significantly limits efficacy.

The vaccinia virus has the ability to replicate within solid tumors and cause necrosis. In addition, thymidine kinase deletion mutants can infect tumor masses and ovarian tissue and express marker genes preferentially in mouse tumor model systems (Gnant et al., 1999). However, as these studies have generally determined tumor tumor based on the expression of the marker gene after 5 days, it is uncertain whether the virus preferentially deposits, expresses genes or replicates in tumor / ovarian tissue (Puhlmann et al., 2000). Regardless of the mechanism, the anti-tumor efficacy of this virus without additional transgenes was not statistically significant (Gnant et al., 1999). In contrast, intratumoral virus injection had significant antitumor efficacy (McCart et al. 2000). Therefore, intravenous efficacy could be improved if the intravenous delivery to the tumor could be improved.

The vaccinia virus replicates in cells and produces intracellular virus (IMV, mature intracellular virus; IEV, intracellular enveloped virus) and extracellular virus (REV, extracellular enveloped virus; CEV, cell-associated extracellular virus) (Smith et al., 1998) . IMV represents approximately 99% of the virus yield after replication by strains of the wild type vaccinia virus. This form of the virus is relatively stable in the environment, and therefore, it is mainly responsible for the spread between individuals; in contrast, this virus does not spread efficiently within the infected host due to inefficient release from cells and sensitivity to complement and / or neutralization of the antibody. In contrast, EEV is released into the extracellular medium and typically represents only approximately 1% of the viral yield (Smith et al., 1998). EEV is responsible for viral spread within the infected host and is degraded relatively easily outside the host. The important thing is that EEV has developed several mechanisms to inhibit its neutralization within the bloodstream. First, EEV is relatively resistant to complement (Vanderplasschen et al., 1998); this characteristic is due to the incorporation of complement inhibitors in host cells within its outer membrane lining plus the secretion of the vaccinia virus complement control protein (PCV) within the local extracellular environment. Second, EEV is relatively resistant to antibody neutralizing effects compared to IMV (Smith et al., 1997). EEV is also released at earlier points in time after infection (for example, 4-6 hours) than IMV (which is only released during / after cell death), and therefore the spread of the EEV form is more (Blasco et al., 1993).

Unfortunately, however, strains of wild-type vaccinia produce only very small amounts of EEV, relatively. In addition, treatment with vaccinia virus (ie, the dose of the virus inlet) has been limited to the forms of the intracellular virus until now. Standard vaccine (W) virus manufacturing and purification procedures lead to inactivation of EEV (Smith et al., 1998), and non-human cell lines are often used to manufacture the virus; EEV from non-human cells will not be protected against complement-mediated clearance (complement inhibitory proteins acquired from the cell by EEV have restricted effects on species). The effectiveness of the vaccinia virus was, therefore, limited by the relative sensitivity of the IMV form to neutralization and by its inefficient dissemination within solid tumor masses; this spread is typically from the cell to the adjacent cell. The spread of IMV to distant tumor masses, either through the bloodstream or through the lymphatic system, is also inefficient.

Therefore, the rare EEV form of the vaccinia virus has naturally acquired characteristics that make it better than the form of the vaccinia virus used in patients so far (IMV); EEV is optimized for rapid and efficient spread through solid tumors locally and to regional or distant tumor sites. As EEV is relatively resistant to the effects of complement, when it is developed in a cell type of the same species, this form of the virus will have greater stability and will retain activity for a longer time in the chain after intravascular administration than standard preparations of the vaccinia virus (which contains only IMV) (Smith et al., 1998). Since EEV is resistant to antibody-mediated neutralization, this form of the virus will retain activity for a longer time in the chain after intravascular administration than standard vaccinia virus preparations (which contain almost exclusively IMV) (Vanderplasschen et al, 1998) . This feature will be particularly important for repeated administration after the neutralizing antibody levels have increased; all approved anti-cancer therapies require repeated administration. Therefore, the EEV form of vaccinia, and other poxviruses, will result in a better distribution of therapeutic viruses and their genetic payload for tumors through the bloodstream. This will lead to greater systemic efficacy compared to standardized poxvirus preparations. Finally, the risk of transmission to individuals in the audience in general should be reduced significantly, as EEV is extremely unstable outside the body. The polypeptides involved in modulating the EEV form of a virus include, but are not limited to, A34R, B5R, and several other proteins that influence the production of the EEV form of poxviruses. A mutation in A34R codon 151 of a lysine for aspartic acid (K151D mutations) makes the A34R protein less able to tie the EEV form to the cell membrane. B5R is a polypeptide attached to the EEV membrane that can bind to the complement. The total deletion of A43R may lead to greater EEV release, but markedly reduced infectivity of viruses, while the K151D mutation increases the release of EEV, while maintaining the infectivity of released viruses. B5R has sequence homology with VCP (anti-complement), but complement inhibition has not yet been proven.

Briefly, a method for identifying a fortified EEV form is as follows. The EEVs are diluted in frozen MEM and mixed (1: 1 by volume) with heat activated or inactivated serum (56 ° C, 30 min, control) diluted in frozen MEM (final serum dilution 1/10, 1/20, or 1/30). After incubation or 75 min at 7 ° C, the samples are cooled on ice, and mAb 5B4 / 2F2 is added to fresh EEV samples to neutralize any contaminants (IMV and broken EEV). The virions are then bound to RK13 cells for an hour on ice, the complement and unbound virions are washed away, and the number of plaques is counted two days later. The greater the number of plaques, the greater the resistance to complement (Vanderplasschen et al., 1998, incorporated here as a reference). Exemplary methods that describe the isolation of EEV forms from the vaccinia virus can be found in Blasco et al, 1992 (incorporated herein by reference).

7. Other Polypeptides

Other viral immunomodulatory polypeptides may include polypeptides that bind to other mediators of the immune response and / or modulate molecular pathways associated with the immune response. For example, chemokine-binding polypeptides such as B29R (this protein is present, but can be inactive in the Copenhagen strain of vaccinia virus), C23L, vCKBP, A41L and polypeptides with similar activities or properties. Other vaccinia virus proteins such as the vaccinia virus growth factor (e.g., C11L), which is a growth factor similar to viral EGF, may also be the target for change in some embodiments of the invention. Other polypeptides that can be classified as viral immunomodulatory factors include, but are not limited to B7R, N1L, or other polypeptides whose activities or properties increase the virulence of a poxvirus.

8. Vaccine Virus-Induced Cell Fusion

In certain embodiments of the invention, an alteration, deletion, or mutation of A56R or K2L, which encodes nucleic genes can lead to cell-cell fusion or Syncya formation induced by W infection. Cellular fusion induced by the vaccinia virus will typically increase the antitumor efficacy of W due to intratumoral viral spread. Intratumoral viral spread by cell fusion will typically allow the virus to avoid neutralizing antibodies and immune responses. The extermination and infection of adjacent uninfected cells (ie, a spectator effect) may be more efficient in W with mutations in one or both of these genes, which may result in better local anti-tumor effects.

D. Other Poxviruses

The vaccinia virus is a member of the family Poxviridae, the subfamily Chordopoxvirinae and the genus Orthopoxvirus. The genus Orthopoxvirus is relatively more homogeneous than other members of the subfamily Chordopoxvirinae and includes 11 distinct but closely related species, which include the species of vaccinia virus, smallpox virus (smallpox causative agent), cow pox virus, virus buffalo pox virus, monkey pox virus, mouse pox virus and horse pox virus, as well as others (see Moss, 1996). Certain embodiments of the invention, as described herein, can be extended to other members of the Orthopoxvirus genus, as well as the genus Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, and Yatapoxvirus. A genus of the poxvirus family is generally defined by serological means, including netralization and cross-reactivity in guinea pigs. Several members of the Orthopoxvirus genus, as well as other members of the subfamily Chordovirinae use immunomodulatory molecules, examples of which are provided here, to counteract the immune responses of a host organism. Accordingly, the invention described herein is not limited to the vaccinia virus, but may be applicable to a number of viruses.

E. Spread of Viruses

The vaccinia virus can be propagated using the methods described by Earl and Moss in Ausubel et al., 1994, which is incorporated herein by reference.

II. Protein and Nucleic Acid Compositions

The present invention relates to poxviruses, including those constructed with one or more mutations in comparison to the wild type, such that the virus has desirable properties for use against cancer cells, and at the same time, being less toxic or nontoxic to non-cancer cells. The teachings described below provide various protocols, by way of example, for implementing the methods and compositions of the invention, such as methods for generating mutated viruses through the use of recombinant DNA technology.

In certain embodiments, the present invention relates to the generation of poxviruses that lack one or more polypeptides or functional proteins and / or generation of poxviruses that have the ability to release more than one specific form of the virus, such as an infectious EEV form . In other embodiments, the present invention relates to poxviruses and their use in combination with a proteinaceous composition as part of a pharmaceutically acceptable formulation.

As used herein, the term protein or polypeptide refers to a molecule that comprises at least one amino acid residue. In some embodiments, a wild-type version of a protein or polypeptide is employed; however, in many embodiments of the invention, a viral protein or polypeptide is absent or altered, in order to make the virus more useful for treating cancer cells or cancer in a patient. The terms described above can be used interchangeably here. A modified protein or modified polypeptide refers to a protein or polypeptide whose chemical structure is altered in relation to the wild-type protein or polypeptide. In some embodiments, a modified protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides can have multiple activities or functions). The modified activity or function can be reduced, decreased, eliminated, enhanced, improved, or otherwise altered (such as specificity) in relation to that activity or function in a wild-type protein or polypeptide. It is specifically contemplated that a modified protein or polypeptide can be altered in relation to an activity or function will still retain wild-type activity or function in other respects. Alternatively, a modified protein may be completely non-functional or its cognate nucleic acid sequence may have been altered in such a way that the polypeptide is no longer expressed in any way, is truncated, or expresses a different amino acid sequence as a result of an offset of the reading frame (frameshift).

In certain embodiments, the size of a mutated protein or polypeptide may comprise, but are not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,

32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180,

190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450,

475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825,

850, 875, 900, 925, 950, 975, 1.000, 1.100, 1.200, 1.300, 1.400, 1.500,

1,750, 2,000, 2,250, 2,500 or more residues of molecules of amino molecules, and any range derivable from them. It is contemplated that polypeptides can be mutated by truncation, making them shorter than their corresponding wild-type shape.

As used herein, the term amino molecule refers to any amino acid, derived from amino acid or mimetic of amino acid as would be known to those skilled in the art. In certain embodiments, the residues of the proteinaceous molecule are sequential, without any non-amino molecules interrupting the sequence of residues of amino molecules. In other embodiments, the sequence may comprise one or more groups of non-amino molecules. In specific embodiments, the residue sequence of the proteinaceous molecule can be interrupted by one or more groups of non-amino molecules.

Consequently, the term proteinaceous composition encompasses sequences of amino molecules that comprise at least one of the 20 amino acids common in naturally synthesized proteins, or at least one modified or unusual amino acid.

Proteinaceous compositions can be manufactured by any technique known to those skilled in the art, including the expression of proteins, polypeptides or peptides through molecular biology techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials. The sequences of nucleotides and proteins, polypeptides and peptides for various genes have been described previously, and can be found in computerized databases known to those skilled in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (www.ncbi.nlm.nih.gov/). The coding regions for these known genes can be amplified and / or expressed using the techniques described herein or as would be known to those skilled in the art.

A. Functional Aspects

When the present patent application relates to the function or activity of viral proteins or polypeptides, this refers to the activity or function of this viral protein or polypeptide under physiological conditions, unless otherwise specified. For example, an interferon-modulating polypeptide refers to a polypeptide that affects at least one interferon and its activity, either directly or indirectly. The polypeptide can induce, intensify, elevate, decrease, weaken, reduce, inhibit or mask the activity of an interferon, directly or indirectly. An example of directly affecting interferon involves, in some embodiments, an interferon-modulating polypeptide that specifically binds to interferon. The determination of which molecules have this activity can be carried out using assays known to those skilled in the art. For example, transfer of genes that encode products that modulate interferon, or its variants, into cells that are induced for interferon activity compared to cells with this gene transfer can identify, due to different levels of a interferon, those molecules that have an interferon modulating function.

It is specifically contemplated that a modulator may be a molecule that affects the expression of proteinaceous compositions involved in the pathway of the assayed molecule, such as ligating a transcript encoding interferon. The determination of which molecules are appropriate modulators of interferon, IL-1 β, TNF, or other molecules of therapeutic benefit can be carried out using assays known to those skilled in the art - some of which are described herein - and may include, for example, use of native and / or recombinant viral proteins.

B. Variants of Viral Polypeptides

Variants of amino acid sequences of the polypeptides of the present invention can be variants by substitution, insertion, or deletion. A mutation in a gene encoding a viral polypeptide can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more non-contiguous or contiguous polypeptide amino acids compared to the wild type . Various polypeptides encoded by the vaccinia virus can be identified with reference to Rosei et al, 1986, Goebel et al, 1990 and GenBank Accession Number NC001559, each of which is incorporated herein by reference.

Deletion variants lack one or more residues of the native or wild-type protein. Individual residues can be deleted or all or part of a domain (such as a catalytic or linker domain) can be deleted. A wall codon can be inserted (by substitution or insertion) into a nucleic acid sequence encoding to generate a truncated protein. Insertion mutants typically involve adding material at a non-terminal point on the polypeptide. This may include the insertion of an immunoreactive epitope or simply one or more residues. Terminal additions, called fusion proteins, can also be generated.

Substitution variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, that is, an amino acid is replaced by one with a similar shape and charge. Conservative substitutions are well known to those skilled in the art and include, for example, changes in; alanine to serine; arginine for lysine; asparagine for glutamine or histidine; aspartate for glutamate; cysteine ​​to serine; glutamine for asparagine; glutamate for aspartate; glycine for proline; histidine for asparagine or glutamine;

isoleucine for leucine or valine; leucine to valine or isoleucine; lysine for arginine; methionine for leucine or isoleucine; phenylalanine for tyrosine, leucine or methionine; serine to threonine; threonine to serine; tyrosine tryptophan; tyrosine for tryptophan or phenylalanine; and valine for isoleucine or leucine. Alternatively, substitutions can be non-conservative, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing a residue with one that is chemically dissimilar, such as a polar or charged amino acid with a supporting or uncharged amino acid, and vice versa.

The term functionally equivalent codon is used here to refer to codons that encode the same amino acid (see Table 1 below).

Table 1: Codon Table

Codon Amino Acids

<td>Alanine</td><td>Allah</td><td>THE</td><td>GCA</td><td>GCC</td><td>GCG</td><td>GCU</td>

<td>Cysteine</td><td>Cys</td><td>Ç</td><td>UGC</td><td>UGU</td><td></td><td></td>

<td>Aspartic acid</td><td>Asp</td><td>D</td><td>GAC</td><td>GAU</td><td></td><td></td>

<td>Glutamic acid</td><td>Glu</td><td>AND</td><td>GAA</td><td>GAG</td><td></td><td></td>

<td>Phenylalanine</td><td>Phe</td><td>F</td><td>UUC</td><td>UUU</td><td></td><td></td>

<td>Glycine</td><td>Gly</td><td>G</td><td>GGA</td><td>GGC</td><td>GGG</td><td>GGU</td>

<td>Histidine</td><td>His</td><td>H</td><td>CAC</td><td>CAU</td><td></td><td></td>

<td>Isoleucine</td><td>Ile</td><td>I</td><td>AUA</td><td>AUC</td><td>AUU</td><td></td>

<td>Lysine</td><td>Lys</td><td>K</td><td>AAA</td><td>THE AG</td><td></td><td></td>

<td>Leucine</td><td>Read</td><td>L</td><td>UUA</td><td>UUG</td><td>AUC</td><td>CUC CUG</td>

<td>Methionine</td><td>Met</td><td>M</td><td>AUG</td><td></td><td></td><td></td>

<td>Asparagine</td><td>Asn</td><td>N</td><td>AAC</td><td>AAU</td><td></td><td></td>

<td>Proline</td><td>Pro</td><td>P</td><td>CCA</td><td>CCC</td><td>CCG</td><td>CCU</td>

<td>Glutamine</td><td>Gin</td><td>Q</td><td>CAA</td><td>CAG</td><td></td><td></td>

Codon Amino Acids

<td rowspan="2">Arginine Serina T reonina Valina T riptofano</td><td rowspan="2">Arg To be Thr Val Trp</td><td rowspan="2">R s T V w</td><td rowspan="2">AGA AGC AGU A CA GUA UGG</td><td rowspan="2">AGG UCA ACC GUC</td><td colspan="2">CGA CGC</td><td rowspan="2">CGG UCU</td>

<td>UCC ACG GUG</td><td>UCG ACU GUU</td>

<td>Tyrosine</td><td>Tyr</td><td>Y</td><td>YOU ACCEPTED</td><td>WOW</td><td></td><td></td><td></td>

It should also be understood that amino acid and nucleic acid sequences can include additional residues, such as amino acids at the N or C terminus or additional 5 'or 3' sequences, and yet are essentially as stated in one of the sequences described herein, provided that the sequence meets the criteria stated above, including the maintenance of the biological activity of the protein, where the expression of the protein is concerned. The addition of terminal sequences applies particularly to nucleic acid sequences which may, for example, include several non-coding sequences flanking the 5 'or 3' parts of the coding region or may include several internal sequences, that is, introns, which are recognized occur within genes.

The following text is a discussion based on exchanging the amino acids of a protein to create an equivalent, or even improved, second-generation molecule. For example, certain amino acids can be replaced by other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antibody antigen binding regions or binding sites on substrate molecules. As it is the interactive capacity and nature of a protein that define this functional biological activity of the protein, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and yet produce a protein with similar properties. Thus, it is contemplated that several changes can be made to the DNA sequences of the genes without appreciable loss of their usefulness or biological activity, as discussed below. Table 1 indicates the codons that encode specific amino acids.

When making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic index of amino acids in conferring an interactive biological function to a protein is generally understood in these techniques (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the protein's interaction with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like .

It is also understood in these techniques that the replacement of similar amino acids can be done effectively on the basis of hydrophilic character. US patent number 4,554,101, incorporated herein by reference, states that the greater the average local hydrophilic character of a protein, as dictated by the hydrophilic character of its adjacent amino acids, correlates with a biological property of the protein. As detailed in US patent number 4,554,101, the following hydrophilic values ​​have been designated for amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+ 3.0 ± 1); glutamate (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); Alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

It should be understood that an amino acid can be replaced by another that has a similar hydrophilic character and still produce a biologically equivalent and immunologically equivalent protein. In these changes, substitution of amino acids whose hydrophilic character values ​​are within ± 2 is preferred, those which are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, for example, their hydrophobic character, hydrophilic character, charge, size, and the like. Exemplary substitutions that take into account the various preceding characteristics are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

III. Nucleic Acid Molecules

A. Polynucleotides That Encode Native Proteins or Modified Proteins

The present invention relates to polynucleotides, isolable from cells, which are capable of expressing all or part of a protein or polypeptide. In some embodiments of the invention, it refers to a viral genome that has been specifically mutated to generate a virus that lacks certain functional viral polypeptides. Polynucleotides can encode a peptide or polypeptide that contains all or part of a viral amino acid sequence or they can be engineered in such a way that they do not encode that viral polypeptide or encode a viral polypeptide that has at least one reduced function or activity, decreased or absent. Recombinant proteins can be purified from cells in expression to produce active proteins. The genome, as well as the definition of the coding regions for the vaccinia virus can be found in Rosei et al., 1986; Goebel et al., 1990; and / or GenBank Accession Number NC_001559, each of which is incorporated herein by reference.

As used herein, the term DNA segment refers to a DNA molecule that has been isolated free of the total genomic DNA of a specific species. Therefore, a segment of DNA that encodes a polypeptide refers to a segment of DNA that contains wild-type, polymorphic or mutant sequences that encode the polypeptide is still isolated, or purified from the total mammalian or human genomic DNA. The term DNA segment includes a polypeptide or polypeptides, DNA segments smaller than a polypeptide, and recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like.

As used in this patent application, the term poxvirus polynucleotide refers to a nucleic acid molecule that encodes a poxvirus polypeptide that has been isolated free of the total genomic nucleic acid. Similarly, a vaccinia virus polynucleotide refers to a nucleic acid molecule that encodes a vaccinia virus polypeptide that has been isolated free of the total genomic nucleic acid. A poxvirus genome or a vaccinia virus genome refers to a nucleic acid molecule that can be supplied to a host cell to produce a viral particle, in the presence or absence of a helper virus. The genome may or may not have been mutated recombinantly compared to the wild-type virus.

The term CDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as a model. The advantage of using a CDNA, instead of genomic DNA or DNA polymerized from a genomic model, unprocessed or partially processed, is that the CDNA contains mainly sequences coding for the corresponding protein. There are occasions when the complete or partial genomic sequence is preferred, such as when noncoding regions are needed for optimal expression or when noncoding regions such as introns must be targeted in an antisense strategy.

It is also contemplated that a specific polypeptide of a given species can be represented by natural variants that have slightly different nucleic acid sequences, but nevertheless, encode the same protein (see Table 1 above).

Similarly, a polynucleotide comprising an isolated or purified or mutant wild-type polypeptide gene refers to a segment of DNA, which includes sequences encoding wild-type or mutant polypeptides and, in certain respects, substantially free, isolated regulatory sequences other naturally occurring genes or protein coding sequences. In this regard, the term gene is used for simplification to refer to a protein, polypeptide, or unit encoding the peptide, functional (including any sequences necessary for transcription, post-translational modification, or appropriate localization). As should be understood by those skilled in the art, this functional term includes genomic sequences, CDNA sequences, and smaller engineered gene segments that express, or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a native or modified polypeptide can contain a sequence of contiguous nucleic acids that encodes all or part of that polypeptide with the following lengths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,

390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520,

530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,

680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,

830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,

980, 990, 1,000, 1,010, 1,020, 1,030, 1,040, 1,050, 1,060, 1,070, 1,080, 1,090, 1,095, 1,100, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 9,000, 10,000, or more nucleotides, nucleosides, or base pairs.

In specific embodiments, the invention relates to isolated DNA segments and recombinant vectors that incorporate DNA sequences that encode a polypeptide or poxvirus peptide of the wild type or mutant that includes within its amino acid sequence a contiguous amino acid sequence according to with or essentially corresponding to a native polypeptide. Therefore, a DNA segment or vector that contains a DNA segment can encode, for example, an INF modulator or TNF modulator polypeptide that can inhibit or reduce INF activity. The term recombinant can be used in conjunction with a polypeptide or with the name of a specific polypeptide, and it generally refers to a polypeptide produced from a nucleic acid molecule that has been engineered in vitro or that is the replicated product of that molecule.

In other embodiments, the invention relates to isolated DNA segments and recombinant vectors that incorporate DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence according to or essentially corresponding to the polypeptide .

The nucleic acid segments used in the present invention, regardless of the length of the coding sequence itself, can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, others coding segments, and the like, so that their total length can vary considerably. It is, therefore, contemplated that a nucleic acid fragment of almost any length can be employed, the total length being preferably limited by ease of preparation and use in the intended recombinant DNA protocol.

It is contemplated that the nucleic acid constructs of the present invention can encode full length polypeptide from any source or encode a truncated version of the polypeptide, for example, a truncated vaccinia virus tripeptide, such that the transcript of the region encoder represents the truncated version. The truncated transcript can then be translated into a truncated protein. Alternatively, a nucleic acid sequence can encode a full-length polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as assertion or efficacy . As discussed above, a heterologous marker or other polypeptide can be added to the modified polypeptide coding sequence, where the term heterologous refers to a polypeptide that is not the same as the modified polypeptide.

In a non-limiting example, one or more nucleic acid constructs can be prepared which include a contiguous elongation of nucleotides identical or complementary to the specific gene, such as the B18R gene. A nucleic acid construct can be at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 , 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000,

5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 30,000, 50,000, 100,000, 250,000, 500,000, 750,000, up to at least 1,000,000 nucleotides in length, as well as larger buildings up to and including sizes chromosomes (including all intermediate lengths and intermediate bands), given the advent of nucleic acid constructions such as an artificial yeast chromosome, known to those skilled in the art. It should be easily understood that the terms intermediate lengths and intermediate ranges, as used herein, refer to any length or range that includes or is among the values ​​cited (that is, all integers including and between these values).

The DNA segments used in the present invention encompass equivalent biologically functional modified polypeptides and peptides, for example, a modified gelonin toxin. Such sequences can come as a sequence of codon redundancy and functional equivalence, which are known to occur naturally within sequences of nucleic acids and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides can be created through the application of recombinant DNA technology, in which changes in protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. Human-designed changes can be introduced by applying site-directed mutagenesis techniques, for example, to introduce improvements in protein antigenicity, to reduce protein toxicity effects in vivo for an individual who received the protein, or to increase the effectiveness of any treatment involving the protein.

In certain other embodiments, the invention relates to isolated DNA segments and recombinant vectors that include within their sequence a sequence of contiguous nucleic acids from that illustrated in the sequences identified herein (and / or incorporated by reference). Such sequences, however, can be mutated to produce a protein product whose activity is altered in relation to the wild type.

It should also be understood that this invention is not limited to nucleic acid and amino acid sequences specific to these identified sequences. Recombinant vectors and isolated DNA segments can, therefore, include the poxvirus coding regions themselves, coding regions carrying selected changes or modifications in the basic coding region, or they can encode larger polypeptides that, however, include poxvirus coding regions or can encode biologically functional equivalent proteins or peptides that have variant amino acid sequences.

The DNA segments of the present invention encompass biologically functional equivalent proteins and peptides from the poxvirus. Such sequences may arise as a consequence of codon redundancy and functional equivalence that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides can be created through the application of recombinant DNA technology, in which changes in protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. The changes designed by Man can be introduced through the application of site-directed mutagenesis techniques, for example, to introduce improvements in the antigenicity of the protein.

B. Poxvirus Polynucleotide Mutagenesis

In several embodiments, the poxvirus polynucleotide can be altered or mutagenized. Changes or mutations may include insertions, deletions, point mutations, inversions, and the like, and may result in the modulation, activation and / or inactivation of certain molecular pathways or mechanisms, as well as alteration in the function, location, or expression of a gene product , particularly making a non-functional gene product. When used, mutagenesis of a polynucleotide that encodes all or part of a poxvirus can be performed by a series of standard mutagenic procedures (Sambrook et al., 1989). Mutation is the process by which changes in the quantity or structure of an organism occur. The mutation may involve modification of the nucleotide sequence of a single gene, gene blocks or integral chromosome. Changes in individual genes may be the result of point mutations that involve the removal, addition, or replacement of a single nucleotide base within a DNA sequence, or they may be the result of changes that involve the insertion or deletion of large numbers of genes. nucleotides.

Mutations can be induced after exposure to chemical or physical mutagens. Such mutation-inducing agents include ionizing radiation, ultraviolet light and a diverse range of chemicals such as alkylating agents and polycyclic aromatic hydrocarbons, all of which are capable of interacting directly or indirectly (usually after some metabolic biotransformations) with nucleic acids. DNA damage induced by such agents can lead to base sequence modifications when the affected DNA is replicated or repaired, and thus a mutation. The mutation can also be targeted at sites through the use of specific methods of assestation.

C. Vectors

To generate mutations in the poxvirus genome, native and modified polypeptides can be encoded by a nucleic acid molecule comprised in a vector. The term vector is used to refer to a carrier nucleic acid molecule within which an exogenous nucleic acid sequence can be inserted for introduction into a cell in which it can be replicated. A nucleic acid sequence can be exogenous ”, which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell, but in a position within the cell's nucleic acid host, in which the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (for example, YACs). Those skilled in the art would be well qualified to construct a vector using standard recombinant techniques, which are described in Sambrook et al., (1989) and Ausubel et al., 1994, both of which are incorporated herein by reference. In addition to encoding a modified polypeptide such as modified gelonin, a vector can encode non-modified polypeptide sequences such as a marker or assay molecule. Useful vectors that encode such fusion proteins include pIN vectors (Inouye et al., 1985), vectors that encode a histidine elongation, and pGEX vectors, for use in the generation of soluble fusion proteins glutathione Stransferase (GST) for purification and subsequent separation or divage. An assay molecule is one that directs the modified polypeptide to an organ, tissue, cell, or other specific location in an individual's body.

The term expression vector refers to a vector that contains a sequence of nucleic acids that encodes at least part of a gene product capable of being transcribed. In some cases, the RNA molecules are then translated to give a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, into the production of antisense molecules or ribozymes. Expression vectors can contain a series of control sequences, which refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a specific host organism. In addition to control sequences that govern transcription and translation, expression vectors and vectors may contain nucleic acid sequences that also serve other functions and are described below.

1. Promoters and Intensifiers

A promoter is a control sequence that is a region of a nucleic acid sequence in which the initiation and the rate of transmission are controlled. It can contain genetic elements to which proteins and regulatory molecules can bind, such as RNA polymerase and other transcription factors. The phrases operatively positioned, operably linked, under control and under transcriptional control mean that a promoter is in a functional location and / or correct orientation in relation to a nucleic acid sequence to control the transcriptional initiation and / or expression of this sequence. A promoter may or may not be used in conjunction with an enhancer, which refers to a regulatory sequence that acts as a cis, involved in the transcriptional activation of a nucleic acid sequence.

A promoter can be one naturally associated with a gene or sequence, as can be obtained by isolating the 5 'non -ocoding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as endogenous ”. Similarly, an enhancer can be one naturally associated with a sequence of nucleic acids, located downstream or upstream of that sequence. Alternatively, certain advantages can be assessed by placing the encoding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and non-naturally occurring promoters or enhancers, that is, containing different elements from different regions. transcriptional regulatory, and / or mutations that alter expression. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences can be produced using recombinant cloning technology and / or nucleic acid amplification, including PCR®, in connection with the compositions described here (see US patent number 4,683,202 , US patent number 5,928,906, each of which is incorporated herein by reference). In addition, control sequences that direct the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can also be used.

Of course, it may be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the type of cell, organelle, and organism chosen for expression. Those skilled in molecular biology techniques generally know the use of promoters, enhancers, and combinations of cell types for the expression of proteins; see, for example, Sambrook et al. (1989), incorporated here as a reference. The promoters employed can be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to target a high level of expression of the introduced DNA segment, as is advantageous in large-scale production of recombinant proteins and / or peptides . The promoter can be heterologous or endogenous.

The identity of promoters or specific tissue elements, as well as the tests to characterize their activity, those skilled in the art are well aware. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin 2 receptor gene (Kraus et al., 1998), the retinoic acid binding gene of the murine epididymis (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998) , a2 collagen (XI) of the mouse (Tsumaki, et al, 1998), the dopamine D1A receptor gene (Lee, et al, 1997), insulin-like growth factor II (Wu et al, 1997), molecule of adhesion of human platelet endothelial cells 1 (Almendro etal., 1996), and the SM22a promoter.

2. Initiation Signs and Internal Ribosome Binding Sites

A specific initiation signal may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translation control signals, including the ATG initiation codon, may be required. Those skilled in the art should be able to readily determine this by providing the necessary signals. It is well known that the initiation codon must be in structure with the reading structure of the desired coding sequence, to ensure the translation of the entire insert. Exogenous translation control signals and initiation codons can be natural or synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancing elements.

In certain embodiments of the invention, the use of elements from internal ribosome entry sites (IRES) is done to create multigens, or polycistronic messages. IRES elements are able to avoid the ribosome scanning model of Cap 5'methylated translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988). The IRES elements of two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES of a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading structures. Multiple open reading structures can be transcribed together, each separated by an IRES, creating polycistronic messages. Because of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be expressed efficiently using a single promoter / enhancer to transcribe a single message (see US patent numbers 5,925,565 and 5,935,819, incorporated herein by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (NCS), which is a nucleic acid region that contains sites with multiple restriction enzymes, any of which can be used in conjunction with standard recombinant technology to digest the vector, (see Carbonelli et al, 1999, Levenson et al, 1998, and Cocea, 1997, incorporated herein by reference). The term restriction enzyme digestion refers to catalytic divage and a nucleic acid molecule with an enzyme that works only at specific locations on a nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Often, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to allow exogenous sequences to be linked to the vector. Bonding refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and binding reactions are well known to those skilled in recombinant technology techniques.

4. Intron Removal Sites

Most of the transcribed eukaryotic RNA molecules will be spliced ​​from the RNA to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see Chandler et al., 1997, incorporated herein by reference).

5. Termination Signals

The vectors or constructs of the present invention must generally comprise at least one termination signal. A termination signal or terminator comprises the DNA sequences involved in the specific termination of an RNA transcript by an RNA polymerase. Therefore, in certain modalities, a termination signal that finalizes the production of an RNA transcript is contemplated. A terminator may be needed in vivo to achieve desirable message levels.

In eukaryotic systems, the terminator region can also comprise specific DNA sequences that allow site-specific dividing of the new transcript in order to expose a polyadenylation site. This signals a specialized endogenous polymerase to add an extension of about 200 A residues (polyA) to the 3 'end of the transcript. RNA molecules modified with this polyA tail appear to be more stable and translated more efficiently. Therefore, in other embodiments involving eukaryotes, it is preferred that this terminator comprises a signal for RNA dividing, and it is more preferred that the terminator signal promotes the polyadenylation of the message. The terminator and / or the polyadenylation site elements can serve to enhance the message levels and / or to minimize reading from the cassette in other sequences.

Terminators contemplated for use in the invention include any known transcription terminator described herein or known to those skilled in the art, including, but not limited to, for example, gene termination sequences, such as, for example, the growth hormone terminator bovine or viral termination sequences, such as, for example, the SV40 terminator. In certain embodiments, the termination signal may be a lack of sequence capable of being transcribed or translated, such as due to a sequence truncation.

6. Signs of Polyadenylation

In the expression, particularly in eukaryotic expression, a polyadenylation signal should typically be included to effect the appropriate polyadenylation of the transcript. It is believed that the nature of the polyadenylation signal is not crucial to the successful practice of the invention, and / or any sequence can be employed. Preferred embodiments include the SV40 polyadenylation signal and / or the bovine growth hormone polyadenylation signal, which is convenient and / or known to work well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

7. Replication Sources

To propagate a vector in a host cell, it can contain one or more origins of replication sites (often called ori), which is a specific nucleic acid sequence in which replication is initiated. Alternatively, an autonomously replicating sequence (ARS) can be used if the host cell is yeast.

8. Selectable and Separable Markers

In certain embodiments of the invention, cells that contain a nucleic acid construct of the present invention can be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell allowing easy identification of cells that contain the expression vector. Usually, a selectable marker is one that confers a property that allows selection. A positive selectable marker is one in which the presence of the marker allows it to be selected, while a negative selectable marker is one in which its presence prevents its selection. An example of a selectable positive marker is a drug resistance marker.

Usually, the inclusion of a drug selection marker assists in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers that confer a phenotype that allows discrimination of transformants, based on the implementation of conditions, other types of markers, including separable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, separable enzymes such as thymidine kinase (herpes simplex virus tk&gt; or chloramphenicol acetyltransferase (CAT) can be used. Those skilled in the art would also know how to employ immunological markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, as long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Other examples of selectable and separable markers are well known to those skilled in the art.

D. Host Cells

As used herein, the terms cell, cell line and cell culture can be used interchangeably. All of these terms also include your progeny, which is any generation and all subsequent generations. It must be understood that every progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, the term host cell refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that are capable of replicating a vector and / or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses (which does not qualify as a vector, if it does not express any exogenous polypeptides). A host cell can be transfected or transformed, which refers to a process by which an exogenous nucleic acid, such as a sequence encoding a modified protein, is transferred or introduced into the host cell. A transformed cell includes the primary cell in question and its progeny.

Host cells can be derived from prokaryotes or eukaryotes, including yeast cells, insect cells, and mammalian cells, depending on whether the desired result is vector replication or expression of part or all of the nucleic acid sequences encoded by the vector. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained from the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc. org). An appropriate host can be determined by those skilled in the art, based on the vector skeleton and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryotic host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and / or expression include DH5a, JM109, and KC8, as well as numerous of the commercially available bacterial hosts such as SURE®se SOLOPACK® Gold Cells Competent Cells (STRATAGENE®, La Jolla , California). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Suitable yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.

Examples of eukaryotic host cells for replication and / or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells of various types of cells and organisms are available and would be known to those skilled in the art. Similarly, a viral vector can be used in conjunction with a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

Some vectors may employ control sequences that allow them to be replicated and / or expressed in prokaryotic and eukaryotic cells. Those skilled in the art would further understand the conditions under which to incubate all the host cells described above, to maintain them and allow replication of a vector. Techniques and conditions that would allow the large-scale production of vectors, as well as the production of nucleic acids encoded by vectors and their polypeptides, proteins, or cognate peptides are also understood and known.

E. Gene Transfer Methods

Suitable methods for delivering nucleic acids to effect the expression of compositions of the present invention are supposed to include virtually any method by which a nucleic acid (for example, DNA, including viral and non-viral vectors) can be introduced into an organelle, a cell, tissue or organism, as described herein or as would be known to those skilled in the art. Such methods include, but are not limited to, direct DNA distribution, such as by injection (US patent numbers 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589 .466 and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; US patent number 5,789,215, incorporated herein by reference); by electroporation (US patent number 5,384,253, incorporated herein by reference); by precipitation with calcium phosphate (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); using DEAE-dextran, and then, polyethylene glycol (Gopal, 1985); by direct sonic charge (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by bombardment of microprojectiles (PCT patent applications No. WO 94/09699 and 95/06128; US patent numbers 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, each of which is incorporated herein by reference; by stirring with silicon carbide fibers (Kaeppler et al., 1990; US patent numbers 5,302,523 and 5,464,765, each of which is incorporated herein by reference); by transformation mediated with Agrobacterium (US patent numbers 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or by PEG-mediated protoplast transformation (Omirulleh et al, 1993; US Patent Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); by desiccation / inhibition-mediated DNA uptake (Potrykus et al, 1985). Through the application of techniques such as these, organelle (s), cell (s), tissue (s) or organism (s) can be transformed in a stable or transient manner.

F. Lipid Components and Groups

In certain embodiments, the present invention relates to compositions that comprise one or more lipids associated with a nucleic acid, an amino acid molecule, such as a peptide, or other small molecule compound. In any of the modalities discussed herein, the molecule can be a poxvirus polypeptide or a poxvirus polypeptide modulator, for example, a nucleic acid that encodes all or part of a poxvirus polypeptide, or alternatively, an amino acid molecule that encodes all or part of the poxvirus polypeptide modulator. A lipid is a substance that is characteristically insoluble in water and is extractable with an organic solvent. The compounds other than those specifically described herein are understood by those skilled in the art as lipids, and are encompassed by the compositions and methods of the present invention. A lipid component and a non-lipid component can be attached to each other, either covalently or non-covalently.

A lipid can be naturally occurring, or synthetic (that is, designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in these techniques, and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with fatty acids linked by ethers and esters and polymerizable lipids, and combinations thereof. .

A nucleic acid molecule or amino acid molecule, such as a peptide, associated with a lipid can be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently attached to a lipid, contained as a suspension in a lipid or otherwise associated with a lipid. An associated lipid or lipid / poxvirus composition of the present invention is not limited to any specific structure. For example, they can also be simply interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. In another example, they may be present in a bilayer structure, such as micelles, or with a collapsed structure. In another non-limiting example, a lipofectamine (Gibco BRL) -poxvirus or Superfect (Qiagen) -poxvirus complex is also contemplated.

In certain embodiments, a lipid composition may comprise about 1%, about 2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or any derivable band between them, of a specific lipid, lipid-type or non-lipid component, such as a drug, protein, sugar, nucleic acids or other material described herein or as would be known to those skilled in the art. In a non-limiting example, a lipid composition can comprise about 10% to about 20% neutral lipids, and about 33% to about 34% a cerebroside, and about 1% cholesterol. In another non-limiting example, a liposome can comprise about 4% to about 12% terpenes, where about 1% of the micelle is specifically lycopene, leaving about 3% to about 11% of the liposome as comprising other terpenes ; and about 10% to about 35% phosphatidylcholine, and about 1% of a drug. Accordingly, it is contemplated that the lipid compositions of the present invention can comprise any of the lipids, types of lipids or other components in any combination or percentage range.

IV. Pharmaceutical Formulations, Distribution and Treatment Schemes

In one embodiment of the present invention, a method of treating a hyperproliferative disease, such as cancer, by distributing an altered poxvirus, such as the vaccinia virus, is contemplated. Examples of cancers contemplated for treatment include liver cancer, lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphomas, pre-neoplastic lung lesions, colon cancer, melanoma, bladder cancer and other cancers or tumors that can be treated.

An effective amount of the pharmaceutical composition is defined herein as the amount sufficient to induce oncolysis, disruption or lysis of a cancer cell, as well as to slow, inhibit or reduce the growth or size of a tumor and includes eradication of the tumor in certain cases. An effective amount may also encompass an amount that results in systemic spread of the therapeutic virus to tumors indirectly, for example, infection of tumors that have not been injected.

Preferably, patients should have adequate bone marrow function (defined as an absolute peripheral granulocyte count&gt; 2,000 / mm³ and a platelet count of 100,000 / mm³), adequate liver function (bilirubin &lt;1.5 mg / dL) and adequate renal function (creatinine &lt;1.5 mg / dL).

The administration

To induce oncolysis, using the methods and compositions of the present invention, a tumor would be contacted with the poxvirus that expresses GM-CSF. Routes of administration will naturally vary with the location and nature of the lesion, and include, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional administration and formulation (for example, in the vicinity of a tumor, particularly with the vasculature or adjacent vasculature of a tumor), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, by inhalation, perfusion, washing, and oral.

Intratumoral injection, or injection directly into the tumor vasculature, is specifically contemplated for distinct, solid, accessible tumors. Local, regional or systemic administration may also be appropriate. In the case of tumors&gt; 4 cm in size, the volume to be administered should be about 4-10 ml (preferably 10 ml), while in the case of tumors &lt;4 cm, a volume of about 1-3 ml should be used (preferably 3 mL). Multiple injections delivered as a single dose comprise volumes of about 0.1 to about 0.5 ml. The viral particles can be contacted by administering multiple injections to the tumor, spaced at intervals of approximately 1 cm. In the case of surgical intervention, the present invention can be used preoperatively, to render a tumor inoperable subject to resection. Continuous administration can also be applied when appropriate, for example, by implanting a catheter into a tumor or within the tumor vasculature. Such continuous infusion can occur for a period between about 1-2 hours, up to about 2-6 hours, up to about 6-12 hours, up to about 12-24 hours, up to about 12 days, up to about 1 -2 weeks or more after starting treatment. Generally, the dose of the therapeutic composition by means of continuous infusion should be equivalent to that given by a single injection or multiple injections, adjusted during the period of time during which the infusion occurs. It is further contemplated that limb perfusion can be used to administer the therapeutic compositions of the present invention, particularly in the treatment of melanomas and sarcomas.

Treatment schedules may also vary, and often depend on the type of tumor, tumor location, disease progression, and the patient's health and age. Obviously, certain types of tumors will require more aggressive treatment, although at the same time, certain patients cannot tolerate more overloaded protocols. The physician is better able to make such decisions, based on the efficacy and toxicity (if any) known from the therapeutic formulations.

In certain modalities, the tumor being treated may not, at least initially, be resectable. Treatments with therapeutic viral constructions may increase the possibility of tumor resection due to contraction in the margins or by the elimination of certain particularly invasive parts. After treatments, resection may be possible. Additional treatments following resection will serve to eliminate microscopic residual disease at the tumor site.

Treatments can include several unit doses. A unit dose is defined as containing a predetermined amount of the therapeutic composition. The quantity to be administered, and the specific route and formulation, are within the capacity of those skilled in clinical techniques. A unit dose does not need to be administered as a single injection, but can comprise continuous infusion over an established period of time. The unit dose of the present invention can be conveniently described in terms of plaque forming units (pfu) for a viral construct. The range of unit doses is from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹°, 10¹¹, 10¹², 10¹³ pfu and higher doses. Alternatively, depending on the type of virus and the attainable titration, one should distribute 1 to 100, 10 to 50, 100-1,000, or up to about or at least about 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10®, 1 x 10⁹, 1 x 10¹°, 1 x 10¹¹, 1 x 10¹², 1 x

10¹³, 1 χ 10¹⁴, or 1 χ 10¹⁵ or more infectious viral particles (pv), including all values ​​and ranges between them, for the tumor or tumor site.

B. Injectable Compositions and Formulations

The preferred method for delivering an expression construct or virus that encodes all or part of the genome of a poxvirus for cancer or tumor cells in the present invention is by means of intratumor injection. However, the pharmaceutical compositions described herein can alternatively be administered parenterally, intravenously, intradermally, intramuscularly, transdermally or even intraperitoneally, as described in US patent number 5,543,158; US Patent No. 5,641,515 and US Patent Number 5,399,363 (each of which is incorporated herein by reference in its entirety).

The injection of nucleic acid constructs can be delivered by syringe or any other method used to inject a solution, provided that the expression construct can pass through the specific needle gauge required for injection. An unusual needle-free injection system has recently been described (US patent number 5,846,233), having a nozzle that defines an ampoule chamber to hold the solution and an energetic device to push the solution out of the nozzle to the dispensing site. A syringe system for use in gene therapy has also been described, which allows multiple injections of predetermined amounts of a solution at precisely any depth (US Patent No. 5,846,225).

Solutions of the active compounds as a free base or pharmaceutically acceptable salts can be prepared in water, suitably mixed with a surfactant, such as hydroxy-propyl-cellulose. Dispersions can also be prepared in glycerin, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of injectable solutions or dispersions (US patent number 5,466,468, specifically incorporated herein by reference in their entirety). In all cases the form must be sterile and must be fluid to the degree that there is ease of use in a syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium that contains, for example, water, ethanol, polyol (for example, glycerin, propylene glycol, and liquid polyethylene glycol, and the like), appropriate mixtures of them and / or vegetable oils. Proper fluidity can be maintained, for example, by using a coating, such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. The prevention of the action of microorganisms can be provided by several antibacterial and antifungal agents, for example, Congratulations, chloro-butanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. The prolonged absorption of the injectable compositions can be provided by the use in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution must be adequately buffered, if necessary, and the liquid diluent must first be made isotonic with sufficient saline or glucose. These specific aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this regard, the sterile aqueous medium that can be employed should be known to those skilled in the art in the light of the present description. For example, a dosage can be dissolved in 1 ml of isotonic NaCI solution and added to 1,000 ml of hypodermoclysis fluid or injected at the proposed infusion site (see, for example, Remington's Pharmaceutical Sciences 15- Edition, pages 10351038 and 1570- 1580). Some variation in dosage will necessarily occur, depending on the condition of the individual being treated. The person responsible for administration should, in any case, determine the dos60 if appropriate for the individual patient. In addition, for human administration, preparations must meet standards for sterility, pyrogenicity, general safety and purity as required by the FDA Division of Biological Standards.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients listed above, as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients in a sterile vehicle that contains the basic dispersion medium and other necessary ingredients among those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus desired additional ingredients from a solution previously sterilized by filtration. .

The compositions described herein can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic acids , tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be obtained from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropyl amine, trimethyl amine, histidine , procaines and the like. After formulating, the solutions should be administered in a manner compatible with the dosage formulation and in the therapeutically effective amount. The formulations are easily administered in a number of dosage forms such as injectable solutions, drug release capsules, and the like.

As used herein, the term vehicle includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic agents and absorption retardants, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in these techniques. Except to the extent that any conventional medium or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase pharmaceutically acceptable or pharmacologically acceptable refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well known in these techniques. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid before injection can also be prepared.

Combined Treatments

The compounds and methods of the present invention can be used in the context of hyperproliferative diseases / conditions, including cancer. In order to increase the effectiveness of a treatment with the compositions of the present invention, such as viruses that express a vaccinia virus that expresses GM-CSF, it may be desirable to combine these compositions with other agents effective in the treatment of such diseases and conditions. For example, the treatment of a cancer can be implemented with the therapeutic compounds of the present invention and other anti-cancer therapies, such as anti-cancer agents or surgery.

Various combinations can be used; for example, a poxvirus, such as the vaccinia virus, is A and secondary cancer therapy is B:

A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

The administration of the poxvirus / vaccine vectors of the present invention to a patient must follow the generic protocols for the administration of this specific secondary therapy, taking into account the toxicity, if any, of the treatment with poxvirus. Treatment cycles are expected to be repeated as needed. It is also contemplated that several standard therapies, as well as surgical intervention, can be applied in combination with the cancer therapy or cancer cell described.

An anticancer agent is able to negatively affect a cancer in an individual, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing the size of tumors , inhibiting the growth of tumors, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the life span of an individual with cancer. Anticancer agents include biological agents (biotherapy), chemotherapeutic agents, and radiotherapy agents. More generally, these other compositions would be provided in a combined amount effective to exterminate or inhibit cell proliferation. This process may involve putting cells in contact with the expression construct and the agent (s) or multiple factors at the same time. This can be accomplished by placing the cell in contact with a single composition or pharmacological formulation that includes both agents, or by placing the cell in contact with two different compositions or formulations at the same time, where one composition includes expression construction and the other includes the second agent (s).

The resistance of cells to chemotherapeutic or radiotherapy agents represents an important problem in clinical oncology. A goal of current cancer research is to find ways to improve the effectiveness of chemotherapy and radiation therapy by combining it with gene therapy. For example, the herpes simplex thymidine kinase gene (HS-tK), when distributed to brain tumors by a retroviral vector system, successfully induced susceptibility to the ganciclovir antiviral agent (Culver et al., 1992). In the context of the present invention, it is envisaged that poxvirus therapy could be used similarly in conjunction with chemotherapy, radiotherapy, immunotherapy or other biological intervention, in addition to other pro-apoptotic or cell cycle regulatory agents.

Alternatively, poxviral therapy can continue or follow treatment with another agent at intervals in the range between minutes and weeks. In certain embodiments in which the other agent and poxvirus are applied separately to the cell, it would generally be ensured that a significant period of time does not expire between the time of each distribution, such that the agent and the poxvirus would still be able to have a beneficially combined effect on the cell. In such cases, it is envisaged that the cell can be brought into contact with both modalities within about 12-24 h between them and, more preferably, within about 6-12 h between them. In some situations, it may be desirable to extend the treatment period significantly; however, when several days (2, 3, 4, 5, 6 or 7) up to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) elapse between the respective administrations.

Chemotherapy

Cancer therapies also include a range of therapies combined with treatments based on chemicals and radiation. Combined chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mecloretamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chloranbucyl, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, mitomycin, plomomycin, hemorrhoicin, plomomycin, mine, tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, navelbine, famesyl transferase inhibitors, transplatin, 5-fluorouracil, vincristine, vinblastine and methotrexate, Temazolomide (an aqueous form of DTIC), or any analog or variant derived from the foregoing. The combination of chemotherapy with biological therapy is known as biochemotherapy.

2. Radiotherapy

Other factors that cause DNA damage and have been used extensively include those commonly known as gamma rays, X-rays and / or the targeted distribution of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves and UV irradiation. It is very likely that all of these factors effect a wide range of damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for extended periods of time (3 to 4 weeks), up to individual doses of 2,000 to 6,000 roentgens. The dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the intensity and type of radiation emitted, and the absorption by neoplastic cells.

The terms put in contact and exposed, when applied to a cell, are used here to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are distributed to a target cell or are placed in direct juxtaposition with the target cell . To achieve cell extermination or stasis, both agents are delivered to a cell in a combined amount effective to exterminate the cell or prevent it from dividing.

3. Immunotherapy

Immunotherapy is generally based on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector can be, for example, an antibody specific for a marker on the surface of a tumor cell. The antibody alone can serve as an effector of therapy or it can recruit other cells to actually exterminate the cells. The antibody can also be conjugated to a drug or toxin (chemotherapy, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a harassing agent. Alternatively, the effector may be a lymphocyte that carries a surface molecule that interacts, directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, that is, direct cytotoxic activity and inhibition or reduction of certain poxvirus polypeptides would provide therapeutic benefit in the treatment of cancer.

Immunotherapy could be used as part of a combination therapy. The generic approach to combination therapy is discussed below. In one aspect of immunotherapy, the tumor cell must support some marker that is susceptible to assay, that is, it is not present in most other cells. There are many tumor markers and any one of them may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, antigen associated with urinary tumor, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, receptor laminin, erb B and p155. An alternative aspect of immunotherapy is for anticancer effects with immunostimulating effects. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL4, IL-12, GM-CSF, IFN-gamma, chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand. Combining immunostimulatory molecules, either as proteins or using gene delivery in combination with a tumor suppressor such as mda-7 has been shown to intensify antitumor effects (Ju et al., 2000).

As discussed earlier, examples of immunotherapies currently under investigation or in use are immune adjuvants (for example, Mycobacteríum bovis, Plasmodium falciparum, dinitro-chloro-benzene and aromatic compounds) (US patent number 5,801,005; US patent number 5,739,169 ; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy (for example, α-interferons, -β and -γ; IL-1, GM-CSF and TNF) (Bukowski etal., 1998; Davidson et al., 1998; Hellstrand etal., 1998) gene therapy (eg TNF, IL-1, IL-2, p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US patent 5,830,880 and US patent 5,846,945) and monoclonal antibodies (e.g., GM2 antiganglioside, anti-HER-2, anti-p185) (Pietras et al., 1998; Hanibuchi et al., 1998; US patent number 5,824,311). Herceptin (trastuzumab) is a chimeric (murine-human) monoclonal antibody that blocks the HER2-neu receptor. It has antitumor activity and has been approved for use in the treatment of malignant tumors (Dillman, 1999). Combined cancer therapy with herceptin and chemotherapy have been shown to be more effective than individual therapies. Therefore, it is contemplated that one or more anticancer therapies can be used with the therapies related to poxviruses described here.

Passive Immunotherapy - There are numerous different approaches to passive cancer immunotherapy. They can be broadly classified into the following categories: injection of antibodies alone; injection of antibodies coupled with toxins or chemotherapeutic agents; injection of antibodies coupled to radioactive isotopes; injection of antiidiotypic antibodies; and finally, purging of tumor cells in the bone marrow.

Preferably, human monoclonal antibodies are used in passive immunotherapy, as they produce few side effects or even no side effects on the patient. Humanized and chimeric monoclonal antibodies are also successfully used in cancer therapy. Monoclonal antibodies used as therapeutic agents for cancer include edrecolomab, rituximab, trastuzumab, gentuzumab, alentuzumab, ibritumomab, tositumomab, cetuximab, bevacizumab, nimotuzumab, and panitumamab.

It may be favorable to administer more than a monoclonal antibody directed against two different antigens or even antibodies with specificity for multiple antigens. Treatment protocols may also include the administration of lympholines or other immune enhancers, as described by Bajorin et al. (1988). The development of human monoclonal antibodies is described in more detail elsewhere in the specification.

Active Immunotherapy - In active immunotherapy, a peptide, polypeptide or antigenic protein, or a composition of autologous or allogeneic tumor cells or vaccine is administered, usually with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993). In immunotherapy of mela67 nomas, patients who elicit high IgM responses often survive better than those who do not elicit or elicit few IgM antibodies (Morton et al., 1992). IgM antibodies are often transient antibodies and the exception to the rule appears to be antiganglioside antibodies or anti-carbohydrates.

Adoptive Immunotherapy - In adoptive immunotherapy, the patient's circulating lymphocytes, or lymphocytes infiltrated in the tumor, are isolated in vitro, activated lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al., 1988; 1989). To accomplish this, an immunologically effective amount of activated lymphocytes in combination with an adjuvant-incorporated antigenic peptide composition, as described herein, should be administered to an animal or human patient. The activated lymphocytes should more preferably be the patient's own cells that were previously isolated from a blood or tumor sample and activated (or expanded) in vitro. This form of immunotherapy produced several cases of regression of melanoma and renal carcinoma, but the percentage of responders was low compared to those who did not respond.

4. Genes

In yet another embodiment, secondary treatment is gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time that an attenuated poxvirus is administered. The distribution of a poxvirus in conjunction with a vector encoding one of the following gene products will have a combined anticancer effect on target tissues. Alternatively, the poxvirus can be engineered as a viral vector to include the therapeutic polynucleotide. Several proteins are encompassed in the invention, some of which are described below. Table 7 lists several genes that can be targeted for gene therapy in some way in combination with the present invention.

Cell Proliferation Inducers - The proteins that induce cell proliferation still fall into several categories depending on function. The common feature of all these proteins is their ability to regulate cell proliferation. For example, a form of PDGF, the oncogene sis, is a secreted growth factor. Oncogenes rarely come from genes that encode growth factors, and today, sis is the only known naturally occurring oncogenic growth factor. In an embodiment of the present invention, it is contemplated that an antisense mRNA directed to a specific cell proliferation inducer is used to prevent expression of the cell proliferation inducer.

The proteins FMS, ErbA, ErbB and neu are receptors for growth factors. Mutations in these receptors result in loss of adjustable function. For example, a point mutation that affects the transmembrane domain of the Neu receptor protein results in the neu oncogene. The erbA oncogene is derived from the intracellular receptor for thyroid hormone. The modified oncogenic ErbA receptor is believed to compete with the endogenous thyroid hormone receptor, causing uncontrolled growth.

The largest class of oncogenes includes signal transducer proteins (for example, Src, Abl and Ras). Src protein is cytoplasmic tyrosine kinase protein, and its transformation from proto-oncogene to oncogene, in some cases, results through mutations in the tyrosine residue 527. In contrast, the transformation of the ras GTPase protein from the proto-oncogene to oncogene, in one example, results from a glycine mutation at amino acid 12 in the sequence, reducing the activity of ras GTPase.

Jun, Fos and Myc proteins are proteins that directly exert their effects on nuclear functions as transcription factors.

Cell Proliferation Inhibitors - Tumor suppressor oncogenes work to inhibit excessive cell proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. The tumor suppressors p53, p16 and C-CAM are described below.

In addition to p53, which has been described above, another inhibitor of cell proliferation is p16. The largest transitions in the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's. A CDK, quina69 if dependent on cyclin 4 (CDK4), regulates progression through Gi. The activity of this enzyme can be to phosphorylate Rb in late Gi. CDK4 activity is controlled by an activating subunit, type D cyclin, and by an inhibitory subunit, p16^{, NK4} it has been biochemically characterized as a protein that specifically binds to CDK4 and inhibits it, and therefore, it can regulate phosphorylation of Rb (Serrano et al., 1993; Serrano et al., 1995). Like the p16 protein^INK4 is a CDK4 inhibitor (Serrano, 1993), the deletion of this gene may increase CDK4 activity, resulting in hyperphosphorylation of the Rb protein. p16 also known to regulate the function of CDK6.

ρ₁θΐΝΚ4 p_ert_ence newly described class of CDK-inhibiting proteins, which also includes p16s, p19, p21, WAF1, and p27^KIP1. The p16 gene maps^{, NK4} for 9p21, a chromosomal region often deleted in many types of tumors. Homozygous deletions and mutations of the p16 gene^INK4 are frequent in human tumor cell lines. This evidence suggests that the p16 gene^{, NK4} it is a tumor suppressor gene. This interpretation was challenged, however, by the observation that the frequency of changes in the p16 gene^INK4 it is much lower in noncultured primary tumors than in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994; Hussussian et al., 1994; Kamb et al., 1994; Kamb et al. , 1994; Mori et al., 1994; Okamoto et al, 1994; Nobori et al, 1994; Orlow et al, 1994; Arap et al, 1995). Restoring p16 function^INK4 wild-type transfection with a plasmid expression vector reduced the formation of colonies by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

Other genes that can be employed in accordance with the present invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-1, MEN-II, zac1, p73, VHL, MMAC1 / PTEN, DBCCR -1, FCC, rsk-3, p27, p27 / p16 fusions, p21 / p27 fusions, antithrombotic genes (eg COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf erb, fms , trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (for example, VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.

Regulators of Programmed Cell Death - Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 families of ICE-like proteins and proteases have been shown to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to various apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et al. , 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bc, -2 protein is now recognized as being a member of a family of related proteins, which can be classified as death agonists and or death antagonists.

After its discovery, Bcl-2 was shown to act to suppress cell death triggered by a series of stimuli. In addition, it is now evident that there is a family of proteins regulating cell death, Bcl-2, which share common structural homologies and sequences. These different members of the family demonstrate functions similar to Bci-2 (for example, BCIx_L, Bcl_w, Bcl_s, Mcl-1, Al, Bfl-1) or counteract the function of Bcl-2 and promote cell death (for example, Bax, Bak, Bik, Bim, Bíd, Bad, Haraliri).

5. Surgery

Approximately 60% of people with cancer will undergo surgery of some kind, including preventive, diagnostic or preparatory, curative and palliative surgery. Curative surgery is a cancer treatment that can be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

Curative surgery includes resection in which all or part of the cancerous tissue is physically removed, excised and / or destroyed. Tumor resection refers to the physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial pre-cancer, or incidental amounts of normal tissue.

After excision of part or all of the cancer cells, tissue, or tumor, a cavity can be formed in the body. Treatment can be carried out by infusion, direct injection or local application of the area with additional anti-cancer therapy. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also be of varying dosages.

6. Other Agents

It is contemplated that other agents can be used in combination with the present invention to improve the therapeutic effectiveness of the treatment. These additional agents include immunomodulatory agents, agents that affect upward regulation of cell surface receptors and GAP junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptotic inducers, or other biological agents . Immunomodulatory agents include tumor necrosis factor; interferon-α, -β, and -γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1.beta., MCP1, RANTES, and other chemolines. It is further contemplated that upward regulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL (Apo-2 ligand) would enhance the apoptosis-inducing capabilities of the present invention by establishing an autocrine or paracrine on hyperproliferative cells. Increases in intercellular signaling by increasing the number of GAP junctions would increase the anti-hyperproliferative effects on the surrounding hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the effectiveness of the present invention. Examples of cell adhesion inhibitors are inhibitors of focal adhesion kinases (FAKs) and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the c225 antibody, could be used in conjunction with the present invention to improve the effectiveness of the treatment.

The Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. TRAIL activates rapid apoptosis in many types of cancer cells, but it is not toxic to normal cells. TRAIL mRNA occurs in a wide range of tissues. Most normal cells appear to be resistant to the cytotoxic action of TRAIL, suggesting the existence of mechanisms that can protect against the induction of apoptosis by TRAIL. The first receptor described for TRAIL, called death receptor 4 (DR4), contains a cytoplasmic death domain; DR4 transmits the apoptosis signal carried by TRAIL. Additional receptors have been identified that bind to TRAIL. One receptor, called DR5, contains a cytoplasmic death domain and signals apoptosis very similar to DR4. DR4 and DR5 mRNAs are expressed in many normal tissues and tumor cell lines. Recently, decoy receptors such as DcR1 and DcR2 have been identified that prevent TRAIL from inducing apoptosis via DR4 and DR5. These decoy receptors thus represent an unusual mechanism for regulating sensitivity to a line of pro-apoptotic cells directly on the cell surface. The preferential expression of these inhibitory receptors in normal tissues suggests that TRAIL may be useful as an anticancer agent that induces apoptosis in cancer cells, while at the same time, sparing normal cells. (Marsters et al., 1999).

There have been many advances in cancer therapy after the introduction of cytotoxic chemotherapeutic drugs. However, one of the consequences of chemotherapy is the development / acquisition of drug-resistant phenotypes and the development of resistance to multiple drugs. The development of drug resistance remains an important obstacle in the treatment of such tumors and, therefore, there is an obvious need to obtain alternative approaches such as gene therapy.

Another form of therapy for use in conjunction with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a procedure in which a patient's tissue is exposed to high temperatures (up to 41 ° C (106 ° F)). External or internal heating devices may be involved in the application of local, regional, or whole body hyperthermia. Local hyperthermia involves applying heat to a small area, such as a tumor. Heat can be generated externally with high frequency waves that target a tumor from a device outside the body. The internal heat may involve a sterile probe, including hollow wires or tubes filled with hot water, implanted microwave antennas, or radio frequency electrodes.

An organ or member of a patient is heated for regional therapy, which is performed using devices that produce high energy, such as magnets. Alternatively, some of the patient's blood can be removed and heated before being infused into an area that will be heated internally. Warming up the entire body can also be implemented in cases where the cancer has spread throughout the entire body. Warm water blankets, hot wax, inducing coils, and thermal chambers can be used for this purpose.

Hormone therapy can also be used in conjunction with the present invention or in combination with another cancer therapy described above. The use of hormones can be used to treat certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases (Table-US-00007 Table 6 Genes Oncogenes Source of Human Disease Functional Growth Factors HST / KS Transfection FGF member of the INT-2 MMTV family FGF promoter Insertion member in the INTI / WNTI MMTV family promoter Factor as Insertion of the Simian Sarcoma SIS PDGF B Tyrosine Receptor Virus ERBB / HER Amplified Avian, EGF / TGF-.alpha. / Erythrocyte

Squamous Amphiregulin / Cancer cell blastosis; Hetacellulin virus; ALV glioblastoma insertion-promoting receptor; human amplified tumors ERBB-2 / NEU / Transfected breast amplification, regulated by HER-2 of the rat ovary, Gastric NDF / Glioblastomas cancers Heregulin and EGF-related factors FMS SM feline CSF-1 sarcoma virus receptor KIT HZ feline MGF / Steel sarcoma virus receptor Hematopoiesis TRK Transfection NGF (human growth factor nerve) colon cancer receptor MET Factor diffuse transfection / HGF RET human osteosarcoma receptor RET Sporadic thyroid translocations Orphan receptors and cancer as a point; hereditary Tyr mutations Medullary thyroid kinase cancer; multiple endocrine neoplasms 2A and 2B ROS URII orphan avian Tyr sarcoma receptor PDGF kinase receptor Chronic translocation TEL (ETS-like myelomonocytic leukemia transcription factor) / PDGF gene fusion receptor).

V. Examples

The following examples are provided for the purpose of illustrating the various embodiments of the invention and are not intended to limit the present invention in any way. Those skilled in the art should readily appreciate that the present invention is well adapted to drive the objectives and obtain the mentioned purposes and advantages, as well as the objectives, purposes and advantages inherent herein. The present examples, together with the methods described herein are currently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes and other uses that are encompassed within the spirit of the invention as defined by the scope of the claims must occur for those skilled in the art.

Example 1

Hepatic Carcinoma Treatment

THE. Objectives (1) To determine the maximum tolerated dose (BAT) and / or the maximum doable dose (MFD) of JX-594 administered by intratumoral injection (IT), (2) To evaluate the safety of JX-594 administered by intratumoral injection, ( 3) A75 assess the replication / pharmacokinetics of JX-594 administered by intratumor injection, (4) Evaluate the immune response to JX-594 and antigens associated with the tumor after intratumoral injection (increased inflammatory infiltration in the sites that received an injection and did not receive an injection; formation of neutralizing antibody; responses to cytokines; and lymphocyte induction T and tumor specific viruses), (5) To assess the antitumor efficacy of JX-594 administered by intratumor injection at sites that received an injection and did not receive an injection

B. Study Design

This is an open phase I study with dose increments in patients with liver carcinoma and injectable superficial tumor nodule (s) under the guidance of images. Patients who have refractory tumors will receive treatment with the following four dose levels in a design with sequential dose increments: Cohort 1: 1 x 10⁸ pfu, Cohort 2: 3 x 10⁸ pfu, Cohort 3: 1 x 10⁹ pfu, Cohort 4: 3 x 10⁹ pfu.

The target period for this study will be 15 months. Enrolled patients will receive 1 treatment per cycle. If a patient receives treatment without dose-limiting toxicity (DLT) and the target tumor has not progressed, the patient will move on to an additional cycle of up to a total of 4 cycles. If a patient has the target tumor progressed, or is withdrawn from the study due to dose-limiting toxicity or other reasons, the patient will conduct an End of Study Consultation and proceed to the follow-up phase. A cycle is defined as 3 weeks. A dose-limiting toxicity will only be seen in the first cycle.

A dose can be distributed into 1 -3 lesions. The total sum of the maximum diameter of the lesion or lesions to be injected must be less than 10 cm. Three patients will be treated and each dose level unless dose-limiting toxicity is observed. Enrollment will proceed to the next dose level if 0 out of 3 patients experience dose-limiting toxicity; if one of the first 3 patients experiences dose-limiting toxicity, then an additional patient will be enrolled until a second dose-limiting toxicity occurs (which is defined as the toxic dose at this time) or up to six patients have been treated. If a second dose-limiting toxicity does not appear in the cohort, the patient advances to the next dose level.

BAT is defined as the immediately preceding dose at which 2 patients experience dose-limiting toxicity after treatment with JX-594. MFD is defined as the maximum dose level when BAT is not defined. When BAT / MFD are defined, six additional patients will be treated to obtain more safety and toxicity data at this dose level. If BAT does not occur in Cohort 4 and the effectiveness of PR develops by more than 2/3 at the dose of the previous cohort, the clinical study of 6 additional patients will be conducted at this dose.

DLT is defined as any of the following, attributed to JX-594: 1. Grade 4 toxicity of any period; 2, Grade 3 Toxicity (excluding flu-like symptoms: fatigue, nausea, myalgia, fever) lasting&gt; 5 days. The Common Toxicity Criteria of The National Cancer Institute in the United States will be used to designate the severity of toxicity that occurs in this study.

1. Decision on Tumor or Control Tumors (Tumor or Tumors that have not received Injection) (Cycle 1) and Injection of JX-594 (Cycle 2+)

During Cycle 1 the investigator will decide the site or sites of control tumors. The tumor or control tumors must be a clear tumor nodule located in the lobes other than the hepatic lobes of the target tumors and outside the lymphatic drainage of the target tumors. Consequently, the target tumors will be located separately in the left and right lobes of the liver. However, if the tumor nodules exist within the boundary on one side of the liver, control tumors may not receive an injection of JX-594 into the tumor nodules; however, a control tumor may not be established if the tumor has a single extensive nodule. This control tumor will be evaluated in the same way as tumors treated with JX-594. this will allow an assessment of the effect of the control on tumor growth toxicity / local activity.

If this patient progresses to Cycle 2, the control tumors will receive an injection of JX-594 at the same dose level as the targeted tumor in Cycle 1. As described above, the dose will be distributed among the tumors proportionally, based on the size of the tumor. tumor.

2. Non-Targeted Responding Tumors (No Injection)

Tumors that did not receive an injection may respond in this study; this phenomenon was reported in a previous phase I trial of JX594 with such patients. It is necessary to understand the mechanism of this effect; the possibilities include spread of the virus from tumors that have received injection and / or induction of tumor-specific cytotoxic T lymphocytes (CTL) (infiltration of T lymphocytes into the tumor and subsequent tumor destruction mediated by T lymphocytes). To better understand the mechanism or mechanisms of this effect, the researchers will conduct the study that follows. If tumors that do not receive an injection respond clinically, core biopsies or fine needle aspirations will be performed at the same points in the collection time as the tumor that received an injection (see Appendix A; biopsies of total non-target tumors do not exceed two sites) . The tumor specimens that did not receive an injection will be analyzed using the same method that will be used for the materials to be obtained from tumors that received an injection.

C. Selection of Patients

1. Inclusion criteria

Typically, patients who meet all of the following criteria: (1) older than 18 years of age, (2) patients with liver carcinoma (primary or metastatic) clinically or histologically confirmed with injectable superficial tumor (£ 10 cm in diameter longer) under progression of image reproduction, which progressed despite standard therapies (ie, refractory to standard therapies), (3) tumor progressed despite standard treatments such as surgical resection, intra-arterial chemoembolization, chemotherapy, and radiation therapy, (4) patients with Karnofsky Performance Scale (KPS)&gt; 70, (5) patients with expected survival of at least 16 weeks, (6) if they are sexually active patients, patients are willing to use a contraceptive method during 3 months after treatment with JX-594, (7) patients with the ability to understand and willingness to sign a written informed consent, (8) patients with the ability to adhere to study procedures and follow-up examinations, (9) patients with adequate bone marrow function: WBC&gt; 3,000 cells / mm³, ANC&gt; 1,500 cells / mm³, hemoglobin&gt; 10 g / dL, and platelet count&gt; 75,000 cells / mm³, (10) patients with adequate renal function: serum creatinine &lt;1.5 mg / dl_, (11) patients with adequate liver function: serum AST (£ 2.5 ULN), ALT (&lt;2.5 ULN), total bilirubin (&lt;2.0 mg / dL); in the case of primary lung cancer, patients should be classified as A or B by the Child-Pugh classification.

2. Exclusion Criteria

Patients must not meet any of the following exclusion criteria: (1) pregnant or nursing an infant, (2) HIV patients, (3) patients classified as C by the ChildPugh classification; (3) patients with total bilirubin&gt; 2 mg / dL among patients classified as A or B (in the case of primary liver cancer), (4) patients with clinically significant active infection or uncontrolled medical condition (for example, respiratory, neurological, cardiovascular, gastrointestinal, urogenital) considered high risk for treatment with new experimental drugs, (5) patients with significant immunodeficiency or family member with the condition due to underlying illness and / or medication taken, (6) patients with a history of eczema requiring systemic therapy, (7) patients with unstable heart disease, including Ml, angina unstable, congestive heart failure, myocarditis, arrhythmias diagnosed and requiring medication within 6 months prior to enrolling the study patient, or any clinically significant condition in cardiac condition, (8) patients who received systemic corticosteroids or any other immunosuppressive medication within 4 weeks prior to treatment with the study drug, (9) patients who received it underwent any other study with drug in investigation, radiotherapy, chemotherapy or surgery within 4 hours prior to enrolling the patient in the study, (10) patients unable or unwilling to provide written consent, (11) patients hypersensitive to the study drug ingredient (s).

D. Study Consultation Procedures

A summary of the study's procedures is presented in the Observations and Tests Program. Usually, a + 1 / -1 window from the scheduled day may be allowed, and weekends and holidays are not counted.

1. Screening Consultation (Day - 14 to 0)

This is a clinical study that uses viruses and the study will continue, discussing with the patient. Any patient who wishes to take part must present a written consent form. After signing an authorization consent, each patient will undergo the following assessments within 14 days before the study begins:

Clinical Assessments include (1) a complete medical and surgical history, including anticancer treatments, (2) weight and vital signs (temperature, pulse rate and blood pressure), (3) physical examination (whole body systems), (4) Karnofsky Performance Score, (5) chest X-rays (posteroanterior and bilateral), (6) 12-lead ECG (acceptable if done within 3 months prior to enrolling the patient in the study), (7) evaluation of concomitant medications (all medications taken within 14 days before the patient's enrollment in the study).

Laboratory Assessments include (1) routine blood tests (including platelet count and differential counts), (2) serum chemistry; sodium, potassium, BUN, creatinine, ALT, AST, alkaline phosphatase, total bilirubin, LDH, calcium, phosphorus, magnesium, random glucose, total protein, albumin and uric acid, (3) coagulation test: prothrombin time (PT) , partial prothrombin time (PTT), and International Normalized Ratio (INR); fibrinogen, (4) HIV, HBV and alpha-fetoprotein testing, (5) neutralizing antibody titration, (6) viral genomes (Q-PCR), (7) routine urine analysis (including microscopic examination), (8) pregnancy test (for women with potential for pregnancy and childbirth), (9) test of appropriate shift markers (CA125, CEA, AFP, PSA, CA19-9, etc.) in the screening test, depending on the type of tumor; when it is increased, the exam will be performed on the 22nd day of each cycle.

Tumor Imaging-Based Assessments and Measurements include measurement of a tumor nodule using CT scan of the abdomen (measurement of the longest diameter); can be replaced by CT performed on Day 1 (before treatment), (acceptable if performed within two weeks prior to enrolling the patient in the study).

Day 1 (Cycle 1 - 4) - It should be noted which assessments will be performed before or after the administration of JX-594.

Day 1; Pre-treatment - Clinical Assessments: Physical examination (integral body systems), weight and vital signs (temperature, pulse rate and blood pressure), Karnofsky Performance Score, identification of concomitant therapies, test and evaluation of tumor (s) - target, measurement of target tumor (s) (n = 1-3); measurement of additional tumor (s) not injected (n = 1-3), biopsy of target tumor (s).

Laboratory Assessments: Blood - 1. Routine blood test (including platelet count and differential counts), 2. Serum chemistry test: sodium, potassium, BUN, creatinine, ALT, AST, alkaline phosphatase, total bilirubin, LDH, calcium , phosphorus, magnesium, random glucose, total protein, albumin and uric acid, (3) coagulation test: prothrombin time (PT), partial prothrombin time (PTT), and International Normalized Ratio (INR); fibrinogen, 4. Cytokines (including GM-CSF), 5. Titer of neutralizing antibody, 6. Viral genomes (Q-PCR).

Laboratory Assessments: Other - 1. Urine test to check for pfu, 2. Smear of throat to check for pfu.

Administration of the Study Drug - 1. Administration of JX594 as described in Chapter 8

Day 1: Post-treatment - 1. Physical examination. Vital signs were checked twice an hour (30 minutes and 60 minutes) for 6 hours and should be checked routinely afterwards, 2. The blood will be drawn for cytokine analysis at the following points in time: one hour and 3 hours after 81 treatment, 3. Blood will be collected for the measurement of circulating JX-594 genomes at the following points in time: 10-15 minutes, 25-35 minutes and 4-6 hours after the start of administration, 4. Urine and throat swab samples to check for viral excretion will be collected 3-4 hours after treatment, 5. Record side effects and concomitant illnesses.

Day 3 (Cycle 1 - 4) - Laboratory Assessments: Blood - 1. Routine blood test (including platelet count and differential counts), 2. Chemical serum test: sodium, potassium, BUN, creatinine, ALT, AST, phosphatase alkaline, total bilirubin, LDH, calcium, phosphorus, magnesium, random glucose, total protein, albumin and uric acid, (3) coagulation test: prothrombin time (PT), partial prothrombin time (PTT), and International Normalized Ratio (INR); fibrinogen, 4. Cytokines (including GM-CSF), 5. Titer of neutralizing antibody, 6. Viral genomes (Q-PCR).

Laboratory Assessments: Other - 1. Urine test to check for pfu, 2. Smear of throat to check for pfu.

Clinical Assessments - Record of side effects and concomitant illnesses.

Assessments based on image reproduction: CT scan of the abdomen when side effects are suspected in Clinical Assessments.

Day 5 (Cycle 1-4) - Clinical Assessments - Record of side effects and concomitant illnesses.

Laboratory Assessments: Blood - 1. Routine blood test (including platelet count and differential counts), 2. Chemical serum test: sodium, potassium, BUN, creatinine, ALT, AST, alkaline phosphatase, total bilirubin, LDH, calcium, phosphorus, magnesium, random glucose, total protein, albumin and uric acid, (3) coagulation test: prothrombin time (PT), partial prothrombin time (PTT), and International Normalized Ratio (INR); fibrinogen, 4.

Day 8 (Cycle 1-4) - Clinical Assessments - Physical examination, CT scan; biopsy of target tumor (s) (the biopsy will also be performed on up to 1 or 2 tumor (s) that have not been injected, indicating a significant change including inflammation, necrosis or contraction, etc.). The biopsy will be performed only in Cycles 1 and 2 by Pl's subjective assessment of the patient's condition. Record of side effects and concomitant illnesses.

Laboratory Assessments: Blood - 1. Routine blood test (including platelet count and differential counts), 2. Chemical serum test: sodium, potassium, BUN, creatinine, ALT, AST, alkaline phosphatase, total bilirubin, LDH, calcium, phosphorus, magnesium, random glucose, total protein, albumin and uric acid, (3) coagulation test: prothrombin time (PT), partial prothrombin time (PTT), and International Normalized Ratio (INR); fibrinogen, 4. Cytokines (including GM-CSF), 5. Titer of neutralizing antibody, 6. Viral genomes (Q-PCR).

Laboratory Assessments: Other - 1. Urine test to check pfu, 2. Smear of throat to check pfu, 3. Aspiration of fine needles for necrosis when necrosis occurs (performed only in Cycles 1 and 2).

Day 15 (Cycle 1-4) - Clinical Assessments - Physical examination.

Laboratory Assessments: Blood - 1. Routine blood test (including platelet count and differential counts), 2. Chemical serum test: sodium, potassium, BUN, creatinine, ALT, AST, alkaline phosphatase, total bilirubin, LDH, calcium, phosphorus, magnesium, random glucose, total protein, albumin and uric acid, (3) coagulation test: prothrombin time (PT), partial prothrombin time (PTT), and International Normalized Ratio (INR); fibrinogen, 4. Viral genomes (Q-PCR).

Laboratory Assessments: Other - 1. Urine test to check for pfu, 2. Smear of throat to check for pfu.

Day 22 (Cycle 1-4) - Clinical Assessments - Physical examination, 2. Assessments based on image reproduction: CT scan of the abdomen (performed only in Cycles 2 and 4); 3. Measurement of target tumor (s) (n = 1-3); measurement of additional tumors that did not receive an injection (n = 1-3); 4. The biopsy of target tumor (s) (the biopsy will also be performed on up to 1 or 2 tumor (s) that have not been injected, which indicates a significant change including inflammation, necrosis or contraction, etc.); 5. Record of side effects and concomitant illnesses. 6. Day 22 can be used as Day 1 before the next cycle. There may be an interval of up to a week between Day 22 and Day 1 of the next cycle.

Laboratory Assessments: Blood - 1. Routine blood test (including platelet count and differential counts), 2. Chemical serum test: sodium, potassium, BUN, creatinine, ALT, AST, alkaline phosphatase, total bilirubin, LDH, calcium, phosphorus, magnesium, random glucose, total protein, albumin and uric acid, (3) coagulation test: prothrombin time (PT), partial prothrombin time (PTT), and International Normalized Ratio (INR); fibrinogen, 4. Neutralizing antibody; 5. Viral genomes (Q-PCR), 6. Test for appropriate tumor markers (CA125, CEA, AFP, PSA, CA19-9, etc.) in the screening test, depending on the type of tumor; when it is increased, the test will be performed on the 22nd day of each cycle.

Laboratory Assessments: Other - 1. Urine test to check for pfu, 2. Smear of throat to check for pfu.

Day 28 or End of Study Consultation - Clinical Assessments Physical examination, and Record of side effects and concomitant illnesses.

Laboratory Assessments: Blood - 1. Routine blood test (including platelet count and differential counts), 2. Chemical serum test; sodium, potassium, BUN, creatinine, ALT, AST, alkaline phosphatase, total bilirubin, LDH, calcium, phosphorus, magnesium, random glucose, total protein, albumin and uric acid, (3) coagulation test; prothrombin time (PT), partial prothrombin time (PTT), and International Normalized Ratio (INR); fibrinogen, 4. Viral genomes (Q-PCR),

Cycle 3-4 - 1. A patient whose tumor at the injection site did not show&gt; 25% growth in the longest diameter on Day 22 of Cycle 2 will advance to Cycle 3-4, 2. A patient whose tumor at the injection site showed a 25-50% increase in the longest diameter on Day 22 of Cycle 2 can advance to Cycle 3-4, 3. A patient whose tumor at the injection site showed&gt; 50% increase in the longest diameter on Day 22 of Cycle 2 will have finished the study.

Patient Review Follow-up - Patients who have completed the clinical study will be routinely followed up for patients with liver cancer for one year after the End of Study consultation. Regardless of the clinical study, living patients should undergo routine examinations, such as serological testing for hepatoma and assessments based on image reproduction when they return for a consultation at the hospital and undergo examinations every 3 months. After the clinical study is completed, if a marked clinical benefit is determined, up to a total of 4 times of additional injection can be administered after obtaining written consent. At this time, all study procedures will proceed in the same way as the first 4 administrations of this study. After the study is completed by Cicio 4, until PI judges that there is a significant clinical benefit (more than stable disease), up to a total of 4 times additional injection of the study drug can be administered. In this case, the PI must discuss with the sponsor in advance and obtain agreement from the sponsor. All study plans will proceed in the same way as this clinical study.

E- Viral Replication, Dissemination and Special Tests

1. Q-PCR Assays and Plasma and Urine Forming Units (Pharmacokinetic Test)

Viral dissemination to the bloodstream will be assessed by quantitative analysis by polymerase chain reaction (Q-PCR). To detect whether viruses are present in urine and throat swabs, samples will be collected after treatment.

2. Tumors and Fine Needle Aspiration Biopsies (Immunity Response Test)

To discover viral replication at the tumor site or sites, biopsies of the nucleus and fine needle aspirations will be conducted (if deemed safe and easy) before and after treatment. These biopsies will be analyzed for evidence of viral replication, infiltration of inflammatory and immune cells, necrosis and apoptosis.

To obtain tissues, the core biopsy needle will be used or the fine needle aspiration biopsy will be performed under image reproduction guidance. However, sometimes these biopsies can cause an emergency or dangerous situation for the patient. Therefore, when biopsy is performed to obtain tissues, patient safety should be the first concern. If the condition of a patient is very likely to lead to danger (liver capsular tumor, etc.), the tissues must be obtained through a safe route.

If the PI believes that tissue biopsy (fine needle aspiration) is likely to cause a hazard to the patient, the biopsy (fine needle aspiration) may not be conducted. In addition, if necessary for the safety of a patient, at the discretion of the PI, patients can be hospitalized and observed for up to 5 days before and after administering a tissue biopsy (fine needle aspiration) and / or intratumoral injection of JX -594.

3. Cytokine Analysis (Immunity Response Test)

Serum concentrations of GM-CSF, IL-1, IL-4, IL-6, 1L-10, IFN-õ and TNF-α will be measured with the ELISA assay.

4. Neutralizing Antibody Assay (Pharmacokinetic Test)

The occurrence of JX-594 neutralizing antibody titration in a patient's serially diluted serum will be identified with a plaque assay.

5. Blood Samples for Pharmacokinetic Examination

The withdrawal for the pharmacokinetic test of 3mL of blood will be stored in a mini-Vacutainer with yellow cap.

F. Research Drug Administration

1. Dose, Administration and Treatment Program

Dose. Doses will typically be as follows: Cohort 1: 1 x 10⁸ pfu, Cohort 2: 3 x 10® pfu, Cohort 3: 1 χ 10⁹ pfu, Cohort 4: 3 χ 10⁹ pfu.

Drug Administration. JX-594 can be administered via intratumor injection. Intratumoral injections should be administered by an experienced physician in the manner described. Using a 21 gauge needle or smaller, tumors will be injected directly with a solution containing viruses whose volume is equivalent to approximately 25% of the total volume of tumors (1-3 tumors) to be injected. Typically, the injection should be conducted under the guidance of image reproduction (for example, with CT). One to three tumors can be injected. Each tumor must receive an equal amount of solution. If 2-3 tumors are injected, the volume of virus solution injected into one tumor must be proportional to the volume of the tumor over the others (that is, if one tumor is twice the volume of the other, the larger tumor will receive 2 / 3 of the total virus solution volume).

Although the target tumor (s) selected in Cycle 1 may stop growing, injections must be continued throughout all cycles. However, if necessary, in Cycle 3, researchers can additionally select non-target tumors that received an injection in Cycle 1 and 2, up to three, including the target tumor (s) in Cycle 1. The sum of the maximum diameters of the tumors that received an injection should be 10 cm. The dose of virus solution injected intratumorally must be proportional to that of the tumor.

Preparation of JX-594. JX-594 is supplied in a frosted disposable glass vial (-60 ° C or below), containing 150 µl of the virus solution (to dispense 0.1 mL). The volume of 100 pL contains 1.9 x 10⁸ pfu of the virus. The bottle should be defrosted vertically at room temperature. JX-594 must not be placed in a hot water bath. Resuspend with a pipette. While being diluted and transported to a patient, the virus can be stored at 4 ° C. Thawed JX-594 must not be injected after 4 hours.

A senior pharmacist and other designated pharmacists should store JX-594 vertically in biologically safe (Class 2) cabinets with care (wearing gloves, safety glasses, an apron, etc.). The initial procedure for all dilutions is as follows: when using a syringe, withdraw the required volume of sterile saline and transfer to a standardized Falcon tube. The final volume of the most diluent virus for injection should be equivalent to approximately 25% of the target tumor volume.

Cohort 1: 1 (one) bottle of JX-594 will be used for patients in Cohort 1. The prescribed volume of JX-594 transferred to sterile saline will be removed with a micropipette / syringe.

Cohort 2: 2 (two) vials of JX-594 will be used for patients in Cohort 2. After mixing, the contents of the first vial will be transferred to the second viral vial. The prescribed volume of JX-594 transferred to sterile saline will be removed with a micropipette / syringe.

Cohort 3 and 4: 4 (fourth) or 11 (eleven) bottles of JX-594 will be used for administration to patients in Cohort 3 or 4, respectively. The entire contents will be transferred to a small mixed polypropylene tube. The prescribed volume of JX-594 transferred to sterile saline will be removed with a micropipette / syringe.

Final procedure for all dilutions: wrap the tube with aluminum foil or place it in a light-proof bag at room temperature. Swirl intensely for 10 seconds before administration. It should not be injected after 30 minutes of exposure to room temperature or after 4 hours of thawing.

Treatment Program. Typically, enrolled patients receive 1 treatment or dose of JX-594 per cycle. A patient whose tumoral tumor received an injection of JX-594 did not progress at the end of a cycle will receive treatment in the subsequent cycle (up to a total of 4 cycles). A patient whose target tumor has progressed will end consultations. A cycle is defined as 3 weeks. A dose can be divided evenly between 1-3 lesions. The sum of the maximum diameters of the injected lesions must be &lt;10 cm.

Dose increment. In the dose increase phase of the clinical study, 2-6 patients will be enrolled for each cohort. If none of the first 3 patients experience DLT, the study will proceed to the next cohort. If a DLT occurs in one of the first 3 patients in a cohort, the study will continue until a total of 6 patients are enrolled in the cohort or 2 patients, including the first, try a DLT.

If fewer than 2 out of 6 patients in Cohort 1 experience a DLT within two weeks after the first injection, and the study will advance to the next cohort. If 2 patients experience a DLT, the dose immediately preceding will be defined as the BAT.

The second patient will not be enrolled until one week after giving the first injection to the first patient in Cycle 1; this rule applies for the entry of the next patient. If a DLT occurs in a cohort, all subsequently enrolled patients will begin treatment within two weeks after completing the first injection in Cycle 1 for all previously enrolled patients. Patients will enter the next dose level cohort at least two weeks after the last patient in the previous cohort completes the first injection in Cycle 1.

If more than 2 patients in Cohort 1 experience DLT, the clinical study will be discontinued.

G. Security

After treatment, systemic side effects may occur: fever, chills, myalgia, fatigue / asthenia, nausea, and vomiting. Side effects at the site of the injected tumor, such as pain, necrosis, ulceration and inflammation may occur. In the light of experience with pre-clinical and clinical study of GM-CSF, a temporary increase in lymphocytes, monocytes, or leukocytes, accompanied by greater neutrophilia may occur. The following may occur at the site of the injected tumor: pain, necrosis, ulceration and inflammation.

Although highly unlikely and not described in the previous phase I trial with JX-594, a disseminated rash associated with vaccinia or encephalitis is theoretically possible; these complications were described in approximately 1 in 10,000 and 1 in 1,000,000 recipients of the vaccine, respectively.

1. Dose-Limiting Toxicity (DLT)

DLT is defined as any Grade 3 or more toxicity attributed to JX-594, excluding flu-like symptoms or symptoms (eg, fatigue, nausea, or myalgia), lasting more than 5 days or any Grade 4 toxicity with any duration assigned to JX-594.

Safety Guarantee for Patients against Procedural Risk. Biopsy can cause complications such as intraperitoneal bleeding and / or shock due to tumor rupture. Although the reported incidence of complication is &lt;0.1% and can be cured with transcatheter embolization, patient safety should be the first concern. Therefore, if the attending physician believes that the biopsy is likely to pose a danger to the patient, the biopsy may not be conducted. In addition, if necessary for the safety of a patient, at the discretion of the PI, patients can be hospitalized and observed for up to 5 days before and after undergoing a biopsy and / or intratumoral injection of JX-594.

H. Effectiveness

The main objective of this study is a phase I clinical study on safety, not a clinical benefit. However, it is expected that this study will cause tumor contraction or tumors that received injection and / or did not receive injection due to the direct viral effect (i.e., oncolysis effect) and / or treatment-induced immune-mediated tumor destruction.

The criterion for evaluating effectiveness is the occurrence of changes in target lesions. If any changes in non-active injuries occur, they will be assessed on the basis of the target injury response by referring to the table below.

Evaluation of target lesions. Complete response (CR): Disappearance of all target injuries; Partial response (PR): A decrease of at least 30% in the LD of target lesions based on the LD baseline sum. Progressive disease (PD): An increase of at least 20% in the LD of target lesions taking as a reference the lowest LD recorded since the beginning of treatment. Stable disease (SD): Neither a sufficient contraction to qualify as PR nor a sufficient increase to qualify as PD taking as reference the lowest sum of LD since the beginning of treatment.

The criteria for assessing the overall response are shown in the table below. The term best overall response means the best response recorded from the starting point of treatment to disease progression / recurrence.

Table 2 - Evaluation of the Best Global Response

<td>Target lesions</td><td>Non-target injuries</td><td>New injuries</td><td>Global response</td>

<td>CR</td><td>CR</td><td>Not</td><td>CR</td>

<td>CR</td><td>CR-no / PD-no</td><td>Not</td><td>PR</td>

<td>PR</td><td>PD-no</td><td>Not</td><td>PR</td>

<td>SD</td><td>PD-no</td><td>Not</td><td>SD</td>

<td>PD</td><td>Any</td><td>Yes or no</td><td>PD</td>

<td>Any</td><td>PD</td><td>Yes or no</td><td>PD</td>

<td>Any</td><td>Any</td><td>Yes</td><td>PD</td>

<td colspan="4">CR = Complete Response; PR = partial response; SD = Stable disease; PD = Progressive Disease</td>

Note: Patients with an overall deterioration in health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having symptomatic deterioration. Every effort should be made to detect the progression of the objective disease, even after discontinuation of treatment.

In some circumstances, it can be difficult to distinguish residual disease from normal tissue. When the assessment of complete response depends on this determination, it is recommended that the residual lesion be investigated (fine needle aspiration biopsy) before confirming the complete response status).

Guidance for assessing measurable injuries. All measurements must be made on the last day of Cycle 2 (Day 22) and on the last day of Cycle 4 (Day 22) by CT or by Magnetic Resonance Imaging (MRI) Reproduction and recorded in metric notation using a ruler or Pachymeter. All baseline assessments should be performed as soon as possible after the start of treatment and no later than 4 weeks before the start of treatment.

Note: Lesions that were previously irradiated are not acceptable as measurable ions. If these injuries are considered acceptable as measurable injuries at the investigator's discretion, the condition for considering these injuries should be described in the protocol. It should also be noted that tumor lesions that are located in an area previously irradiated could be considered measurable. If the investigator considers it appropriate as measurable injuries, the conditions under which such injuries should be considered should be defined in the protocol.

The same evaluation method and technique should be used to characterize each lesion identified and reported at baseline and during follow-up. Based assessment is preferred for assessment by clinical examination when both methods were used to assess the antitumor effect of a treatment.

Conventional CT should be performed with contiguous cuts of 10 mm or less in the slice thickness. Spiral CT should be performed using a 5 mm contiguous reconstruction algorithm. If applicable, PET-CT can be performed in the screening consultation and in this case PET-CT should be used in the evaluation on Day 22 of Cycle 2. If necessary, PET-CT can be repeated on Day 22 of Cycle 4.

Measurement confirmation / Response time. Confirmation: To be assigned a PR or CR status, changes in tumor measurements must be confirmed by repeated evaluations that must be performed within 8 weeks after the criteria for the response are first met. In the case of DS, follow-up measurements with an interval of at least 16 weeks must meet the SD criteria at least once after entering the study.

Response duration: The duration of the overall response is defined as the first documented CR or PR date (whichever is first documented) until the most recent date of objectively confirmed recurrence or progressive disease (taking the lowest recorded measurements as a reference for progressive disease) from the start of treatment). The duration of the complete global response is defined as the time between the date of the first documented CR and the oldest objectively confirmed recurrence date.

Stable disease duration: SD is defined as the time between the date of the first documented DS after treatment and the oldest objectively confirmed DP date (taking the smallest measurements recorded since the beginning of treatment as a reference).

Reassessment of tumor response. If necessary, independent radiologists in this study will evaluate the tumor response. However, the results of the evaluations will be used for the purpose of the study only and will not affect the clinical outcome.

I. Statistical Methods and Data Analysis

1. Sample size

The estimated sample size will be 18 patients and the possible range will be 2-30 patients. The main objectives of the study will determine the safety and BAT or MFD of JX-594 by intratumor injection. This study represents the 2² clinical trial of JX-594 in humans. Due to the fact that there are no previous clinical studies in humans, based on expressive statistical calculations, the sample size for this study is selected based on clinical safety considerations. The study results can be used to provide estimates of variability to determine sample size requirements for future clinical studies.

Patients in each cohort have a chance to leave the study before reaching the actual BAT, as well as a chance to move beyond the actual BAT. The tables below indicate the statistical probability of each outcome based on the incidence of true DLT. The table below presents the probabilities (several true incidences provided for each patient population) of each outcome in a cohort of the first 3 patients.

Table 3

<td>N⁹ of DLTs in</td><td>Action</td><td colspan="5">Incidence of DLT in the Patient Population</td>

<td>a Cohort of 3</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Patients</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 0,1</td><td> 0,2</td><td> 0,3</td><td> 0,4</td><td> 0,5</td>

<td></td><td></td><td colspan="5">Probability of each result</td>

<td> 0</td><td>Advance to next cohort</td><td> 0,729</td><td> 0,512</td><td> 0,343</td><td> 0,216</td><td> 0,125</td>

<td> 1</td><td>Enroll 3 additional patients</td><td> 0,243</td><td> 0,384</td><td> 0,441</td><td> 0,432</td><td> 0,375</td>

<td> &gt;2</td><td>Stop treatment, set BAT</td><td> 0,028</td><td> 0,104</td><td> 0,216</td><td> 0,352</td><td> 0,500</td>

The table below indicates the probabilities of each outcome in a cohort of 6 patients. After observing 1 DLT in the first 3 patients in the cohort and adding 3 more patients to the cohort, it represents several true incidences in the given patient population.

Table 4

<td>N³ of DLTs in a Cohort of 6 Patients</td><td>Action</td><td colspan="5">Incidence of DLT in the Patient Population</td>

<td></td><td></td><td colspan="3"> 0,1 0,2 0,3</td><td> 0,4</td><td> 0,5</td>

<td></td><td></td><td colspan="5">Probability of each result</td>

<td> 0</td><td>Not applicable</td><td>AT</td><td>AT</td><td>AT</td><td>AT</td><td>AT</td>

<td> 1</td><td>Enroll 3 additional patients</td><td> 0,177</td><td> 0,197</td><td> 0,151</td><td> 0,093</td><td> 0,047</td>

<td> &gt;2</td><td>Stop treatment, set BAT</td><td> 0,066</td><td> 0,187</td><td> 0,290</td><td> 0,339</td><td> 0,328</td>

* 1 patient in the first 3 and 1 patient in the second 3 patients 2. Statistical Methods / Data Analysis

The population to be summarized will be an intended population 10 to treat (ITT), defined as all patients who received at least one treatment with JX-594. In addition, a population of evaluable patients will also be assessed as a subset of the ITT population. Evaluable patients are those who have received at least one cycle of therapy with the appropriate tumor measurement being performed at an appropriate pre-treatment and post-treatment time period.

This study will continue with four treatment cohorts to have two to six patients according to the cohort. The data for each cohort will be summarized with descriptive statistics, frequency tabs, graphs, and appropriate data listings. Data from treatment cohorts will be combined for selected data presentations. The presentations of specific data to be generated are described below.

Age, weight and height will be summarized with descriptive statistics (mean, median, standard deviation, minimum and maximum), while gender and race will be summarized with frequency tabs. Data for treatment cohorts will be summarized separately for each patient, as well as combined. To do this, individual patient listings will be produced. The physical medical history data will be separated for each treatment cohort and will be combined to summarize the frequency tables. The administration of treatment will be summarized with descriptive statistics (mean, median, standard deviation, minimum and maximum). Any patients who receive the study drug will be included in the safety analysis. Safety data, including adverse events, laboratory results, toxicity, vital signs and withdrawal information, will be summarized separately at the end of each treatment cohort. AEs will be coded and tabulated using the COSTART body system classification scheme. The number and percentage of individuals who have AEs will be tabulated by treatment cohort and treatment purpose; in addition, the data will be stratified by the severity of the AE and relationship with JX-594 specified by the investigator.

The laboratory results will be summarized, at the time of completion, with deviation tables showing the numbers of patients with changes from pre-treatment to post-treatment. The laboratory results of selected variables will be presented graphically.

In addition to the rates of global tumor responses, rates of tumor responses at target and non-target sites will be reported. The time to tumor progression at target and non-target sites will be reported and overall survival will also be reported. As this is an uncontrolled, non-randomized study, with a small number of patients in each group, the hypothesis95 to test the data from this study alone is not assumed. To assess differences between treatment cohorts, parametric or nonparametric methods can be used to compare each group, as appropriate.

Example 2

Treatment of Non-Resectable Malignant Melanoma

A. Dose and Program

1. Rationale for Dose and Program

A total dose per treatment of 1 x 10⁸ pfu will be given. This dose is lower than the maximum weekly dose of 1.6 x 10⁸ pfu, which was safely administered in the first phase I study of JX-594 for the treatment of surgically incurable cutaneous melanoma (Mastrangelo et al., 1998). In addition, 1 x 10® pfu is ten times lower than the maximum dose that has been safely administered so far (n = 2 patients) in the ongoing phase I intratumor (IT) trial with JX-594 and three times lower than the maximum dose level cleared so far. In that trial, IT injection treatments within 1-3 liver tumors are administered every three weeks. Preliminary results from this study reveal that flu-like symptoms and haematological parameters typically recover to baseline levels within 4 days (ie, Day 5) after treatment with JX-594.

A weekly dosing schedule was chosen because patients in all cohorts recovered from mild to moderate treatment-related toxicities on Day 5 in the ongoing liver IT study described above. In addition, data from Mastrangelo et al (1998) indicate that IT injections twice a week of up to 8 x 10⁷ pfu per treatment are safe and effective.

As evidenced by the initial Phase 1 / ll melanoma study (Mastrangelo et al., 1998), patients were shown to have developed a significant humoral immune response to the vaccinia virus within 14-21 days after revaccination. Antibody titrations have been shown to reach a plateau in 4-6 weeks after exposure despite continued treatments. Therefore, this protocol investigates weekly IT administration for six weeks to provide maximum possible distribution and anti-tumor effects of JX-594 before the development of high antibody and T-cell titers.

2. Rationality for Study

Melanoma may be the ideal target for immunotherapy with JX594 because of the relatively high rate of disease accessible for injection, the positive melanoma response seen with IL-2 immunotherapy, and the lack of effective tolerable therapy for patients with metastatic melanoma. In addition, JX-594 replication is contemplated to support the EGFR pathway, which is highly expressed in melanocytes.

The results of an initial phase I / II study suggest that intratumoral injection of JX-594 is safe and effective for treating distant and injected disease in patients with surgically incurable metastatic melanoma. The response of tumors that received an injection (in 5 of 7 patients) and the response of at least one tumor that did not receive an injection (in 4 of 7 patients) were demonstrated, including two patients who achieved a partial response (6+ months) and a complete response (4+ months) to treatment with JX-594. Particularly noteworthy is that efficacy and gene expression occurred despite pre-treatment vaccination (and therefore pre-existing anti-vaccine immunity) in all patients.

This study design was selected to expand on the initial phase I / II study described above and to evaluate the response of tumors that received injection in up to 15 evaluable patients with Stage 3 or Stage 4 non-resectable metastatic melanoma. safety of JX-594, pharmacokinetics, pharmacodynamics, immune response to JX-594, and the expression of the GM-CSF transgene in blood and tumor tissues will be assessed. The investigators will also assess whether JX-594 is able to spread intravenously and infect regional disease from a distant region that has not received an injection, suggesting that it may be able to confer antitumor effects similar to those experienced at the direct intratumoral injection site. This discovery, in addition to adding to the overall clinical experience of JX97

594 administered via IT, would strongly support treatment with JX-594 by IV administration for the treatment of advanced / metastatic disease, particularly in the treatment of advanced malignant melanoma.

B. Description of the Research Product

JX-594 is a selective replication vaccine virus, targeted against cancer, derived from the commonly used Wyeth vaccine strain (Dryvax®, Wyeth laboratories). The virus is derived from a vaccine strain with the inactivated thymidine kinase (TK) gene. JX-594 contains the gene and promoter for hGM-CSF, a potent cytokine involved in an immune response. JX-594 is further modified with the insertion of the lacZ gene to allow the virus to be traced in tissues.

C. Objectives

Objectives include assessing (a) the objective response rate of tumors that received an injection, (b) the safety and toxicity of JX594 administered by IT injection, (c) the objective response rate of the entire disease burden after administration of JX-594 by IT injection (RECIST criteria), (d) the progression-free survival time (PFS), and (e) the response rate of tumors that did not receive an injection.

D. Study Design

1. Study Overview

This is an open phase I / II trial in patients with non-resectable Stage 3 or Stage 4 malignant melanoma. Patients will receive a total of 6 (six) intratumoral injections of JX-594 over a period of 6 weeks. A total dose of 1 x 10® plaque-forming units (pfu) will be administered in each treatment and will be evenly divided between up to 5 (five) tumors. If patients experience a partial response of tumors that received an injection to IT treatment with JX-594 after completing 6 treatments, 3 additional treatments given weekly can be given.

2. Study Results

The primary results for clinical studies are typically the response rate of tumors that received an injection, including the complete response rate, partial response rate, and duration of response. Secondary results from such studies may include safety, determined by the incidence of treatment-related adverse events, serious adverse events (SAEs), and clinically significant changes from baselines in routine laboratory parameters, including complete response rate, rate of partial response, duration of response, progression-free survival (PFS), response rate of tumors that did not receive an injection, including complete response rate, partial response rate, and response duration. Other results may include overall survival, clinical benefit (including weight gain from improved performance status), evaluation of JX-594 (eg, viral genome (Q-PCR) in plasma and / or whole blood; viral infectious virus in plasma and / or whole blood, optional (plaque assay)), immunological assessment (neutralizing antibodies to JX-594 in serum; plasma GM-CSF measurements (ELISA assay)), histological assessment (viral gene expression in tissue; GM-CSF; lac-Z expression; infiltration of inflammatory cells; necrosis; apoptosis; virus replication factories within the cytoplasm; state of the EGFR pathway; and thymidine kinase status of the vector).

3, Dose

Typically, the virus will be diluted in sterile normal saline as described herein. A total dose of 1 x 10⁸ plaque forming units (pfu) will be administered in each treatment and will be divided between up to five (5) tumors.

4. Global Study Duration and Follow-up

A study period will typically consist of patient consultations for screening, study treatment, and post-treatment follow-up assessments.

Screening. Patients' eligibility for a study will be determined within 14 days prior to the first treatment with JX-594.

Treatment. Eligible patients will be treated with a dose of 1 x 10⁸ pfu administered by weekly intratumor injection (Days 1, 8,

15, 22, 29, and 36) for a total of 6 treatments given over 6 weeks. Patients must continue to meet all eligibility criteria before treatment is repeated. If a treatment is missed for any reason, the missed treatment will be given the following week as long as the eligibility criteria are met, and the consultation schedule will be adjusted and patients will be followed up accordingly, so that the patient receives a total of 6 treatments. Injections can be delayed for a cumulative maximum of 4 weeks. Patients who have delayed treatment will still complete all 6 treatments and will be assessed for response one week after their 6² treatment. Response assessment will be conducted initially one week after the final dose is administered (ie, Day 43). If patients experience partial response of tumors that received an injection to IT treatment with JX594 after completing 6 treatments, 3 additional treatments given weekly can be given.

Post-treatment follow-up. All patients will return for a follow-up visit 28 days after the last treatment with JX-594 (ie Day 64). For 6 months after therapy is completed or until the patient has progressive disease at the injection site, a new cancer therapy begins, or dies. The patient will return to the clinic every three weeks after the last injection, for tumor measurement by physical examination (PE) (if possible) and response assessment. Every 6 weeks, the patient will also have a response assessment by PE and / or CT / MRI. After 6 months of follow-up, the patient will return to the clinic every 3 months for tumor measurement and response assessments (including CT / MRI) until progressive disease at the injection site, death, or until the start of new therapy cancer.

Long Term Monitoring of Products for Gene Therapy. After disease progression at the injection site or initiation of new cancer therapy, the patient can continue to be monitored for survival and for the potential long-term effects of gene therapy, according to current FDA guidelines. If patients are no longer 100 returning to the clinic for treatment or post-treatment follow-up, these data can be collected by post or telephone.

E. Study population

1. Inclusion criteria

Patients will typically meet all of the following criteria: histologically confirmed, Stage 3 or Stage 4 malignant melanoma; tumor mass at least measurable by CT / MRI and / or physical examination that can receive injection by direct visualization or by ultrasound guidance; expected survival of at least 16 weeks; cancer is not surgically resectable for a cure; KPS score £ 70; age £ 18 years; men and women with reproductive potential must be willing to follow accepted contraceptive methods during treatment and for 3 months after the last treatment with JX-594; understand and condescendingly sign a written consent form approved by the Institutional Review Board (IRB) / Independent Ethics Committee (IEC); able to undergo study procedures and follow-up exams; adequate liver function (total bilirubin 2.0 x ULN; AST, ALT 2.0 x ULN); proper bone marrow function (WBC&gt; 3,500 cells / mm³ and &lt;50,000 cells / mm³; ANC&gt; 1,500 cells / mm³, hemoglobin&gt; 10 g / dL; platelet count&gt; 125,000 platelets / mm³); acceptable coagulation state (INR &lt;(ULN + 10%)); and acceptable renal function (serum creatinine &lt;2.0 mg / dL).

2. Exclusion Criteria

Typically, patients should not meet any of the following exclusion criteria: adherent tumor (s) and / or invaders of an important vascular structure (eg, carotid artery); pregnant or breastfed an infant; known HIV infection; use of systemic corticosteroid medication or other immunosuppressive medication within 4 weeks of the first treatment with JX-594; significant clinically active infection or uncontrolled medical condition (eg, pulmonary, neurological, cardiovascular, gastrointestinal, urogenital) considered high risk for treatment as a new drug under investigation; immuno101 significant deficiency due to underlying disease and / or medication (for example, systemic corticosteroids); history of eczema that at some stage required systemic therapy; clinically significant and / or rapidly cumulative ascites, pericardial and / or pleural effusions (eg, requiring drainage to control symptoms; severe or unstable heart disease that includes, but is not limited to, any of the following within 6 months of screening: myocardial infarction, unstable angina, congestive heart failure, myocarditis, arrhythmias diagnosed and requiring medication or any significant change in cardiac status; treatment of the target tumor (s) with radiotherapy, chemotherapy, surgery, or a drug under investigation within 4 weeks of screening (6 weeks in the case of mitomycin C or nitrosoureas); experienced a severe reaction or side effect as a result of a previous smallpox vaccination; inability or reluctance to provide consent or acquiescence to procedures required in this protocol; patients who have contact at home with pregnant or nursing mothers or infants, children &lt;5 years of age, have a history of eczema that at some stage required systemic therapy, or have significant immunodeficiency due to an underlying disease (eg HIV) and / or medication (for example, systemic corticosteroids) will be excluded unless alternative life arrangements can be made during the patient's active dosing period and for three weeks after the last study medication.

3. Other Eligibility Criteria Considerations

Deviations from Eligibility Criteria. Patients with minimums of the inclusion / exclusion criteria criteria above (for example, laboratory values ​​outside the pre-specified range) may be allowed in the study, if these deviations are not expected to affect patient safety, the conduct of the study, or the interpretation of the study results. Written approval by the sponsor or sponsor representative to enroll patients with minimal deviations must be requested.

4. Patient Registration Procedures

After the investigator conducted the screening and screening

102 confirms patient eligibility, the sponsor typically reviews screening and eligibility information and provides written verification to the investigator for each patient's enrollment. After confirming enrollment, the patient will assign an identifier using a predefined numbering scheme. The patient identifier will be a composite of study number, site number, patient number and patient initials.

F. Research Product

JX-594 will be provided by Jennerex Biotherapeutics. Typically, JX-594 is formulated as a liquid and is stored frozen in glass bottles designed for single use. Each vial contains 0.15 mL. The virus solution is a colorless to slightly yellow solution that is clear to slightly opaline. The concentration of JX-594 and T, 9 x 10⁹ pfu / ml.

JX-594 is considered a Biosafety Level 2 (BSL-2 ) infectious substance . The BSL-2 designation and associated guidelines apply to agents with moderate potential risk to personnel and the environment. Examples of other BSL-2 agents include measles virus, salmonella and Hepatitis B virus. Institutional infection control policies should be consulted.

JX-594 is typically stored in a secure monitored freezer with restricted access. JX-594 should be stored in bottles clearly labeled in the secondary packaging at -60 ° C or below with biohazard labeling (indicating the nature of the agent) on the freezer door and the room door. Freezers must have a fin limit set at -65 ° C to allow response before the freezer temperature rises to -60 ° C. An extended time at&gt; -60 ° C will require quarantining the affected material until the titration can be reconfirmed.

Instructions for ensuring proper handling and preparation of JX-594 should be provided to a study site with supplementary information about the study. Institutional infection control policies for the preparation, transport, and disposal of viral vectors [Biosafety Level 2 (BSL-2)] should be consulted and followed. Gloves, apron

103 and eye protection must be worn at all times. All work with JX-594 must be conducted in a vertical biological safety cabinet (class 2), according to the BSL-2 handling guidelines in a pharmacy / laboratory under the direction of an accredited pharmacist / scientist. The chapel itself must be cleaned with 70% ethanol before and after each use.

Defrosting. Thawing should take place at room temperature with the bottle upright. JX-594 must not be placed in a hot water bath. After thawing, the vial is placed in a conical 15 ml polypropylene centrifuge tube (for example, Corning or Falcon), capping the tube, and centrifuging at 100 xg for 2 minutes. Remove the JX-594 vial from the polypropylene tube with forceps or equivalent. The virus formulation must be stored on ice or refrigerated (2 - 8 ° C) until diluted and distributed to the patient. The infusion should not be started more than 4 hours after the virus formulation has been thawed.

Preparation. After centrifuging a vial, gently put it back into suspension with a micropipettor (200 pL micropipettor set as a suggestion to 100 pL). Care must be taken not to cause bubbles to form into the formulation. Approximately 2.75 ml of virus solution (JX-594 + saline) are typically prepared, which will be distributed within 5 syringes of 0.5 ml each. Using a micropipettor, transfer 2.64 mL of sterile normal saline to an appropriately sized polypropylene tube (for example, 5 mL Falcon tube). Remove one (1) bottle of JX-594 116 pL of JX-594 and transfer the Falcon tube containing the saline solution. Replace the cap on the tube, protect the tube from light (with aluminum foil or place in a light-proof receptacle), and immediately place the covered tube at 2 - 8 ° C (refrigerate or place on ice / water).

Within 30 minutes before administration, swirl intensely for 10 seconds. After swirling, withdraw 0.5 mL of the virus solution (JX-594 + saline) into each 5 syringes. Cap the syringes and deliver to the investigator for injection. Do not start the injection

104 more than 4 hours after the virus formulation was thawed. The virus formulation must be stored on refrigerated ice (2 - 8 (C) until diluted and distributed to the patient.

1. Administration of JX-594

JX-594 must be administered by intratumor injection each week for a total of 6 (six) injections over six weeks. Administration should be done on Days 1, 8, 15, 22, 29, and 36. Patients should receive a dose of 1 x 10® pfu per treatment divided into 25 lesions. Only lesions accessible for treatment through percutaneous injection (eg, palpable skin nodules or lymph node metastases) or ultrasound-guided injection will be eligible for treatment.

The investigator will determine in each treatment which lesions (tumors) to receive injection. Tumors will receive an injection based on size; larger lesions should be injected with each treatment. At the investigator's discretion, one or more syringes can be used to treat a tumor.

After aseptic preparation of the skin at the site or sites of needle entry, a local anesthetic will be administered. An 18-22 gauge needle will be used for the injection. The injection needle should be inserted into the tumor as described below. The injections will be done by the chief investigator or assistant investigator.

The injection into each tumor should be done by injecting the entire volume of the syringe (0.5 mL) into 4 needle sections equally spaced by a tumor that spreads out from the central puncture site. As an example, injection of the virus can be performed as follows: (1) insert the needle (gauge 18 - 22) into the center of the tumor, (2) extend the needle towards the edge of the tumor (up to within 1 - 3 mm from the edge of the tumor), (3) inject about 25% of the syringe volume (approximately 0.125 mL) while pulling back towards the central puncture site, (4) without removing the needle completely from the tumor, repeat the steps above in 90 ° spacing for a total of 4 stretches the needle.

105

Expected Toxicities. The following systemic toxicities are expected after treatment: fever, chills, anorexia, myalgia, fatigue / asthenia and / or headache. Decreases in neutrophils, lymphocytes, platelets and hematocrits are expected. Haematological parameters typically returned to baseline levels on Day 5 (typical duration 2-3 days). For Cycle 1 only, an increase in leukocytes within the first four days after the initial injection is possible. Leukocyte counts of 24,000 / pL and 118,000 / pL were reported in two patients in Cohort 3 within 5-8 days after the dose. An increase in eosinophils is also expected after treatment and typically remains elevated until Day 8. In the injected sites, the following toxicities are likely: pain, necrosis, ulceration and inflammation. In other sites of viral replication (for example, distant tumors), pain, necrosis, ulceration and inflammation are possible.

Although highly unlikely and not observed after any treatment or exposure to JX-594, a disseminated rash associated with vaccinia or encephalitis is possible; these complications have been described in approximately 1 in 10,000 and 1 in 1,000,000 of measles vaccine recipients, respectively. In addition, a statistically significant increased risk of myocarditis (1-2 per 10,000 vaccinees) was demonstrated in a recent vaccination program with the NYCBOH vaccine strain (Arness et al., 2004).

G. Statistics

1. Results Definitions

The following are the definitions of the results in relation to the statistical analyzes. The codification and definition of progressive disease toxicities, complete response, partial response, duration of total responses, related to evaluable patients are described elsewhere in the protocol.

Progression-Free Survival. The time from the first treatment with JX-594 to the date of diagnosis of progression evaluated by the investigator, or the date of death without progression. Patients who are known to have been last alive without progression will be re-registered at the time of their last progression assessment. Patients who receive therapy other than the protocol prior to documenting progressive disease will also be assigned for statistical analysis.

Global survival. Time from the first treatment with JX-594 until the date of death or date known to be alive; patients admittedly alive last are designated as listed in statistical analyzes.

2. Analysis or Population Sets

All patients receiving JX-594 will be analyzed for demographic characteristics in the screening and subsequently for safety, efficacy, pharmacokinetics and pharmacodynamics. The population to be summarized will be an intended to treat (ITT) population, defined as all patients who receive at least one treatment with JX-594. In addition, an evaluable patient population will also be assessed (a subset of the ITT population). A patient will be considered an evaluable patient if the patient receives at least one treatment with JX594 and has an appropriate tumor measurement at baseline and at the first point at the appropriate time after treatment.

3. Analysis method

Continuous variables will be summarized using descriptive statistics (n, mean, standard deviation, median, minimum, and maximum). The categorical variables will be summarized indicating the number and percentage (n,%) of patients within each classification. The analyzes will be made based on evaluable patients, as well as on the population with intention to treat. Global analyzes will be conducted; in addition, safety and efficacy analyzes will be correlated with the stage of the disease.

Security: Analysis Methods. Patients receiving any study medication will be included in the safety analysis. Safety data, including adverse events, laboratory results, toxicity, vital signs and withdrawal information will be summarized over time. The patients' age, weight and height will be summarized with descriptive statistics, while gender and race will be summarized with frequency tabs. Medical history data will be summarized

107 with frequency tabs.

Adverse episodes will be coded and tabulated using the MedDRA classification scheme. The incidence of AEs emerging from treatment will be tabulated; in addition, the data will be stratified by severity (degree) of adverse episode and specified relationship with the investigator with JX-594. The safety analysis will focus on Grade 3 or 4 hematological adverse episodes and Grade 4 hematological adverse episodes. A list of SAEs will be produced.

The results of hematological and serum chemistry will be summarized using descriptive statistics for continuous variables. In addition, a nadir analysis of selected hematological parameters will be performed and summarized. Laboratory results will be summarized over time in deviation tables that show the numbers of patients with changes after dosing from baseline values ​​in relation to the reference range. Laboratory results for selected variables will also be presented graphically.

KPS performance scores will be summarized using descriptive statistics for categorical variables. The maximum deviation in KPS performance scores compared to screening and / or baseline values ​​will also be summarized. The remaining security variables will be summarized using descriptive statistics.

Pharmacokinetics / Pharmacodynamics: Methods of Analysis. Over time, viral replication and excretion into the blood will be assessed by the following genomic concentrations in the blood. Blood concentrations of JX-594 and GM-CSF levels will be measured in all patients and pharmacokinetic parameters will be estimated.

The pharmacodynamic parameters to be analyzed will include the effect of JX-594 and GM-CSF on peripheral blood counts, MIA, and tumor biopsy tissue. The immune response to JX-594 after IT injection will be evaluated and summarized, including changes from baseline values ​​in leukocyte subsets (absolute count of eosinophils, ANC, lymphocytes), cytokines, and formation of neutralizing antibodies to JX-594.

108

The change from baseline to histological results (tumor tissue and normal control tissue), including infiltration of inflammatory cells, viral gene expression, GM-CSF expression, lac-Z expression and tumor necrosis will be evaluated and summarized . Apoptosis, viral replication plants within the cytoplasm, state of the EGFR pathway, and state of tumor thymidine kinase can also be evaluated.

Effectiveness: Methods of Analysis. The response rate to treatment based on RECIST criteria will be assessed for the overall response, response of tumors that received an injection, and response of tumors that did not receive an injection. The rates of complete response, partial response, stable disease, and progressive disease will be summarized. Progression-free survival, time to progression, duration of response, and overall survival will also be reported. The correlation with the stage of the disease will be assessed.

Progression-free survival and response duration will be estimated using the Kaplan-Meier method. The median, 95% (bilateral) confidence interval for the median, minimum, and maximum duration, as well as the number of patients enrolled, will be presented. Descriptive statistics and the curve will be made. The assessment of the clinical benefit for patients will also be done by assessing weight gain and improving the performance status over time after treatment with JX594. The change over time in the protein with melanoma inhibitory activity (MIA) can be evaluated. MIA can also be compared against response to treatment.

Independent Review of Response Assessment. The locations where the exams were performed may be asked to provide copies of all radiology data in selected patients (digital or on CD-ROM are preferred) to an independent radiology reviewer (IRR). In the case of patients with skin lesions, the photographs must also be sent to the independent radiology reviewer. The results of the locations where the exams were performed and the independent radilogy reviewer will be reported. No disagreement assessment among readers will be conducted.

109

Example 3

Treatment of Refractory Liver Tumors

In a phase I pilot trial with JX-594, seven melanoma patients received increasing doses injected into superficial skin metastases (Mastrangelo et al., 1999). No maximum tolerated dose (BAT) has been reported; tumor responses were reported. The objectives of the present trial were to define the following: safety and BAT at significantly higher doses (100 times), without pre-immunization (as done in the pilot study), specifically after treatment within a solid organ; pharmacokinetics, including replication-dependent excretion into the blood for three weeks; effectiveness against a wide spectrum of cancers. In this phase I trial, the inventors therefore treated patients with liver tumors (primary or metastatic) by intratumor injection. For the first time, the inventors reported BAT, plus high-level replication of JX-594 and GM-CSF expression, efficacy and assay of distant tumors in well-tolerated doses. The results reported here support future intratumor and intravenous trials with JX-594 and products in this class.

A. Materials and Methods

1. Study Design

The main objectives were to determine the safety and BAT of JX594. Secondary goals included pharmacokinetics, replication and excretion (urine, throat swabs), immune responses (neutralizing antibodies, cytokines) and tumor response. Patients received one of four dose levels (10⁸, 3x10®, 10⁹, 3 x 10⁹ plaque-forming units, pfu) in a sequential dose increment design per group (2-6 patients per dose level). BAT was defined as the dose level immediately preceding that at which two or more dose-limiting toxicities (DLT) were observed. DLT was defined as any grade 4 toxicity, or grade 3 toxicity that lasted&gt; 5 days. An independent Data Security Monitoring Board (DSMB) reviewed all dose increase decisions and key security assessments.

110

2. Patient Selection

Patients signed an authorization consent, according to the guidelines of Good Clinical Practice (GCP). Inclusion criteria included non-resectable solid injectable tumors within the liver, which had progressed despite treatment with standard therapies, normal hematopoietic function (WBC count&gt; 3,000 mm³, hemoglobin&gt; 10 g / dL, platelet count&gt; 75,000 / mm³ and organic function (including creatinine &lt;1.5 mg / dl_, AST / ALT &lt;2-5 of ULN, Child-Pugh class A or B), life expectancy&gt; 16 weeks, and Karnofsky Performance Status (KPS) £ 70. Exclusion criteria included increased risk of complications from vaccination (eg, immunosuppression, eczema), treatment with immunosuppressive agents or for treatment of cancer within 4 weeks, pregnancy, or breastfeeding.

3. Manufacture and Preparation of JX-594

JX-594 is a Wyeth vaccine strain modified by inserting the human GM-CSF and lacZ genes into the TK gene region under the control of the early-synthetic synthetic promoter and the p7-5 promoter, respectively. The clinical trial material was generated according to GMP guidelines in Vero cells and purified by sucrose gradient centrifugation. The genome to pfu ratio was approximately 70: 1. JX594 was formulated in phosphate buffered saline with 10% glycerin, 138 mM sodium chloride at pH 7.4. The final product's QC release tests included tests for sterility, endotoxin and potency. JX-594 was diluted in normal 0.9% saline to a volume equivalent to 25% of the estimated total volume of target tumor (s).

4. Treatment Procedure

JX-594 was administered through intratumoral injection guided by image reproduction, using PEIT needles (percutaneous ethanol injection, with multiple pores; HAKKO Medicais; Tokyo, Japan) with 21 gauge. The tumors (n = 1-3) received injection every three weeks over two stretches of the needle during needle withdrawal through the tumor. The course of initial treatment was 2 cycles; up to 6 additional cycles were allowed if the tumor response had occurred.

5. Monitoring of Patients

Patients were monitored as described in Table 5. Patients were monitored after treatment at the hospital for at least 48 hours, and for four weeks as outpatients. Table 5 Study Procedures____

<td>Study Day</td><td>Day 14-0</td><td>Day 1 Pre</td><td>Day 1 Post</td><td>Day 3</td>

<td>Injection of JX-594 (under CT guidance)</td><td></td><td></td><td>X</td><td></td>

<td colspan="5">Clinical Assessments</td>

<td>Physical examination, ECOG Performance status</td><td>X</td><td>X</td><td>x¹</td><td>X</td>

<td colspan="5">Laboratory Safety Assessments²</td>

<td>Hematology³/Coagulation</td><td>X</td><td>X</td><td></td><td>X</td>

<td>Serum chemicals</td><td>X</td><td>X</td><td></td><td>X</td>

<td colspan="5">Viral assays</td>

<td>Plasma / blood levels of JX-594: Q-PCR</td><td></td><td>X</td><td>X⁴</td><td>X</td>

<td>Excretion (throat smear, urine): plaque assay</td><td></td><td>X</td><td>X⁴</td><td>X</td>

<td colspan="5">Immunological Evaluation</td>

<td>Neutralizing antibodies</td><td></td><td>X</td><td></td><td></td>

<td>Cytokines (including GM-CSF)</td><td></td><td>X</td><td>X⁵</td><td>X</td>

<td colspan="5">Pathological assessments</td>

<td>Tumor biopsy</td><td></td><td>x^B</td><td></td><td></td>

<td>Effectiveness Assessments</td><td></td><td></td><td></td><td></td>

<td>TC scan PET-TC (optional)</td><td>X</td><td></td><td></td><td></td>

<td>Serum tumor markers</td><td>X</td><td></td><td></td><td></td>

<td>Study Day</td><td>Day 5</td><td>Day 8</td><td>Day 15</td><td>Day 22</td><td>Completion of Consultation of Study / Day 28</td>

<td>Injection of JX-594 (under CT guidance)</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="6">Clinical Assessments</td>

<td>Physical examination, ECOG Performance status</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td colspan="6">Laboratory Safety Assessments²</td>

<td>Hematology³/Coagulation</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Serum chemicals</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

112

<td colspan="6">Viral assays</td>

<td>Plasma / blood levels of JX- 594: Q-PCR</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Excretion (throat smear, urine): plate test</td><td></td><td>X</td><td>X</td><td>X</td><td></td>

<td colspan="6">Immunological Evaluation</td>

<td>Neutralizing antibodies</td><td></td><td></td><td></td><td>X</td><td></td>

<td>Cytokines (including GM-CSF)</td><td></td><td>X</td><td></td><td></td><td></td>

<td colspan="6">Pathological assessments</td>

<td>Tumor biopsy</td><td></td><td>X⁵</td><td></td><td>χ^ΰ</td><td></td>

<td colspan="6">Effectiveness Assessments</td>

<td>TC scan PET-TC (optional)</td><td></td><td></td><td></td><td>X</td><td></td>

<td>Serum tumor markers</td><td></td><td></td><td></td><td>X</td><td></td>

6. Neutralizing Antibody Titrations (NAb)

NAb titrations were determined by cytopathic inhibition assay. The heat inactivated serum was serially diluted in the medium using half log dilutions. 50 µl samples were incubated with 1,000 pfu of JX-594 for two hours, and then inoculated onto A2780 cells. After 3 days, cell viability was determined using the Cell Counting Kit 8 (Donjindo Laboratories, Kumamoto, Japan). NAb titration was defined as the reciprocal of the highest serum dilution that resulted in £ 50% cell viability.

7, Quantitative PCR for JX-594

Quantitative PCR (Q-PCR) was used to measure the genomes of JX-594 in the blood serially due to its reproducibility and ability to detect the product independently of the antibody and / or complement neutralization. The JX-594 DNA was purified from the samples using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). Q-PCR was operated as previously described (Kulesh et al., 2004). The lower limits of JX-594 detection and quantification were 666 and 3,333 copies / mL of plasma, respectively.

8. Detection of JX-594 Excretion

A plaque-forming assay was used to detect any excretion of infectious JX-594 into the environment; unit excretion

113 infectious disease would be relevant to public health. Urine and saliva samples were centrifuged, resuspended in 10 mM Tris (pH 9.0), and titrated on A2780 cells by plate assays. The detection limit was 20 pfu / ml of sample.

9. Cytokine Assays

GM-CSF was detected by the ELISA kit (BioSource International; Carlsbad, CA, USA) following the supplier's instructions. The serum levels of IL-Ιβ, IL-6, IL-10, TNF-alpha, and interferon-gamma were evaluated using the LINCOplex kit, according to the manufacturer's instructions (LINCO; St. Charles, MO).

10. Histopathological Staining for Vacinia and LacZ Proteins in Blood and Tumor Samples

Biopsies embedded in paraffin and fixed in formalin were stained with hematoxylin and eosin for histology. For immunohistochemistry, murine monoclonal antibodies to B5R (Vac-14, a-B5R, 46 pg / mL; Dr. Gary Cohen, University of Pennsylvania; diluted 1: 50 or 1: 100) were used, and then incubation with polymer HRP-tagged anti-mouse DAKO EnVision + ® (DAKO, Carpinteria, CA) prior to development using DAB (Kirkegaard &amp; Perry Laboratories; Gaithersburg, MD). For LacZ staining, the cells were centrifuged at 900 rpm for 1 minute, rinsed, and fixed with 0.5% glutaraldehyde on glass slides. The cells were then washed and stained with X-gal solution for 4 hours until the entire night.

11. Tumor Response Assessment

The tumor response was assessed after every two cycles. Enhanced contrast-enhanced CT scanning was the standard (unless contraindicated). The maximum diameters of the tumors and Hounsfield units (HU; density estimate) were obtained. RECIST and Choi criteria for response were applied (Choi et al., 2007). Tumor markers were monitored if they were elevated at baseline.

12. Statistical Issues

The study sample size was determined by questions

114 of security. The population intended to treat (&gt; 1 dose) and the standard dose increase project were used. The possibility of dose increase, given the varying rates of DLT in the treated population, was calculated according to the Phase I dose increment testing routine.

B. Results

1. Patient Characteristics

Fourteen patients were enrolled (characteristics listed in Table 6; profile of the trial in Figure 20). Three patients were treated in cohorts 1-2, six in the third and two in the highest. Six patients were treated in cohort 3 at the request of the DSMB due to an unrelated patient's death, attributed to tumor progression. Two patients (cohorts 1, 3) had treatment stopped after a cycle due to unrelated adverse events, and patients at the highest dose received one due to DLT (see below).

Table 6 Patient Demography

<td>Average age (years)</td><td> 56 5 (38 - 66)</td>

<td>Sex</td><td>11 men, 3 women</td>

<td>Average previous therapies</td><td> 5 6 (2-12)</td>

<td>Tumor size (cm)</td><td> 6'9 (3 5 - 9 8)</td>

<td>JX-594 cycles received</td><td> 3 4 (1-8)</td>

<td>Tumor types</td><td>colon (4), HCC (3), melanoma (2), RCC (1), Thymic SCC (1), SCC-lung (1), gastric (1), extragonadal germ cell (1)</td>

Treatment-Related Toxicity

Adverse Episodes (AE)

JX-594 was well tolerated until BAT (10⁹ pfu). No treatment-related deaths occurred in the study. All patients experienced grade 1-2 flu-like symptoms (4-16 hours after treatment). Drug-related hypotension (grade 2, no organ dysfunction) occurred within 4-12 hours. Table 7 lists the most common AEs possibly related to JX-594. Only one case of severe AE (anorexia and abdominal pain) was judged to be related to treatment. Ten AEs

115 severe and unrelated (according to the PI) were attributed to complications associated with tumor progression. Four patients died due to the progression of tumors during the reporting period of AEs.

Two patients in cohort 4 experienced DLTs. Both experience enhanced Grade 3 direct hyperbilirubinemia due to tumor expansion and intrahepatic bile duct obstruction, plus anorexia and abdominal pain Grade

III.

B. Laboratory Data

Transient decreases related to treatment in lymphocytes, platelets and hematocrits were noted during the first 3 days. Nine patients had a significant increase in absolute neutrophil counts (ANC) within the first four days (seven increased&gt; 100%; Figure 2A). Increases in ANCs were dose-related and often associated with detection of GM-CSF in the blood. ANC increased significantly (£ 5,000 / pL) in 75% of patients in cohorts 3 and 4 (versus 17% in cohorts 1, 2; Figure 21 A); increases in monocytes and eosinophils were observed. Thrombocytopenia was also dose dependent (Figure 22A), but independent of the cycle (Figure 22B). The increases in ANCs were greater in cycle 1 (Figure 22C). Lymphopenia and leukopenia occurred in 20 patients in 2 patients (Table 7). Significant transaminitis did not occur in BAT (Figure 21B).

116

Table 7: Most Common Adverse Episodes (including Grade 1/2 AEs Experienced by&gt; 3 Patients and Grade 3/4 AEs Experienced by&gt; 1 Patient) Possibly Related to JX-594

<td colspan="3">Patients Totals (n = 14)</td><td> (14) 100%</td><td>(14) 100% i</td><td>(6) 43% I_</td><td> (10)71%</td><td> (3)21%</td><td>_; (4) 29% !</td><td> (2) 14%</td><td> (2) 14%</td><td> (3) 21%</td><td> (2) 14%</td><td> (2) 14%</td><td> (1)7%</td><td> (2) 14%</td><td> (4)29%</td><td> (3) 21%</td><td> (3)21%</td><td> (2) 14%</td><td> (4)29%</td>

<td rowspan="12">Number of Patients per Cohort</td><td rowspan="4">Grade 4 (5)</td><td>CM II ç, xT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>co II Ç &lt;* r</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>2 (n = 3)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>1 (n = 3)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td rowspan="4">Grade 3</td><td>4 (n = 2)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td><td> -</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>3 (n = 6)</td><td> -</td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td><td></td><td> -</td><td></td><td>5T “</td><td></td><td> -</td><td></td><td></td><td></td><td></td>

<td>2 (n = 3) '</td><td></td><td> -</td><td></td><td></td><td></td><td></td><td></td><td>T "</td><td></td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td><td>CM</td><td></td>

<td>1 (n = 3)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> -</td><td> -</td><td> -</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td rowspan="4">= 3 β O</td><td>4 (n = 2)</td><td>CM</td><td>CM</td><td> -</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> -</td><td></td><td></td><td></td><td></td>

<td>3 (n = 6)</td><td>IO</td><td>CO</td><td>co</td><td>OU</td><td> -</td><td>CM</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td> -</td><td> -</td><td></td><td>CM</td>

<td>I 2 (n = 3) I_</td><td>CO</td><td>CM</td><td>CM</td><td>CM</td><td> -</td><td> -</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> -</td><td></td><td> -</td>

<td>CO II ç</td><td>co</td><td>CO</td><td></td><td>CM</td><td> -</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> -</td><td></td><td></td><td></td><td></td><td> -</td>

<td colspan="3">Episode</td><td>Fever</td><td>Chills</td><td>Fatigue</td><td>Anorexia</td><td>Nausea</td><td>Headache</td><td>^! Hyponatremia</td><td>Increased alkaline phosphatase</td><td>Hyperbilirubinemia</td><td>ALT increased</td><td>Increased AST</td><td>Hypophosphatemia</td><td>Decreased fibrinigen</td><td>Leukocyte count increased</td><td>Platelet count decreased</td><td>Leukopenia</td><td>Neutrophil count decreased</td><td>Generalized pain</td>

<td colspan="3">System Corporal</td><td colspan="3">General</td><td colspan="2">Gastroin- testinal</td><td>System Nervous</td><td colspan="7">Metabó- lico / La- boratory</td><td colspan="4">Hema- tological</td><td>Ache</td>

117

3. Pharmacokinetic and Pharmacodynamic Results

The. GM-CSF Serum

Thirteen patients were negative when serum GM-CSF at baseline. Three patients in BAT had detectable GM-CSF&gt; 48 hours (46 - 16,000 pg / mL) after injection of JX-594 (Figure 21A), concentrations that were higher than those reported after subcutaneous injection of GM protein -CSF in patients (Cebon et al, 1992). GM-CSF concentrations correlated with WBC induction (Figure 21A).

B. Neutralizing Antibodies (NAb)

Low (&lt;10) or undetectable levels of antiJX-594 antibody (NAb) were noted in 79% of patients. All patients developed NAb within 22 days. NAb titrations peaked after the first dose in 45% of patients, and increased further by 55%.

No correlation was observed between baseline or post-treatment NAb titrations and any clinical or laboratory results, including pharmacokinetics, JX-594 replication, GM-CSF expression or efficacy. Three patients with objective RECIST tumor responses had detectable baseline NAb titrations and high titers after treatment (32,000, 32,000, and 10,000). In addition, two patients had newly developed neck metastases, treated after induction of high levels of NAb, and both tumors underwent objective responses (below; Table 8 and Figure 24B).

118

Table 8. Target tumor responses and duration of survival

<td colspan="2">Survival⁵</td><td>CO AND oo 2nd&gt;</td><td colspan="2">11 + m</td><td colspan="2">122 + m I</td><td>to AND tn</td><td>AND σ&gt; oo</td><td>AND O</td><td> 8^-2 m</td><td>AND in is-</td><td>T8m</td><td>CO AND Ό- Ó&gt;</td><td>AND ÇO</td><td>TD O</td><td>3 + m</td><td>18 + d</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>k_ O</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &gt;5 ©*»</td><td>xp © * “</td>

<td>XJ</td><td>CD</td><td></td><td>CO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>m</td>

<td>CD</td><td>u. O</td><td></td><td>CO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>co</td><td>co</td>

<td>O</td><td rowspan="2">AND 3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> ·*—*·</td>

<td> (0</td><td>CD</td><td>QC</td><td></td><td>CD</td><td></td><td>CD</td><td>Q</td><td>CD</td><td>CD</td><td>O</td><td>O</td><td>Ω</td><td>CD</td><td>CD</td><td>X</td><td>x</td>

<td> 2</td><td>H-</td><td>d</td><td>CL</td><td></td><td>d</td><td></td><td>d</td><td>to</td><td>d</td><td>d</td><td>CO</td><td>Q.</td><td>CO</td><td>ç</td><td>d</td><td>CL</td><td>CL</td>

<td></td><td></td><td></td><td></td><td></td><td>σ '·</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>σ&gt; φ</td><td>xp o ^ *</td><td> 29</td><td>xo θ '*</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>ç</td><td>CO</td><td></td><td>CM</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>r-</td><td>L- '</td><td>xr</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>O</td><td></td><td>O</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>PET</td><td></td><td>at</td><td>Fig.</td><td>Neck:</td><td>Fig.</td><td>Neck:</td><td> +40%</td><td>s ° O** +</td><td>at</td><td>1 at</td><td> 1 -6%</td><td> +55%</td><td>CO LO +</td><td>at</td><td>CD d</td><td> -41%</td><td>! at</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>CD</td><td>CD</td><td>CD</td><td>CD</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>TD</td><td>TD</td><td>TD</td><td>TD</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>xp o ^ *</td><td>&gt; p</td><td>xp σ '</td><td></td><td>&gt; p O**</td><td>&gt; e σ '</td><td>&gt; p © * ·</td><td>σ '</td><td>&gt; p O**</td><td>xp σ '</td><td>xp © **</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>O</td><td>Ç-</td><td>CO</td><td></td><td>CM</td><td>m</td><td>CO</td><td></td><td>O</td><td>oo</td><td>co</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>m</td><td>CO</td><td>in</td><td>CO</td><td>in</td><td>^ J ·</td><td>T "</td><td></td><td>CO</td><td>xr</td><td>CM</td><td>co</td><td>co</td><td></td><td></td><td></td>

<td>- £ Z</td><td></td><td> 7-&gt;</td><td> 7&gt;</td><td> 7-&gt;</td><td></td><td></td><td> —&gt;</td><td> 7*</td><td></td><td> 7~&gt;_</td><td> 72.</td><td>• Ç77</td><td> -7»</td><td>CD</td><td>CD</td><td>CD</td><td>CD</td>

<td>O</td><td></td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td>T</td><td>T</td><td> +</td><td> +</td><td>Τ '</td><td>d</td><td>d</td><td>d</td><td>d</td>

<td></td><td></td><td></td><td>or</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>CN</td><td></td><td></td><td>CL</td><td>C £</td><td>CL</td><td>O</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>CL</td><td></td><td rowspan="2">to</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>to</td><td></td><td></td><td>O</td><td></td><td>O</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>O</td><td></td><td></td><td>ad</td><td>O</td><td>ad</td><td>O</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>co</td><td></td><td></td><td></td>

<td>LU</td><td></td><td>The:</td><td>σ&gt;</td><td>Φ</td><td>cn</td><td>Φ</td><td>O</td><td>O</td><td>O</td><td>O</td><td>Q</td><td>O</td><td>d</td><td>CD</td><td>CD</td><td>CD</td><td>here</td>

<td>oc</td><td></td><td>CL</td><td>LL</td><td>z</td><td>LL</td><td>z</td><td>CO</td><td>to</td><td>co</td><td>to</td><td>CO</td><td>CO</td><td>CL</td><td>d</td><td>d</td><td>d</td><td>d</td>

<td></td><td> (/)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>O</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>O</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>O O</td><td>_O</td><td>CD</td><td>ÇO</td><td></td><td>ÇO</td><td></td><td rowspan="2"></td><td></td><td></td><td>ÇO</td><td>IO</td><td>ÇM</td><td>ÇM</td><td>y-</td><td></td><td></td><td></td>

<td>O</td><td>CD</td><td></td><td>CM</td><td></td><td>CO</td><td></td><td></td><td>CM</td><td></td><td>CM</td><td>CO</td><td>CO</td><td></td><td>co</td><td></td><td></td>

<td>1 ω</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>AND</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>AND</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Φ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>AND</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>AND</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>&lt;cd</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>O</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Ό</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> *”“</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>O AND</td><td></td><td>in</td><td></td><td></td><td> £2</td><td></td><td></td><td></td><td>m / 4)</td><td></td><td>(O AND</td><td></td><td></td><td></td><td>m / 7)</td><td></td><td></td>

<td> 3</td><td></td><td>AND</td><td></td><td></td><td>AND</td><td></td><td></td><td></td><td>O</td><td></td><td>φ</td><td></td><td></td><td></td><td> 0</td><td></td><td></td>

<td>Φ TD</td><td>res)</td><td>O oo look&gt;</td><td>m / 5)</td><td></td><td>O 00 S- m</td><td></td><td>m / 5)</td><td rowspan="2">n // 90cm / 6]</td><td>1- çn 73</td><td>cm / 4)</td><td rowspan="2">co dog TD CD ç O O)</td><td>cm / 5)</td><td>cm / 11</td><td>5cm / 6</td><td>a&gt; O CD</td><td>m / 12)</td><td>cT AND</td>

<td>O Q.</td><td>ytterium</td><td>O) Ç 3</td><td>O CM co</td><td></td><td>! IUOU</td><td></td><td>O r ^. •B</td><td>thyn</td><td>ç</td><td>r ~ - ç</td><td>00 «N ~ c</td><td>ric / 8 ·</td><td>AND 0 ç</td><td>0 in co</td><td>0 00 σ&gt;</td>

<td rowspan="2">Φ</td><td>CD</td><td>O</td><td>O</td><td></td><td>CD</td><td></td><td>O</td><td>O</td><td>O</td><td>O</td><td>CD u.</td><td>O</td><td> 0</td><td>to</td><td>CD</td><td>O</td><td>O</td>

<td rowspan="2">CO O</td><td>O</td><td>O</td><td></td><td>Φ</td><td></td><td>O</td><td>O</td><td>O</td><td>O</td><td>R</td><td>O</td><td> 0</td><td>co</td><td>Φ</td><td>O</td><td>O</td>

<td rowspan="2">Ç Φ O</td><td>to</td><td>X</td><td></td><td> 2</td><td></td><td>çc</td><td>P</td><td>co</td><td>P</td><td>Ψ</td><td>P</td><td>P</td><td>P</td><td>Z</td><td>X</td><td>X</td>

<td rowspan="2">ç Φ</td><td>CO</td><td></td><td></td><td>Tf</td><td></td><td>t—</td><td>CM</td><td>CM</td><td>CM</td><td>CO</td><td>m</td><td>co</td><td>V *</td><td> &lt;0</td><td></td><td>CM</td>

<td>CD</td><td>O</td><td>O</td><td></td><td>O</td><td></td><td>O</td><td>O</td><td>O</td><td>O</td><td>O</td><td>O</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td>O</td>

<td>CL</td><td>AND</td><td></td><td>CM</td><td></td><td>CO</td><td></td><td>CO</td><td>CO</td><td>CM</td><td></td><td>CM</td><td>co</td><td>co</td><td></td><td>co</td><td>^ r</td><td></td>

119 ¹ First number reflects the dose level (for example, 103 was at dose level 1) ² RECIST criteria: partial response (PR) is a maximum decrease in diameter&gt; 30%; progressive disease (PD) is an increase&gt; 20%; stable disease is a change in diameter between these two hops for PR and PD³ Choi criteria: maximum decrease in diameter&gt; 10% or decrease in density&gt; 15%; + indicates answer⁴ Definition of tumor marker response: decrease of&gt; 50%: PR; increase&gt; 25%: PD; decrease &lt;50% or increase of 25%: SD; the marker was alpha-fetoprotein (AFP) in patients 201, 301, 402; PIVKA2 to 401; carcinoembryonic antigen (CEA) to 302, 305, 306.

⁵ Survival: + indicates no cancer-related deaths; m: months; d: days⁶ still alive ⁷ HU: Hounsfield Units ⁸ CT scans performed at week 3 showed tumor progression

ç. Cytokines interleukin-6, IL-10, and TNF-α peaked in 3 hours. Later peaks (day 3 - 22) were also observed. Cytokine induction was greater in cycles 2-8 than in cycle 1. Interleukin-6 induction correlated with GM-CSF in serum. The induction of IL-1 β and IL-4 was not noticed.

d. Pharmacokinetics of JX-594

All patients had JX-594 genomes detected immediately after the injection (49 out of 50 cycles). The concentrations correlated with the dose (Figures 23A and 23B), decreasing -50% within 15 minutes and -90% within 4-6 hours. The initial clearance rates were neither dose-dependent nor antibody-titer dependent. After the initial release and clearance of JX-594 injected into the blood, the delayed resurgence of circulating JX-594 was frequently detected, consistently with replication. Twelve out of 15 (80%) of the patients had detectable genomes (blood or plasma) between days 3 - 22. Secondary peak concentrations were generally correlated with the dose, and

120 pharmacokinetics were similar (Figure 23B). Lower concentration peaks were detected after repeated dosing in cycles 2-7 (4 out of 11 patients). Representative pharmatics are shown in Figure 23C.

and. Dissemination of JX-594, replication within distant tumor sites that have not received an injection

JX-594 was detected in tumor tissues that did not receive an injection, indicating selective infection and replication in distant tumors (Figure 23D-F). For example, a patient's malignant ascites and pleural effusion had higher concentrations of the genomes (17 and 12 times higher, respectively) and GM-CSF concentrations (24 and 13 times higher, respectively) than in blood in the same time point (Figure 3D). LacZ (+) cells in the pleural effusion confirmed infection with JX-594 (Figure 23E). Another patient had a distant neck tumor that underwent biopsy and a replicating JX-594 was demonstrated histologically (Figure 23F).

f. Excretion of JX-594

No infectious JX-594 was detected in any throat or urine sample.

4. Antitumor Efficacy of JX-594

Ten patients were evaluable for target tumor responses; non-evaluable patients had contraindications for contrast (2) or no post-treatment scan (2). Nine (90%) had an objective response (30%) or stable disease (60%) by RECIST criteria with CT. Eight (80%) had objective responses using the Choi criteria (Table 8). Patients responding to the RECIST and Choi criteria had non-small cell lung cancer (Figure 24A), HCC and melanoma (Table 8). The objective responses were durable; Regrowth in responding tumor sites did not occur (4-18 months of follow-up). Direct injection into tumors that had not previously been injected into the neck in two patients, after 4 previous cycles in the liver, led to Choi and / or RECIST responses despite high-level neutralizing antibodies for JX594 (Table 8, Figure 24B); therefore, the effectiveness of retreatment was feasible.

121

Responses in distant tumors that did not receive an injection were also evaluated. Among seven patients with distant tumors who did not receive an injection, six patients had distant stable disease according to RECIST criteria; the time for progression of these distant tumors was 5 in the range between 6+ and 30+ weeks. Three of these patients had responses according to Choi (n = 2) or PET-CT criteria (n = 1; decrease of 25-100%; Table 9). Table 9. Distant Tumor Responses in Patients with Targeted Tumor Control (RECIST PR or SD)

<td>Patient (dose 9P¹)</td><td>Size of distant tumor (cm) / location</td><td>RECIST</td><td>Choi</td><td>PET</td><td>Time for progression tumor²</td>

<td> 202</td><td>5'9 / Liver</td><td>SD</td><td>+ (4-35% HU)</td><td>not available</td><td>30+ weeks</td>

<td> 103</td><td>8'5 / Liver</td><td>SD</td><td>+ (4-22% HU)</td><td>Not available</td><td>7+ weeks</td>

<td> 304</td><td>7 / face, mediasti- at the</td><td>at</td><td>at</td><td>CR - SC tumor PR - PA tumor</td><td>6+ weeks</td>

<td> 102</td><td>4 3 / Liver</td><td>SD</td><td>- (4-10% HU)</td><td>at</td><td>9 weeks</td>

<td> 203</td><td>8'3 / LNs</td><td>SD</td><td>- (5%)</td><td>-6% (12 weeks)</td><td>15 weeks</td>

<td> 201</td><td>3'7 / Liver</td><td>SD</td><td> -(4-6%)</td><td>SD</td><td>18 weeks</td>

<td> 301</td><td>11'8 / Liver</td><td>PD</td><td>- (Í22%)</td><td> +10%</td><td>6 weeks</td>

First number reflects the dose level (for example, 103 was at dose level 1) ² by CT RECIST; + indicates no cancer-related deaths

HU: Hounsfield Units; LN: lymph nodes; SC: supraclavicular; PA: pre-auricular; CR: complete answer; PR: partial response; SD: stable disease; PD: progressive disease

So far, eight patients (57%) have survived for at least 8 months, four for more than a year and one for up to 20+ months. The median survival was 9 months.

All compositions and / or methods described and claimed herein can be made and performed without undue experimentation in the light of this description. Although the compositions and methods of this invention have been described in terms of preferred embodiments, it should be evident to those skilled in the art that variations can be applied to the compositions and / or methods and in the steps or sequence of steps of the method described here without escaping of the concept, spirit and scope of the invention. More specifically, it should be evident that certain agents that are chemically and also physiologically related can replace the agents described herein, and at the same time, the same or similar results would be achieved. All such substitutes and similar modifications evident to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

The following references, to the extent that they provide details of procedures or other exemplary details in addition to those set out herein, are hereby specifically incorporated by reference.

US Patent 4,554,101

US patent 4,683,202

US Patent 4,684,611

US patent 4,952,500

US Patent 5,073,627

US Patent 5,302,523

US patent number 5,322,783

US patent number 5,384,253

US patent number 5,399,363

US patent number 5,464,765

US patent number 5,466,468

US patent number 5,538,877

US patent number 5,538,880

US patent number 5,543,158

US patent number 5,550,318

US patent number 5,563,055

US patent number 5,580,859

US patent number 5,589,466

123

US patent number 5,591,616

US patent number 5,610,042

US patent number 5,633,016

US patent number 5,641,515

US patent number 5,656,610

US patent number 5,702,932

US patent number 5,736,524

US patent number 5,739,169

US Patent 5,780,448

US patent 5,789,215

US patent 5,798,339

US patent number 5,801,005

US patent number 5,824,311

US patent number 5,824,348

US patent number 5,830,880

US patent number 5,846,225

US patent number 5,846,233

US patent number 5,846,945

US patent number 5,925,565

US patent number 5,928,906

US patent number 5,935,819

US patent number 5,945,100

US patent number 5,981,274

US patent number 5,994,624

Alcami and Smith, Cell., 71 (1): 153-167, 1992.

Alcami et al., J. Gen. Virol., 80: 949-959, 1999.

Alcami etal., Sem. Virol., 5: 419-427, 1998.

Alcami etal., Virology, 74 (23): 11230-11239, 2000.

Almendro etal., J. Immunol., 157 (12): 5411-5421, 1996.

Arap et al., Cancer Res., 55 (6): 1351-1354, 1995.

Arness et al., Am. J. Epidemiol., 160: 642-51, 2004.

Austin-Ward and Villaseca, Revista Medica de Chile, 126 (7): 838-845, 1998.

124

Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley &amp; Sons, Inc, NY, 16.15.1-16.18.10, 1996.

Bajorin etal., J. Clin. Oncol., 6 (5): 786-792, 1988.

Bakhshi etal., Cell, 41 (3): 899-906, 1985.

Blasco and Moss, J. Virology, 66 (7): 4170-4179, 1992.

Blasco et al., J. Virology, 67 (6): 3319-3325, 1993.

Boyd et at., Cell, 79: 341-351, 1994.

Brizel, Semin. Radiat. Oncol., 8 (4): 237-246, 1998.

Bukowski etal., Clinicai Câncer Res., 4 (10): 2337-2347, 1998.

Caldas et al., Cancer Res., 54: 3568-3573, 1994.

Carbonelli etal., FEMS Microbiol. Lett., 177 (1): 75-82, 1999.

Cebon et al., Br. J. Haematol., 80 (2): 144-150,1992.

Chandleref al., Proc. Natl. Acad. Know. USA, 94 (8): 3596-601, 1997.

Chen and Okayama, Mol. Cell Biol., 7 (8): 2745-2752, 1987.

Cheng etal., Cancer Res., 54 (21): 5547-5551, 1994.

Choi etal., J. Clin. Oncol., 25 (13): 1753-1759, 2007.

Christodoulides etal., Microbiology, 144 (Pt 11): 3027-3037, 1998.

Cleary and Sklar, Proc. Natl. Acad. Know. USA, 82 (21): 7439-7443, 1985.

Cleary etal., J. Exp. Med., 164 (1): 315-320, 1986.

Cocea, Biotechniques, 23 (5): 814-816, 1997.

Colamonici etal., J. Biol. Chem., 270: 15974-15978, 1995.

Culveref al., Science, 256 (5063): 1550-1552, 1992.

Curran, Seminars Radiation Oncol., 8 (4Sup 1): 2-4, 1998.

Davidson etal., J. Immunother., 21 (5): 389-398, 1998.

Dillman, Cancer Biother. Radiopharm., 14 (1): 5-10, 1999.

Dobbelstein and Shenk, J. Virology, 70: 6479-6485, 1996.

Durrant and Spendlove, Curr. Opin. Investig. Drugs, 2 (7): 959-66, 2001. Eliopoulos etal., Oncogene, 11 (7): 1217-28, 1995.

Erlandsson, Cancer Genet. Cytogenet., 104 (1): 1-18, 1998.

Fechheimer et al., Proc Natl. Acad. Know. USA, 84: 8463-8467, 1987.

Fraley etal., Proc. Natl. Acad. Know. USA, 76: 3348-3352, 1979.

GenBank Accession Number NC001559

125

Gnant et al., Ann Surg, 230 (3): 352-360, 1999.

Gnant etal., CancerRes., 59 (14): 3396-403, 1999.

Gnant et al., J. Natl. Cancer Inst., 91 (20): 1744-1750, 1999.

Goebel etal., Virology, 179 (1): 247-266,517-563, 1990.

Gopal, Mol. Biol Cell., 5: 1188-1190, 1985.

Graham and Van Der Eb, Virology, 52: 456-467, 1973.

Graham etal., Virology, 229 (1): 12-24, 1997.

Gross etal., Genes Dev., 13 (15): 1899-911, 1999.

Gross etal., J. Biol. Chem., 274: 1156-1163, 1999.

Hanibuchi etal., Int. J. Cancer, 78 (4): 480-485, 1998.

Harland and Weintraub, J. Cell Biol., 101 (3): 1094-1099, 1985.

Heise etal., Cancer Gene Ther., 6 (6): 499-504, 1999.

Heise etal., Cancer Res., 59 (11): 2623-2628, 1999.

Hellstrand et al., Acta Oncologica, 37 (4): 347-353, 1998.

Hermiston, J. Clin. Invest., 105: 1169-1172, 2000.

Ho etal., J. Biol. Chem., 27.77Q5-77Q9, 1998.

Homey et al., Nature. Rev. Immunol., 2: 175-184, 2002.

Hui and Hashimoto, Infection Immun., 66 (11): 5329-5336, 1998. Hussussian et al., Nat. Genet., 8 (1): 15-21, 1994.

Ikeda etal., Nat. Med., 5 (8): 881-7, 1999.

Inouye and Inouye, Nucleic Acids Res., 13: 3101-3109, 1985.

Irie and Morton, Proc. Natl. Acad. Know. USA, 83 (22): 8694-8698, 1986. Irie etal., Lancet., 1 (8641): 786-787, 1989.

Isaacs et al., Proc. Natl. Acad. Know. USA, 89 (2): 628-32, 1992. Johnson and Hamdy, Oncoí Rep., 5 (3): 553-557, 1998.

Ju etal., Gene Ther., 7 (19): 1672-1679, 2000.

Ju et a!., J. Neuropathol. Exp. Neurol., 59 (3): 241-250, 2000. Kaeppler et al., Plant Cell Reports, 9: 415-418, 1990.

Kamb etal., Nat. Genet., 8 (1): 23-26, 1994.

Kamb etal., Science, 2674: 436-440, 1994.

Kaneda etal., Science, 243: 375-378, 1989.

Katoetal, J. Biol. Chem., 266: 3361-3364, 1991.

126

Kay et al., Proc. Natl. Acad. Know. USA, 94 (9): 4686-4691, 1997.

Kerr et al., Br. J. Cancer, 26 (4): 239-257, 1972.

Kettle etal., J. Gen. Virology, 78: 677-685, 1997.

Kirn etal., Nat. Med., 7: 781-787, 2001.

Kolmel, J. Neurooncol., 38 (2-3): 121-125, 1998.

Kraus et al. FEBS Lett., 428 (3): 165-170, 1998.

Kulesh et al., J. Clin. Microbiol., 42 (2): 601-609, 2004.

Kyte and Doolittle, J. Mol. Biol., 157 (1): 105-132, 1982.

Lareyre etal., J. Biol. Chem., 274 (12): 8282-8290, 1999.

Lee et al, Biochem. Biophys. Res. Commun., 238 (2): 462-467, 1997. Levenson etal., Hum. Gene Ther., 9 (8): 1233-1236, 1998.

Liebermann, Oncogene, 17 (10): 1189-94, 1998.

Macejak and Sarnow, Nature, 353: 90-94, 1991.

Magi-Galluzzi etal., Anal. Quant. Cytol. Histol., 20 (5): 343-350, 1998.

Mangray and King, Front Biosci., 3: D1148-1160, 1998.

Marsters et al., Recent Prog. Horm. Res., 54: 225-234, 1999.

Mastrangelo etal., Cancer Gene Ther., 6 (5): 409-422, 1999.

Mastrangelo et al., Cancer Treat Res., 94: 35-50,1998.

Mayer etal., Radiat. Oncol. Investig., 6 (6): 281-288, 1998.

McCartefa /., Gene Ther., 7 (14): 1217-23, 2000.

Mitchell et al., Ann. NY Acad. Sci., 690: 153-166, 1993.

Mitchell et al., J. Clin. Oncol., 8 (5): 856-869, 1990.

Mori et al., Cancer Res., 54 (13): 3396-3397, 1994.

Morton et al, Arch. Surg., 127: 392-399, 1992.

Moss, In: Fields Virology., Fields (Ed.), Lippincott-Raven Publishers: Philadelphia, 2637-2672, 1996.

Mossman et al., Virology, 215 (1): 17-30, 1996.

Mougin et al., Ann. Biol. Clin., (Paris) 56 (1): 21-8, 1998.

Mumby and Walter, Cell Reguí, 2 (8): 589-98, 1991.

Natoli et al, Biochem. Pharmacoí, 56 (8): 915-20, 1998.

Nicolau and Sene Nobori et al, 1994 Nomoto etal., Gene, 236 (2): 259-271, 1999.

127

Ochi etal., Am. J. Gastroenterol., 93 (8): 1366-1368, 1998.

Ohara, Gan To Kagaku Ryoho, 25 (6): 823-828, 1998.

Okamoto etal., Proc. Natl. Acad. Know. USA, 91 (23): 11045-11049, 1994. Omirulleh et al., Plant Mol. Biol., 21 (3): 415-428, 1993.

Orlow etal., Cancer Res, 54 (11): 2848-2851, 1994.

PCT patent application n⁹ WO 94/09699

PCT patent application n⁹ WO 95/06128

Pelletier and Sonenberg, Nature, 334 (6180): 320-325, 1988.

Pietras etal., Oncogene, 17 (17): 2235-2249, 1998.

Puhlmann et al., Cancer Gene Ther., 7: 66-73, 2000.

Qin etal., Proc. Natl. Acad. Know. USA, 95 (24): 14411-14416, 1998. Ravindranath and Morton, Intem. Rev. Immunol., 7: 303-329, 1991. Remington's Pharmaceutical Sciences 15- Edition, 1035-1038 and 15701580, 1990.

Rippe, etal., Mol. Cell Biol., 10: 689-695, 1990.

Rosei etal., J. Viroi, 60 (2): 436-449, 1986.

Rosenberg et al, Ann. Surg. 210 (4): 474-548, 1989.

Rosenberg etal., N. Engí. J. Med., 319: 1676, 1988.

Sambrook et al, In: Molecular cloning: a laboratory manual, 2^na Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Saraiva and Alcami, J. Virology, 75 (1): 226-33, 2001.

Serrano et al, Nature, 366: 704-707, 1993.

Serrano et al., Science, 267 (5195): 249-252, 1995.

Sinkovics and Horvath, J. Clin. Viro., 16: 1-15, 2000.

Smith et al., Immunol. Rev., 159: 137-154, 1997.

Smith et al., J. Clin. Oncoí, 18: 2046-2052, 2000.

Smith etal., Neuron., 20: 1093-1102, 1998.

Solyanik et al, Cell. Prolif., 28 (5): 263-278, 1995.

Spriggs et al., Cell., 71 (1): 145-52, 1992.

Stokke et al, Cell. Prolif., 30 (5): 197-218, 1997.

Symons et al, Cell, 81: 551-560, 1995.

Todo et al., Cancer Res., 61: 153-161, 2001.

128

Tsujimoto and Croce, Proc. Natl. Acad. Know. USA, 83 (14): 5214-5218, 1986. Tsujimoto etal., Nature, 315: 340-343, 1985.

Tsujimoto etal., Science, 228 (4706): 1440-1443, 1985.

Tsumaki etal., J. Biol. Chem., 273 (36): 22861-22864, 1998.

Upton et al., Science, 258 (5086): 1369-1372,1992.

Upton etal., Virology, 184 (1): 370-82, 1991.

Vanderplasschen et al, Proc. Natl. Acad. Know. USA, 95 (13): 7544-7549, 1998. Vicari and Caus, Cytokine Growth Factor Rev., 13: 143-154, 2002.

Vogelstein and Kinzler, Cell, 70 (4): 523-6, 1992.

Wallach et al, Annu. Rev. Immunoí, 17: 331-367,1999.

Wold et al, J. Viroí. 52: 307-313, 1984.

Wold et al., Trends Microbioí, 2: 437-443, 1994.

Wong et al, Gene, 10: 87-94, 1980.

Wu etal., Biochem. Biophys. Res. Commun., 233 (1): 221-226, 1997.

Zhao-Emonet et al, Biochim. Biophys. Acta, 1442 (2-3): 109-119, 1998.