WO1999020311A1

WO1999020311A1 - METHODS FOR CANCER IMMUNOTHERAPY USING RETROVIRAL VECTORS EXPRESSING η INTERFERON

Info

Publication number: WO1999020311A1
Application number: PCT/US1998/022198
Authority: WO
Inventors: Tim Fong; Douglas J. Jolly; James Merritt; Dale Ando
Original assignee: Chiron Corporation
Priority date: 1997-10-21
Filing date: 1998-10-21
Publication date: 1999-04-29
Also published as: AU1107199A

Abstract

The present invention provides methods for inhibiting the growth of solid tumors utilizing hη-IFN recombinant viral vectors. Briefly, within one aspect of the present invention, a method for inhibiting the growth of a solid tumor is provided comprising the step of the directly administering to a patient a vector construct which directs the expression of the hη-IFN gene, such that the growth of said tumor is inhibited.

Description

METHODS FOR CANCER IMMUNOTHERAPY USING RETROVIRAL VECTORS EXPRESSING INTERFERON

Technical Field

The present invention relates generally to the field of cancer immunotherapeutics, .and more specifically, to methods of inhibiting the growth of a solid tumor utilizing vector constructs.

Background of the Invention

Cancer accounts for one-fifth of the total mortality in the United States, and is the second leading cause of death. Cancer is typically characterized by the uncontrolled division of a population of cells. This uncontrolled division typically leads to the formation of a tumor, which may subsequently metastasize to other sites.

Primary solid tumors can generally be treated by surgical resection. However, the majority of patients which have solid tumors also possess micrometastases beyond the primary tumor site. If treated with surgery alone, approximately 70% of these patients will experience recurrence of the cancer. In addition to surgery, many cancers are now also treated with a combination of therapies involving cytotoxic chemotherapeutic drugs (e.g., vincristine, vinblastine, cisplatin, methotrexate, 5-FU, etc.) anoVor radiation therapy. One difficulty with this approach however, is that radiotherapeutic and chemotherapeutic agents are toxic to normal tissues, and often create life-threatening side effects. In addition, these approaches often have extremely high failure/remission rates (up to 90% depending upon the type of cancer).

In addition to chemo- and radiation therapies, many have attempted to bolster or augment an individual's own immune system in order to eliminate the cancer cells. Several immunotherapies have utilized bacterial or viral components as adjuvants, in order to stimulate the immune system to destroy the tumor cells. Examples of such components include BCG, endotoxin, mixed bacterial vaccines, interferons (α, β, and γ), interferon inducers (e.g., Brucella abortus, and various viruses), and thymic factors (e.g., thymosin fraction 5, and thymosin alpha- 1) (see generally "Principles of Cancer Biotherapy," Oldham (ed.), Raven Press, New York, 1987). Such agents have generally been useful as adjuvants and as nonspecific stimulants in animal tumor models, but have not yet proved to be generally effective in humans.

Lymphokines have also been utilized in the treatment of cancer. Briefly, lymphokines are secreted by a variety of cells, and generally have an effect on specific cells in the generation of an immune response. Examples of lymphokines include TNFα, IFN-α,β,γ, Interleukins (IL)-l, -2, -3, -4, .and -12 as well as colony stimulating factors such as G-CSF, GM-CSF, and M-CSF. Recently, one group has utilized IL-2 to stimulate peripheral blood cells in order to expand and produce large quantities of cells which are cytotoxic to tumor cells (Rosenberg et al., N. Engl. J. Med. 575:1485-1492, 1985).

Others have suggested the use of antibody-mediated anti-cancer therapies. Briefly, antibodies may be developed which recognize certain cell surface antigens that are either unique, or more prevalent on cancer cells compared to normal cells. These antibodies, or "magic bullets," may be utilized either alone or conjugated with a toxin in order to specifically target and kill tumor cells (Dillman, "Antibody Therapy," Principles of Cancer Biotherapy, Oldham (ed.), Raven Press, Ltd., New York, 1987). For example, Ball et al. (Blood (52:1203-1210, 1983) treated several patients with acute myelogenous leukemia with one or more of several monoclonal antibodies specific for the leukemia, resulting in a marked decrease in circulating leukemia cells during treatment. Similarly, others have utilized toxin-conjugated antibodies therapeutically to treat a variety of tumors, including, for example, melanomas, colorectal carcinomas, prostate carcinomas, breast carcinomas, and lung carcinomas (see Dillman, supra). One difficulty however, is that most monoclonal antibodies are of murine origin, and thus hypersensitivity against the murine antibody may limit its efficacy, particularly after repeated therapies. Common side effects include fever, sweats and chills, skin rashes, arthritis, and nerve palsies.

Therefore, compositions and methods which augment natural host defenses against tumor induction or progression without the cytotoxic side effects of prior methods, may increase remission rates and enhance survival of patients with cancer. The present invention provides such compositions and methods, and further provides other related advantages.

Summary of the Invention

Briefly stated, the present invention is directed towards methods for inhibiting the growth of a solid tumor. Within one aspect of the invention, a method is provided for inhibiting the growth of a solid tumor in a patient, comprising the step of directly administering to the tumor 10⁶ to 10⁸ CFU of a human γ-interferon (hγ-IFN) construct such that the growth of the tumor is inhibited. Within one embodiment of the invention, the vector is administered daily for 5 days. Within another embodiment of the present invention, 5 x 10⁶ CFU, 10⁷ CFU, 5 x 10⁷ CFU, or 10⁸ CFU of the retroviral vector is administered daily. Within yet another embodiment of the invention, a pBA-5a, pBA-5b, and pBA-5c recombinant retroviral vector expressing hγ-IFN are provided.

Also provided by the present invention are pharmaceutical compositions comprising the above described hγ-IFN recombinant retroviral vector in combination with a pharmaceutically acceptable carrier or diluent.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.

Brief Description of the Drawings

Figure 1 is a graph which illustrates tumor growth following intratumoral injection of mγ-IFN retroviral vector into established B 16F 10 tumors in mice.

Figure 2 illustrates the correlation between tumor volumes of subcutaneous lesions .and lung metastases. Mice were given an injection of mγ-IFN retroviral vector into the subcutaneous lesion subsequent to a subcutaneous and intravenous inoculation with parental CT26 tumor cells.

Detailed Description of the Invention

As noted above, the present invention is directed generally towards methods of inhibiting the growth of a solid tumor utilizing a retroviral vector which directs the expression of hγ-IFN. Briefly, the ability to recognize and defend against foreign pathogens such as tumor cells is central to the function of the immune system. This system, through immune recognition, is capable of distinguishing "self from "nonself (foreign), and is essential to ensure that defensive mechanisms are directed towards invading entities rather than against host tissues. The methods which are described in greater detail below provide an effective means of inducing MHC unrestricted response, potent Class I-restricted or Class II-restricted protective .and therapeutic CTL responses, as well as humoral responses.

In particular, the present invention provides methods for inhibiting the growth of a solid tumor in a patent comprising the step of directly administering to the tumor a retroviral vector which directs the expression of hγ-IFN, such that the growth of the tumor is inhibited. Within the context of the present invention, "inhibiting the growth of a selected tumor" refers to either (1) the direct inhibition of tumor cell division, or

(2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells. Inhibition of tumor growth by either of these two mechanisms may be readily determined by one of ordinary skill in the art based upon a number of well known methods. For example, tumor inhibition may be determined by measuring the actual tumor size over a period of time. Alternatively, tumor inhibition may be determined by estimating the size of a tumor (over a period of time) utilizing methods well known to those of skill in the art. More specifically, a variety of radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Oncology," Winkler, C. (ed.) Springer- Verlag, New York, 1986), may be utilized to estimate tumor size. Such methods may also utilize a variety of imaging agents, including for example, conventional imaging agents (e.g., Gallium-67 citrate), as well as specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging (e.g., radiolabeled monoclonal antibody specific tumor markers). In addition, non-radioactive methods such as ultrasound (see, "Ultrasonic Differential Diagnosis of Tumors", Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984), may also be utilized to estimate the size of a tumor.

In addition to the in vivo methods for determining tumor inhibition discussed above, a variety of in vitro methods may be utilized in order to predict in vivo tumor inhibition. Representative examples include lymphocyte mediated anti-tumor cytolytic activity determined for example, by a ⁵¹Cr release assay, tumor dependent lymphocyte proliferation (Ioannides, et al., J Immunol. 146(5): 1700-1707, 1991), in vitro generation of tumor specific antibodies (Herlyn, et al., J. Immunol. Meth. 75:157-167, 1984), cell (e.g., CTL, helper T cell) or humoral (e.g., antibody) mediated inhibition of cell growth in vitro (Gazit, et al., Cancer Immunol. Immunother 55:135-144, 1992), and, for any of these assays, determination of cell precursor frequency (Vose, Int. J. Cancer 50:135-142 (1982).

Alternatively, for other forms of cancer, inhibition of tumor growth may be determined based upon a change in the presence of a tumor marker. Examples include prostate specific antigen ("PSA") for the detection of prostate cancer (see U.S. Patent No. Re. 33,405), and Carcino-Embryonic Antigen ("CEA") for the detection of colorectal and certain breast cancers. For yet other types of cancers such as leukemia, inhibition of tumor growth may be determined based upon the decreased numbers of leukemic cells in a representative blood cell count. A variety of tumors may be selected for treatment in accordance with the methods described herein. In general, solid tumors are preferred, although leukemias and lymphomas may also be treated if they have developed a solid mass, or if suitable tumor associated markers exist such that the tumor cells can be physically separated from nonpathogenic normal cells. For example, acute lymphocytic leukemia cells may be sorted from other lymphocytes with the leukemia specific marker " CALL A" .

Representative examples of suitable tumors include melanomas, colorectal carcinomas, lung carcinomas (including large cell, small cell, squamous and adeno-carcinomas), renal cell carcinomas, cervical carcinoma, head and neck carcinoma, prostate carcinoma, hepatic carcinoma, pancreatic carcinoma, breast adeno-carcinomas mesothelioma, and sarcomas of the soft tissue. Gamma interferon is an anti-tumor agent which acts as an immune activator to inhibit the growth of a selected tumor as discussed above. Briefly, immune activators function by improving immune recognition of tumor-specific antigens such that the immune system becomes "primed." Priming may consist of lymphocyte, activation, proliferation, differentiation, or evolution to higher affinity interactions. The immune system thus primed will more effectively inhibit or kill tumor cells. Immune activation may be subcategorized into immune modulators (molecules which affect the interaction between lymphocyte and tumor cell) and lymphokines, that act to proliferate, activate, or differentiate immune effector cells. Other representative examples of immune modulators include CD3, ICAM-1, ICAM-2, LFA-1, LFA-3, -2-microglobulin, chaperones, alpha interferon and gamma interferon, CD80, CD86 (B7.1 , B7.2), CD40, CDD40L, and major histocompatibility complex (MHC). Representative examples of lymphokines include, tumor necrosis factor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, GM-CSF, CSF-1, and G-CSF.

The hγ-IFN gene is inserted into a vector construct which directs its expression. Within the context of the present invention, a "vector construct" is understood to refer to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. The vector construct must include transcriptional promoter element(s), and preferably includes a signal which directs polyadenylation. In addition, the vector construct must include a sequence which, when transcribed, is operably linked to the sequence(s) or gene(s) of interest and acts as a translation initiation sequence.

Optionally, the vector construct may also include a selectable marker such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence. In addition, if the vector construct is placed into a retrovirus, the vector construct must include a packaging signal, long terminal repeats (LTRs), and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present).

As noted above, within one aspect of the present invention recombinant retroviruses are provided which carry a vector construct capable of directing the expression of hγ-IFN. The construction of such recombinant retroviral vectors is described in greater detail in an application entitled "Recombinant Retroviruses" (U.S.S.N. 07/586,603, filed September 21, 1990, which is hereby incorporated by reference in its entirety). These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S.S.N. 07/800,921).

Vector constructs of the present invention may also be developed and utilized with other viral carriers including, for example, poliovirus (Evans et al., Nature 559:385- 388, 1989, and Sabin, J. ofBiol. Standardization 7:115-118, 1973); rhinovirus; pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 56:317- 321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 5:17-21, 1990; U.S. Patent Nos. 4,603,112 and 4,769,330; WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114, 1979); influenza virus (Luytjes et al., Cell

59:1107-1113, 1989; McMicheal et al., The New England Journal of Medicine 509:13- 17, 1983; .and Yap et al., Nαtwre 275:238-239, 1978); adenovirus (Berkner, Biotechniques 6:616-627, 1988, and Rosenfeld et al., Science 252:431-434, 1991); parvovirus such as adeno-associated virus (S-amulski et al., Journal of Virology 65:3822- 3828, 1989, and Mendelson et al., Virology 766:154-165, 1988); heφes (Kit, Adv. Exp. Med. Biol. 275:219-236, 1989); SV40; HIV; lentivirus (Trono, et al. Science 236: 263, 1996) measles (EP 0 440,219); and Sindbis virus (Xiong et al., Science 234:1188-1191, 1989), .and corona virus. In addition, viral carriers may be homologous, non-pathogenic (defective), replication competent virus (e.g., Overbaugh et al., Science 259:906-910, 1988), .and nevertheless induce cellular immune responses, including CTL.

As noted above, a vector construct which directs the expression hγ-IFΝ is directly administered to the tumor. Various methods may be utilized within the context of the present invention in order to directly administer the vector construct to the tumor, such methods are described in greater detail in an application entitled "Recombinant Retroviruses" (U.S.S.Ν. 07/586,603), which is herein expressly incorporated by reference. For example, within one embodiment a small metastatic lesion may be located, and the vector injected several times in several different locations within the body of tumor. Alternatively, arteries which serve a rumor may be identified, and the vector injected into such an artery, in order to deliver the vector directly into the tumor. Within another embodiment, a tumor which has a necrotic center may be aspirated, and the vector injected directly into the now empty center of the tumor. Within yet another embodiment, the vector construct may be directly administered to the surface of the tumor, for example, by application of a topical pharmaceutical composition containing the vector construct, or preferably, a recombinant retroviral vector carrying the vector construct.

Within another aspect of the present invention recombinant retroviral vector constructs carrying the hγ-IFN gene can be administered to a solid tumor in a patient at various doses ranging between 10⁶ and 10¹⁰ CFU per day. Titers of retroviral vector range between 10⁷ and 10⁹ CFU/ml. Patients can be injected with 0.3 ml of hγ-IFN retroviral vector for 5 days. The tumor lesion may vary up to 10 to 12 cm in size, for example, in the case of soft tissue sarcoma, multiple myeoloma, or head and neck squamous cell carcinoma. Patients can receive escalating doses of 0.3, 0.5 or 1.0 ml/injection for 5 days. However, since the dose is dependent on tumor size, up to 10 mis of vector can be administered per day. The dose can be administered in a single injection or in multiple injections within the same tumor site over a time period of one day. Alternatively, a dose consisting of up to 10 mis per day, can be administered over a time period of 5 days in order to establish one course. Patients can receive as many courses as necessary in order to establish a response without proving toxic. Courses can be given, for example, weekly or every other week (week on/week off).

As noted above, within preferred embodiments of the present invention, pharmaceutical compositions are provided comprising a recombinant retrovirus carrying the hγ-IFN vector construct, in combination with a pharmaceutically acceptable carrier or diluent. The composition may be prepared either as a liquid solution, or as a solid form (e.g., lyophilized) which is suspended in a solution prior to administration. In addition, the composition may be prepared with suitable carriers or diluents for either surface administration, injection, oral, or rectal administration.

Pharmaceutically acceptable carriers or diluents are nontoxic to recipients at the dosages and concentrations employed. Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin. A particularly preferred composition comprises a vector or recombinant virus in 10 mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2, .and 150 mM NaCl. In this case, since the recombinant vector represents approximately 1 g of material, it may be less than 1% of high molecular weight material, and less than 1/100,000 of the total material (including water). This composition is stable at -70°C for at least six months.

Pharmaceutical compositions of the present invention may also additionally include factors which stimulate cell division, and hence, uptake and incorporation of a recombinant retroviral vector. Representative examples include Melanocyte Stimulating Hormone (MSH), for melanomas or epidermal growth factor for breast or other epithelial carcinomas. Pharmaceutical compositions of the present invention may be injected via a variety of routes (e.g., intradermally ("i.d."), intracranially ("i.e."), intraperitoneally ("i.p."), intrathecally ("i.t."), intravenously ("i.v."), subcutaneously ("s.c"), intramuscularly ("i.m."), or preferably, directly into the tumor. The individual doses normally used are 10⁷to 10⁹ CFU. (colony forming units of neomycin resist.ance titered on HT1080 cells). These can be administered in a variety of intervals, for example, at one to four week intervals for three or four doses initially. Subsequent booster shots may be given as one or two doses after 6-12 months, and thereafter annually.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1

PREPARATION OF MURINE RETROVIRAL PROVECTOR DNA AND

PACKAGING CELL LINES

Preparation of the retroviral backbone KT-3 and cloning of the mγ-IFN and hγ-

IFN genes into the KT-3 backbone was prepared as described in PCT WO 94/21792 which is hereby incorporated by reference. Packaging cell lines were prepared and transduction of mouse and human cell lines were perfomed using the mγ-IFN and hγ- IFN gene respectively. MHC Class I expression and γ-IFN activity was determined as also described in PCT WO 94/21792 which is hereby incorporated by reference.

Example 2

CONSTRUCTION OF PBA-5A, PBA-5B, AND PBA-5C RETROVIRAL

VECTOR BACKBONES CONTAINING THE Hγ-IFN GENE

This eχ.ample describes several modifications of the retroviral vector pKT-1 resulting in decreased sequence homology to the retroviral gag/pol and envelope expression constructs. The construction of the pKT-1 starting material used in the present example is further described in detail in PCT WO 95/30763 and in co-owned U.S. Serial No. 08/721,327, both of which are hereby incorporated by reference in their entirety. In addition, two stop codons were introduced in the DNA sequence of the packaging signal sequence in order to increase the safety of these vectors. All modifications are summarized in co-owned U.S. Serial No. 08/869309 and the resulting retroviral crossless backbones are called pBA-5a, pBA-5b, and pBA-5c. Further details on the construction of pBA-5a, pBA-5b, and pBA-5c are provided in co-owned U.S. Serial No. 08/721,327 and co-owned application U.S. Serial No. 08/850,961 which is also hereby incorporated by reference.

In order to construct a crossless retroviral vector containing the hγ-IFN gene, the Xho I-Cla I hγ-IFN fragment is removed from the SK hγ-IFN vector as described in Example 1 above and ligated into the pBA-5a, pBA-5b, or pBA-5c crossless retroviral vector backbones.

EXAMPLE 3

DIRECT ADMINISTRATION OF VECTOR INTO TUMOR BEARING ANIMALS

A. Direct Administration of Vector into Mice

Mouse tumor systems may be utilized to show that cell mediated immune responses can be enhanced by direct administration of a vector construct which expresses hγ-IFN. For example, six to eight week old female Balb/C or C57B1/6 mice are injected subcutaneously with 1 x 10^ to 2 x 10^ tumor cells which are allowed to grow within the mice for one to two weeks. The resulting tumors can be of variable size (usually 25-

40 mm- in volume) as long as the graft is not compromised by either infection or ulceration. One-twentieth to two-tenths of a milliliter of the hγ-IFN vector construct,

(minimum titer 10^ CFU/ml) is then injected intratumorally. Multiple injections of the vector are given to the tumor daily, twice d ly, or every two to three days.

Depending on the parameters of the particular experiment, the nature of the vector preparations can be variable as well. The vector can be from filtered or unfiltered supernatant from vector producing cell lines (VCL), or may be processed further by filtration, concentration or dialysis and formulation. Other standard purification techniques, such as gel filtration and ion exchange chromatography, may also be utilized to purify the vector. For example, dialysis can be used to eliminate -interferon that has been produced by the VCL itself (and which, if administered, may effect tumor growth). Dialysis may also be used to remove possible inhibitors of transduction. Another option is to perform intratumor injections of the -interferon VCL itself, in order to more extensively introduce the vector. Briefly, cells are injected after being spun down from culture fluid and resuspended in a pharmaceutically acceptable medium (e.g., PBS plus 1 mg/ml HSA). As few as 10^ cells may be used within this aspect of the invention.

Efficacy of the vector construct may be determined by measuring the reduction in primary tumor growth, the reduction in tumor burden (as determined by decreased tumor volume), or by the induction of increased T-cell activity against tumor target cells (as measured in an in vitro assay system using lymphocytes isolated from the spleens of these tumor beting cells). In a metastatic murine tumor model, efficacy may also be determined by first injecting tumor cells that are metastatic, and, when the tumor is 25-40 mm3 in volume, injecting vector several times into that tumor. The primary tumor graft may or may not be surgically removed after 2-3 weeks, and the reduction in metastases to the established target organ (lung, kidney, liver, etc.) counted. To measure the change in metastases in a target organ, the organ can be removed, weighed, and compared to a non-tumor bearing organ. In addition, the amount of metastases in the target organ can be measured by counting the number of visible metastatic nodules by using a low powered dissecting microscope.

B . Direct Administration of Vector into Humans

For humans, the preferred location for direct administration of a vector construct depends on the location of the tumor or tumors. The human γ-interferon gene can be introduced directly into solid tumors by vector administration (the vectors may be purified as previously described). They may also be delivered to leukemias, lymphomas or ascites tumors. For cutaneous lesions such as melanomas, the vector may be directly injected into or around the lesion. At least 10^ CFU of vector particles should be administered, preferably more than 10" CFU in a pharmaceutically acceptable formulation (e.g., 10 mg/ml mannitol, 1 mg/ml HSA, 25 mM Tris pH 7.2 and 105 mM NaCl). For internal tumor lesions, the effected tumor can be localized by X- ray, CT scan, antibody imaging or other methods known to those skilled in the art of tumor localization. Vector injection can be through the skin into internal lesions, or by adaptations of bronchoscopy (for lungs), sigmoidoscopy (for colorectal or esophageal tumors), intra-arterial or intra-blood vessel catheter (for many types of vascularized solid tumors), or CT scan (for renal cell carcinomas, cervical carcinoma, prostate carcinoma, hepatic carcinoma, head and neck carcinoma, or pancreatic carcinoma). The injection can be into or around the tumor lesion. The efficiency of induction of a biological response may be measured by CTL assay or by delayed type hypersensitivity (DTH) reactions to the tumor. Efficacy and clinical responses may be determined by measuring the tumor burden using X-ray, CT scan, or antibody imaging or other methods known to those skilled in the -art of tumor localization.

EXAMPLE 4 INTRATUMORAL INJECTION OF MΓ-IFN RETROVIRAL VECTOR INTO TUMORS IN MICE

This ex.ample demonstrates that mγ-IFN retroviral vector administered intratumorally in mice significantly inhibits tumor growth of established B16F10 tumors. Briefly, B16F10 cells (a tumor cell line obtained from Dennert, University of Southern California, Comprehensive Cancer Center, Los Angeles, CA) were thawed in a 37 C water bath. Cells were maintained in log phase growth conditions in a 37 C incubator at 10% CO2. The B16F10 cells were prepared for injection into C57 BL/6 mice (Harlan Sprague Dawley, Inc., Indianapolis, IN) as follows:

i. Cells were harvested from tissue culture flasks using trypsin and counted for viability using the trypan blue exclusion method.

ii. The cells were then resuspended in IX HBSS for injection into 1 x 10⁵ cells per mouse in 100 μl dosages, subcutaneously on the abdomen.

Thirteen days following injection, tumors were measured and grouped equally by tumor volume into 4 groups of 10 mice each. The tumors were treated by intratumoral injection of mγ-IFN retroviral vector with 100 μl of 2 x 10⁸ cfu/mL each day for 7 days. Group 1 (n=10) was injected with mγ-IFN retroviral vector containing 8 μg/mL polybrene, Group 2 (n=10) was injected with a control vector containing 8 μg/mL polybrene, Group 3 (n=10) was injected with formulation buffer containing 8 μg/mL polybrene and Group 4 (n=10) received no treatment.

Tumor nodules were measured every 3-4 days and the volume was calculated according to the following equation: width² x height x π/6. Statistical significance was determined using a Student's paired T-test.

The results presented in Figure 1 (tumor volume versus time) demonstrate a significant inhibition of tumor growth observed when mγ-IFN retroviral vector is injected intratumorly into established B16F10 tumors in mice as compared to the injection of a control vector or the injection of formulation buffer containing no vector.

EXAMPLE 5

REGIONAL TREATMENT BY DIRECT INJECTION OF Mγ-IFN

RETROVIRAL VECTOR INTO TUMOR BEARING MICE

This example demonstrates that regional treatment by direct injection of mouse γ- IFN retroviral vector into a small subcutaneous tumor will result in a significant regional and anti-tumor protection as measured by subcutaneous tumor volume and systemic anti- tumor protection as measured by lung weight;

CT26 cells tumor cells (M. Brittain, Baylor College of Medicine, Houston, TX) were grown in vitro maintaimng log phase growth conditions. Female BALB/c mice 6- 8 weeks old (Harlan Sprague Dawley, Indianapolis, IN) were divided into 3 groups:

On day, 2 x 10⁵ CT26 were inoculated subcutaneously into the ventral abdominal area and the subcutaneous inoculations were allowed to form palpable tumors, reaching a volume of 20 - 40 mm³ in 7-10 days. On the same day, 1 x 10⁵ CT26 cells were injected intravenously into the tail vein to seed the lungs with micrometasteses. On day 10 post tumor cell inoculation, the subcutaneous tumor nodule was directly injected with formulation buffer or processed and purified mγ-IFN retroviral vector at a titer of 2 x 10⁸ cfu/ml as described above

The subcutaneous tumor nodules were measured every 3 - 4 days and recorded for 25 days post inoculation for CT26 at which time the mice were sacrificed. The lungs were removed and weighed. Tumor volume was calculated according to the following equation: width x length x height x π/6. The subcut-aneous tumor volume is a measurement of regional efficacy. Statistical significance between the control and mγ- IFN retroviral vector treated groups was determined using ANOVA. A measurement of the wet weight of the lungs at the time of sacrifice is directly correlated to systemic tumor burden. Statistical correlation between the volume of the subcutaneous tumor and lung weight was determined using Spe-arman's Rho analysis.

The results are presented in figure 2. Tumor volumes of the subcutaneous lesion are shown on the Y-axis. The corresponding lung weight is shown as a function of the size of the data point (i.e., the larger the point, the greater the lung weight). Animals with small subcutaneous tumor volumes (small Y-axis value) are predicted to have smaller lung weights (small points). The average volume of the subcutaneous tumors in mice in the Neo control vector treated groups was large (1218 ± 256 mm )(Figure 2). The average tumor volume in mice in the group treated with mγ-IFN retroviral vector was significantly smaller (624 ± 161 mm ) (p=0.05). In those mice in which significant regression of the subcutaneous tumor was demonstrated ( 250 mm³), a highly correlated decrease in pulmonary tumor burden was observed (p<0.0005).

The results demonstrated that significant retardation of the subcutaneous tumor size and growth rate were observed in the group receiving injections of mγ-IFN retroviral vector. Additionally, significant reduction in pulmonary tumor burden in those mice with regional responses was highly correlated. The data from this study demonstrated that direct injection of mγ-IFN retroviral vector into a solid tumor nodule resulted in significant regression of the treated tumor. And, in those animals with significant regional responses, a significant systemic anti-tumor response was also observed based on the tumor burden in the lungs. EXAMPLE 6 CLINICAL TREATMENT OF CUTANEOUS MELANOMA

Malignant melanoma is the most rapidly increasing cancer in the United States with an apparent doubling every ten years. Current treatment of melanoma includes a combined approach with surgical excision, staging of disease, and chemotherapy in patients with metastatic disease. Results of treatment are poor in patients with metastasized melanoma. Dacarbazine (DTIC) is the most active single agent but response rates are generally <20% and only rare patients achieve long-term survival (Balch et al Cutaneous Melanoma 39:498-508. Philadelphia Second Edition J.B.

Lippincott Co 1992). Combination chemotherapy with other chemotherapy agents (i.e., Cisplatin, Carmustine) have shown increased activity against melanoma with partial and complete response rates between 30% and 55%, respectively. Combination of four drugs; DTIC, Cisplatin, BCNU, and Tamoxifen has been shown to have a 50% response rate in patients with metastatic melanoma. Tamoxifen may increase thromboembolic phenomena, but it is a critical component of this regimen (Balch et al 1992, Supra; Del Prete et al, Cane Treat Rep 1984; 68[ll]:1403-05, 1984). Unfortunately, median survival rates have not been significantly prolonged (10-11 months). However, the majority of patients (more than 90%) are initially diagnosed with a disease that is localized to a primary site or regional lymph nodes. This group of patients is characterized by variable recurrence (Balch et al 1992, Supra;).

Recommended Chemotherapy Treatment Prior To γ-IFN Treatment:

Patients will be offered standard treatment with chemotherapy. The following is the recommended chemotherapy for metastatic melanoma (Del Prete et al 1984, Supra). CDDP 25 mg/m² IVQD Days 1-3 and 21-23

DTIC 220 mg/m² 1 VQD Days 1 -3 and 21 -23

BCNU 150 mg/m²1. V. Day 1 , repeat q 6 wks

Tamoxifen 10 mg/bid P.O. Dl -31

Patients responding to standard chemotherapy will continue treatment until there is evidence of stable disease or disease progression. Patients must be off treatment for one month prior to study participation. There have been extensive studies on the use of immunotherapy in melanoma. In a review of 111 published studies of cancer vaccines (DeVita et al, Biologic Therapy of Cancer 6:87-119., 1991), 37 studies have shown efficacy of either tumor regression or prolongation of survival. Of the 37 studies that have shown efficacy, 18 have been in melanoma. This has resulted in the general impression that melanoma appears to be a tumor that is responsive to immunotherapy.

γ-IFN Transduced Autologous Tumor Cells

A Phase I study treating metastatic melanoma patients with γ-IFN transduced autologous melanoma cells (IT AT) has been completed at Duke University by Dr.f Hillli-ard Seigler. Patients underwent tumor biopsy and autologous melanoma cell lines were derived ex vivo. These cell lines were then transduced with the human γ-IFN retroviral vector in vitro.

ITAT cell lines were successfully derived for twenty patients from 175 patient biopsies. Treatment consisted of a single subcutaneous injection of 2 million cells at weeks 1 and 3, 7 million cells at weeks 5 and 7, and 18 million cells at weeks 9 and 11. A total of fifteen patients were treated with ITAT. Eleven patients completed the full course of therapy. Five patients died during the treatment period due to progressive disease. There were no serious adverse events associated with ITAT injection. Clinical responses included one complete response with regression of a left neck metastasis. One mixed response with resolution of a 3.3 x 4.5 cm left axillary mass, but concurrent development of liver metastasis, and one stable disease. There have been a total of eight deaths due to progressive disease.

Immunologic studies showed the development of tumor specific IgG antibodies and CTL responses in patients (see Table below). The development of the anti-tumor response was apparent after at least five immunizations. The patients with some evidence of anti-tumor responses had both humoral and cellular immune response to autologous tumor. These studies show that γ-IFN transduced autologous tumor immunizations are safe, elicit both humoral and cellular anti-tumor responses, and may induce a systemic anti-tumor response. The low yield and duration of time required for manufacturing of the ITAT cell lines, however, make this approach impractical. This has led to the development of the strategy for injection of the γ-IFN retroviral vector directly into tumors to assess the anti-tumor immune responses and clinical benefit. Summary of Clinical Response and Immunologic Assays:

Direct Intratumoral Injection of γ-IFN retroviral vector

A Phase I study of direct intratumoral injection of γ-IFN retroviral vector in patients with metastatic melanoma has been completed (Dr. John Nemunaitis, Texas Oncology PA, Dallas, Texas). Eleven patients received intratumoral injections of 0.3 ml of γ-IFN retroviral vector every day for 5 days. Tumors were excised on day 8. No clinical tumor responses were seen. No significant injection related complications were seen. Five patients died of progressive disease. Three out often tumor s-amples were positive for γ-IFN secretion by Elispot analysis. There was no increase in expression associated with co-injection of retroviral transduction enhancing polycations. Safety of direct injection of γ-IFN retroviral vector was established with this Phase I study. Rationale and Study Objectives

The Phase I ITAT study provides evidence in humans that γ-IFN transduced autologous tumor cells induce tumor specific immune responses and may provide systemic anti-tumor responses (3 MR 15 patients). The Phase I γ-IFN direct retro vector injection shows that direct intratumoral injection is feasible and safe. The onset of both cellular and humoral immune responses in the ITAT study required 5 immunizations which may be important to achieve systemic anti-tumor effects.

The prim.ary objective is to investigate the safety, immunogenicity and tolerability of intratumoral injection of γ-Interferon retroviral vector in patients with metastatic melanoma. Additionally, the study will evaluate the safety and tolerability of three dose levels and two dose regimens (one cycle and six cycles) of the γ-IFN retroviral vector.

Study Design

This is an open label outpatient phase I interpatient dose-escalation study to be conducted at a single site. Patients will receive direct intratumoral injection of the hγ- IFN retroviral vector. There will be six groups consisting of three patients per group for an anticipated total of eighteen patients. The first three groups will receive a daily intratumoral injection of hγ-IFN retroviral vector for five consecutive days with an interpatient dose escalation of > 1 x 10⁷ CFU/mL at volumes of 0.3, 0.5 and 1 mL. The second group of patients will be treated with the same dose levels for six cycles. A cycle is defined as five daily injections every two weeks. All protocol participants will be on study for a total of sixteen (16) weeks. All three patients must be enrolled and receive one cycle of treatment prior to treating patients at the next dose level.

Dose escalation will be conducted in the following m-anner: a) If none of the three patients experience treatment limiting toxicity that is considered to be therapy related during the first cycle of therapy, then dose escalation will be allowed. b) If one of the three patients experience a therapy related treatment limiting toxicity, then an additional three patients will be evaluated at the same dose level. If at least one of the additional three patients experience a treatment limiting toxicity that is therapy related, then there will be NO further dose escalation. The previous dose level will be considered the maximum tolerated dose (MTD). c) If at least two of the three patients experience therapy related treatment limiting toxicity, then there will be NO treatment at this dose level. The previous dose level will be considered the maximum tolerated dose (MTD).

A clinically significant, treatment-limiting toxicity will be defined as one which, in the investigatoris opinion, is drug related and treatment-limiting for a monitored outpatient regimen. The National Cancer Instituteis Common Toxicity Criteria will be used to assess toxicity as summarized below.

TOXICITY GRADING SCALE: ESTIMATING SEVERITY GRADE

GRADE 1 Transient or mild discomfort; no limitation in activity; no medical intervention/therapy required.

GRADE 2 Mild to moderate limitation in activity - some assistance may be needed; no or minimal medical intervention/therapy required.

GRADE 3 Marked limitation in activity, some assistance usually required; medical intervention therapy required, hospitalizations possible.

GRADE 4 Extreme limitation in activity, significant assistance required; significant medical intervention/therapy required, hospitalization or hospice care probable.

All patients injected with hγ-IFN retroviral vector on study will be evaluated for safety, immunologic, and clinical responses.

If a subject develops progressive disease while on study, continuation of treatment will be based on the following criteria: 1) the subject has not experienced grade 3 or 4 drug related toxicity, and 2) the patient does not require systemic chemotherapy for optimal management of his/her disease. Surgical resection or small field radiotherapy to sites of progressive disease are allowed and do not require discontinuation of protocol treatment.

Patient Selection Criteria Approximately 18 patients with metastatic melanoma will be entered into this study. Patients will be monitored on study for 16 weeks. It is requested that patients agree to participate on the long term follow-up protocol after completion of this study.

A. Inclusion Criteria

Patients must meet all of the following criteria to be considered for study entry:

I . Patient must have histologically confirmed metastatic melanoma, either: 1) Stage 4 or recurrent metastatic disease or 2) Stage 3 (node positive) disease with 2 or more involved nodes or history of an ulcerated primary lesion. 2. Patients must have at least one measurable metastatic tumor site.

3. The tumor of the patient that will be injected must be at least 2 grams (2f ml volume) and accessible by direct transcutaneous injection.

4. Must be at least 18 years of age. 5. Must have an expected survival of at least nine months.

6. Patients must be HIV negative as confirmed by ELISA AB test.

7. Must have a performance status ECOG 0 or 1.

8. Patients must have a reactive skin test (> 2mm induration) to at least one of the seven following antigens, tetanus, diphtheria, streptococcus, tuberculin, Candida, trichophyton, and proteus by

(Multi-test CMI, Merieux, Lyon, France).

9. Patients must be able to give informed consent according to IRB and FDA guidelines.

10. Women of child bearing age should have a negative pregnancy test. Male and female patients should commit to use effective contraception for the duration of the study.

I I . Patients must have the following laboratory parameters: a) Adequate clotting studies .and hematocrit to allow intratumoral injection b) WBC count > 3000/mm³ c) Platelet count > 100,000/mm³ d) Bilirubin < 2.5 mg/dl e) Creatinine < 2.5 mg/dl f) Absolute lymphocyte count > 1000/mm³ g) Hbg ≥ 9.0 gm dl h) Transaminases < 3x high normal 12. Patients must be completely recovered from effects of prior major surgery before initiating treatment.

13. Patient must have negative anti-nuclear antibodies.

B. Exclusion Criteria

1. Pregnant or lactating women are discouraged from protocol participation.

2. Prior tumor vaccines or steroids within 4 weeks prior to study.

3. Cytotoxic agents or steroids within 4 weeks of study treatment

4. Major surgery within 4 weeks of study treatment.

5. Radiotherapy within 4 weeks of study treatment. 6. Prior treatment with biologic agents (e.g., Interferons, Interleukin-

2) within 4 weeks of study treatment except G-CSF.

7. Concurrent use of other investigational agents.

8. Known hypersensitivity to components of the formulations

9. Patients with a history of secondary malignancies (within 5 years), other than in situ carcinomas of the cervix or epidermal skin cancer. 10. Patients with documented autoimmune systemic diseases such as, but not limited to, inflammatory bowel disease or multiple sclerosis.

11. Patients with organic brain syndrome or significant psychiatric abnormality which would preclude participation in the full treatment protocol and follow-up.

12. Patients with active systemic infections or other major medical illnesses of the respiratory and/or cardiovascular systems (NYHA class 3 or 4 or myocardial infarction within the past 6 months). 13. Patients with acute or chronic active viral hepatitis, antigenemia, or cirrhosis.

14. Patients with previous history of a) brain metastasis, b) radiation therapy or surgical treatment of CNS metastases or c) clinical evidence of brain metastases. 15. The patient is unwilling or unable to give written informed consent to participate in the study.

Treatment Procedures

A. Investigational Product Description:

The hγ-IFN vector (in lactose buffered vehicle lyophilized vials stored at -20°C) will be supplied at a concentration of > lxl 0⁷ CFU/mL. To reduce the possibility of bacterial contamination, reconstituted solutions should be stored under refrigeration and used within 48 hours. Patients who respond to therapy may be re-treated at the same dose level on the multi-dose group regimen. A biopsy of tumor sites and additional blood samples for research purposes may be requested prior to re-treatment. The same schedule of procedures used for the multi-dose regimen will be followed. Patients will be monitored on study for a total of 16 additional weeks.

B . Administration of hγ-IFN vector

Patients will receive an intratumoral injection of the γ-IFN retroviral vector daily for five consecutive days (days 1-5). The first three groups of patients will receive an injection of γ-IFN retroviral vector at doses of 0.3, 0.5 or 1.0 ml for five (5) consecutive days for a single cycle (two weeks). The second three groups will receive the same dose of γ-IFN retroviral vector but will be treated every other week for 6 cycles of therapy (12 weeks). Patients will be monitored on study for a total of 16 weeks.

The dose and schedule is shown below. Treatment may be discontinued at any point either at the discretion of the patient and/or Investigator, in cases of treatment emergent toxicity (Grade 3-4, National Cancer Instituteis Common Toxicity Criteria as described above), and/or tumor progression requiring alternative therapy.

Dosage and Schedule

* Titer: > 1 x 10⁷ CFU/ml

C. Procedures For Administration Of hγ-Interferon Vector

Two individuals (principle and backup operators) will be trained to perform the final formulation procedure at Texas Oncology Physicians Association (T.O.P.A.). A single tumor will be identified and injected on each treatment day. The entire volume of study product should be injected intratumorally. Topical or subcutaneous local anesthesia may be utilized at the investigatoris discretion.

Vital signs (BP, P, T) will be obtained prior to each injection and 15 and 30 minutes following each injection. Injection site(s) will be inspected for any bleeding or other drainage immediately after and at 15 and 30 minutes post treatment. These sites should be observed each injection day for local reactions.

Patients may receive concomitant medications to control the side effects of treatment. These include: acetaminophen, Indomethacin, and ranitidine throughout the course of treatment. Hydroxyzine hydrochloride may be given to treat pruritus. Steroids are contraindicated for study participants. If systemic steroids are required, then the patient will immediately be taken off protocol therapy. Concurrent therapy with other investigational agents or biologic agents (except G-CSF) are not allowed while patient is receiving study treatment.

Study Procedures A. Pre-Study Screening

The following clinical, immunology and laboratory testing is to be completed within 30 days prior to treatment.

1. Complete history and physical examination.

2. Tumor assessment by radiologic technique noting in detail the exact size and location of any lesions that exist (measurements should be reported on the case report form).

3. CT/MRI of head, chest and abdomen.

4. Electrocardiogram.

5. CXR. 6. Immunologic tests (tumor specific antibody, lymphocyte proliferation and research assays): 28 mis of blood to be collected and shipped Monday through Thursday to Chiron. a) Immunologic tests: Twenty (20) mLs to be collected in CPT blue tiger top tube, .and five (5) mL to be collected in a red top tube. b) hγ-IFN antibodies: 3 mLs of blood to be collected in red top tube spun down and frozen in Nunc tubes at -70°C.

7. Replication Competent Retrovirus (RCR) Sampling: 23 mL of blood will be collected and shipped to Chiron. a) Peripheral blood mononuclear cells: twenty (20) mLs of blood to be collected in CPT blue tiger top tube. (The CPT sample technique preparation will be provided with the tubes.) b) Serum: 3 mLs of blood will be collected in a red top tube spun down and frozen in Nunc tubes at -70 C.

8. Blood specimen for anti-nuclear antibodies. 9. Blood specimen for CBC, DIFF, PLT, PT/PTT, and chemistry.

10. Urinalysis/culture.

11. Blood specimen for HIV ELISA.

12. Blood specimen for Hepatitis A, B, and C screening. 13. Serum pregnancy test for female participants.

14. Merrieux multi-skin test (CMI).

On Study Evaluations

On injection days, blood specimens should be drawn prior to the study drug injection. All participants should be scheduled for the same day each week if at all possible. However, a 48 hour window is allowed for scheduling problems. All adverse events will be documented and graded when appropriate National Cancer Instituteis Common Toxicity Criteria as described above.

1. Week l a) History and physical examination b) Blood specimen for CBC, DIFF, PLT, PT/PTT, and chemistry c) Designation of target tumor for γ-IFN injection. The tumor site should be included on the appropriate CRF. Care must be taken to inject only the designated tumor on each treatment day. Days 1 - 5

d) Test article administration e) Vital signs prior to injection and at 15 and 30 minutes post-injection

Weeks 3, 5, 7, 9, 11 - Treatment Visits

a) History and physical examination b) Blood specimen for CBC, DIFF, PLT, PT/PTT, and chemistry c) Blood specimen for immunologic tests. d) Blood specimen for γ-IFN antibodies

Day 1 - 5 of each week e) Test .article administration into the previously designated target tumor (Patients receiving 6 cycles only) f) Vital signs prior to injection and at 15 and 30 minutes post-injection

C Termination

1. Week 16 a) History and physical examination b) Tumor assessment (to include radiologic technique for evaluation of all lesions) c) CT/MRI Head, chest and abdomen d) Blood specimen for CBC, DIFF, PLT, PT/PTT, and chemistry e) Urinalysis/Culture f) Blood specimen for immunologic tests. g) Blood specimen for RCR h) Blood specimen for γ-IFN .antibodies i) Blood specimen for anti nuclear antibodies j) Participation in the long term follow-up protocol is explained and consent is obtained.

D. Follow-up

Post-treatment evaluations will be performed under a long-term follow-up protocol. Patients will be followed for survival.

Criteria for evaluation All patients who receive at least one injection of γ-IFN are evaluable for safety and efficacy.

Efficacy Endpoints

• Objective Response Rate (CR + PR) • Complete Response Rate

• Response Duration

• Progression Free Survival

• Overall Survival

Response Criteria

Complete Response (CR): Complete disappearance of all tumor with resolution of all symptoms and all laboratory abnormalities attributable to tumor, documented on two evaluations using identical methodology, separated by at least 28 days constitutes a durable CR. Pertinent organ systems must be evaluated to rule out the presence of tumor lesions. The first day of CR is the first date on which all evaluable lesions have disappeared. Tumors should be measured bi-dimensionally.

Partial Response (PR): > 50% reduction in the sum of the products of tumor dimensions for measurable lesions compared to baseline, without increase in size of any lesion > 25%. There must be improvement or stabilization of evaluable lesions and improved or stabilization of patient symptoms and laboratory abnormalities attributable to tumor. Documentation is to be obtained on evaluations using identical methodology separated by at least 28 days. Every site of known disease must be evaluated to rule out progression of tumor lesions in sites of evaluable but not measurable disease. Minor Response (MR): > 25% BUT < 50% decrease in size of lesions without appearance of new lesions, or PR > 50% that lasts < 28 days. Stable Disease (SD): No change m size of lesions, or increase or decrease of <f 25%. There must be no appearance of new lesions.

Progressive Disease (PD): >25% increase in size of any single lesion, or appearance of any new lesion, or worsening of unmeasured but evaluable lesions.

Response Duration

Response duration will be calculated beginning from the first day of documented regression to PR or CR status (assuming confirmation >28 days later) and ends on the day progressive disease is documented or death. In the case of patients lost to follow-up, the last day the patientis response was documented will be used as the last day of response duration.

Progression Free Survival (PFS)

PFS is the interval from the day the first dose of treatment is given to date of progression or date of last follow-up if progression has not occurred. Documentation of progressive disease will be submitted verifying the end of progression free survival on the Case Report Form.

Survival

Survival duration is the interval from first dose of treatment to the date the patient was last known alive or date of death.

Surgical Resection of Residual Disease Two clinical endpoints for this trial are assessment of the rate and duration of objective response. Resection of disease to achieve surgical NED status is permitted only after full documentation of objective response. The duration of response in resected patients will be censored as of the day resection is performed. If the Investigator and the patient choose surgical resection, these patients will continue to be followed for survival and resolution of adverse events. Statistical methods Sample Size

Approximately, eighteen patients will be enrolled for treatment in this dose escalating study. There are no formal statistical comparisons planned for this Phase I study. Therefore, the sample size is not based on statistical considerations.

Safety

1. All patients treated will be evaluated for clinical and laboratory safety. This will include standard clinical and laboratory safety assessments, serum and PBMC for RCR analysis.

2. Adverse events recorded on the case report forms will be mapped to a modified COSTART preferred terms and body systems. The number and percent of patients experiencing adverse events will be tabulated by dose group, preferred term, and worst grade. Laboratory measures will be compared with their corresponding normal ranges. The number and percent of patients with abnormally low and abnormally high results will be presented for each relevant laboratory test by dose group. Also, mean changes from baseline in these laboratory tests will be calculated for each dose group.

3. All treated patients will be followed for replication competent retrovirus RCR and survival under a long-term follow-up protocol.

Effectiveness

1. Serum and peripheral blood lymphocytes will be obtained for evaluation of anti-tumor immune response, and summarized by dose group.

Clinical endpoints will include radiographic measurements of tumor size when possible. The change in tumor size will be summarized by dose group.

*** While the present invention has been described above both generally and in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art in light of the description, supra. Therefore, it is intended that the appended claims cover all such variations coming within the scope of the invention as claimed.

Additionally, the publications and other materials cited to illuminate the background of the invention, and in particular, to provide additional details concerning its practice as described in the detailed description and examples, are hereby incorporated by reference in their entirety.

Claims

We claim:

1. A method of administering a h╬│-IFN retroviral vector to a solid tumor in a patient, comprising directly administering to said tumor 10⁶ to 10⁸ CFU of said retroviral vector.

2. The method of claim 1 wherein said vector is administered for at least 3 days.

3. The method of claim 1 wherein said vector is administered for 5 days.

4. The method of claim 1 wherein 5 x 10⁶ CFU of said retroviral vector is administered.

5. The method of claim 1 wherein 10⁷ CFU of said retroviral vector is administered.

6. The method of claim 1 wherein 5 x 10⁷ CFU of said retroviral vector is administered.

7. The method of claim 1 wherein 10⁸ CFU of said retroviral vector is administered.

8. A method for inhibiting the growth of a selected tumor in a patient, comprising directly administering to said tumor 10⁶ to 10⁸ CFU of said retroviral vector such that the growth of said tumor is inhibited.

9. The method of claim 8 wherein said vector is administered for at least 3 days.

10. The method of claim 8 wherein said vector is administered for 5 days.

11. The method of claim 8 wherein 5 x 10⁶ CFU of said retroviral vector is administered.

12. The method of claim 8 wherein 10⁷ CFU of said retroviral vector is administered.

13. The method of claim 8 wherein 5 x 10⁷ CFU of said retroviral vector is administered.

14. The method of claim 8 wherein 10⁸ CFU of said retroviral vector is administered.

15. A pB A-5a recombinant retroviral vector expressing h╬│-IFN.

16. A pBA-5b recombinant retroviral vector expressing h╬│-IFN.

17. A pB A-5c recombinant retroviral vector expressing h╬│-IFN.

18. A pharmaceutical composition comprising 10⁶ to 10⁸ CFU of a h╬│-IFN recombinant viral vector in combination with a pharmaceutically acceptable carrier or diluent.