WO1999036094A2 - Composition and method for treating metastatic tumors or cancer induced by cells expressing sv40 tumor antigen - Google Patents

Abstract

A prophylactic and therapeutic method for vaccinating and treating a mammal against metastatic tumors comprising cells which express large SV40 tumor antigen. The disclosure further describes an experimental murine pulmonary model for metastatic cancer and a method for quantitatively and automatically analyzing metastatic tumor foci.

Description

COMPOSITION AND METHOD FOR TREATING METASTAΗC TUMORS OR CANCER INDUCED BY CELLS EXPRESSING SV40 TUMOR ANTIGEN

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of U.S. Provisional Application Serial No. 60/071,327, filed January 14, 1998, which is hereby incorporated by reference herein in its entirety.

BACKGROUND The present invention is related to methods of vaccinating against cancer, and more particularly to methods of vaccinating against metastatic cancers which express SV40 large tumor antigen.

The primary tools used in cancer treatment include surgery, chemotherapy, and radiation therapy or combinations thereof. These modalities represent highly invasive and relatively nonspecific treatments, thus alternative methods to treat or prevent cancer are under active investigation. Included in these alternative methods are prophylactic and therapeutic vaccination strategies that target tumor specific or tumor associated antigens expressed on the cancer cell. Although a number of investigations have described the utilization of cancer vaccines as a means to treat cancer (reviewed in 9) , several problems have been identified that have limited the progress in this field of investigation. Progress in the identification of tumor antigens that can be employed as tumor vaccines has been slow. Tumor associated antigens may be expressed both normal cells and/or tumor cells, such that the induction of immune responses to self antigens is problematic. There may also be difficulty in obtaining large quantities of the highly purified tumor antigen because the complex nature of the antigen does not allow one to implement the newer technologies presently employed in the development of vaccines against infectious agents (reviewed in 3) . Finally, the lack of experimental animal models in which tumor formation can be evaluated and accurately quantitated has hampered efforts to examine the effectiveness of certain tumor vaccines within an in vivo setting. Surgical removal of the primary tumor can be curative in many cancer patients. However, surgery may result in exposure of tumor cells to the circulation which can lead to metastasis and tumor establishment at distal sites. Chemotherapy and/or radiation therapy are normally used to eliminate metastatic cells, however, this may only control disease in a temporal manner. Immunotherapeutic strategies may exhibit differing effects in controlling a localized primary tumor versus metastatic lesions (e.g., Refs . 2, 23, 42). In these studies, models of experimental metastasis appeared to be more difficult for inducing evidence of tumor immunity when compared to primary solid tumor models (23, 42) .

Previous studies employing murine models for simian virus (SV40) tumors have used the intraperitoneal route for introduction of SV40 tumorigenic cells into a syngeneic murine system causing induction of primary solid tumor and lethal tumor burden serving as a reference point (5, 54) . Additionally, intradermal inoculation of SV40 tumorigenic cells results in a visible primary tumor mass that can be measured for quantitative purposes (25). Neither of these two routes (i.p. and i.d.) of inoculation represent models for tumor metastasis. Other animal models of SV40-induced tumors have employed newborn hamsters challenged with SV40 or hamsters inoculated with SV40 tumorigenic cells (reviewed in 38) . However, the lack of availability of immunologic reagents specific for the hamster has hampered the development of this animal model .

BRIEF DESCRIPTION OF THE INVENTION A recombinant SV40 tumor antigen vaccine preparation is shown herein as able to prevent the establishment of experimental pulmonary metastasis. Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-ag) vaccine was injected into BALB/c mice followed by challenge with an intravenous injection of syngeneic SV40-transformed tumorigenic cells . The experimental murine pulmonary metastasis model also described herein enables accurate measure of metastatic lesions in the lungs at various days post-challenge using computer assisted video image analysis as described in more detail below. Animals immunized with recombinant SV40 T-ag showed no detectable sign of lung metastasis and survived greater than 120 days post-challenge. Antibodies specific for SV40 T-ag were detected in the serum of immunized mice by ELISA. Splenocytes obtained from recombinant SV40 T-ag immunized mice did not lyse syngeneic tumor cells indicating that no cytotoxic T lymphocyte response was induced. Control mice developed extensive lung metastasis and succumbed to lethal tumor within 4 weeks post-challenge . These data indicate that immunization with the recombinant SV40 T-ag induces in mammals a protective immunity against metastatic cancers which express SV40 T-ag.

In the experimental murine pulmonary metastasis model employing syngeneic SV40 tumor cells described herein, the intravenous (i.v.) route of SV40 tumorigenic cell administration results in tumorigenic cell metastasis to the lungs and eventual pulmonary tumor formation. Additional reference points for use in monitoring tumor induction and progressive tumor development in organs and tissues other than the lung have also been defined within this model . This in vivo tumor model is useful for the screening and evaluation of immunologic based therapies with the goal of preventing the development of metastatic foci in the lungs and subsequent metastatic events. This newly developed SV40 murine tumor metastasis model is used herein to examine the ability of recombinant SV40 large tumor antigen (T-ag) immunization to produce protective immunity against SV40 tumorigenic cell induced pulmonary metastasis. Results described herein indicate that recombinant SV40 T-ag represents an effective prophylactic and therapeutic cancer vaccine strategy.

As noted above, quantification of the SV40 tumorigenic cell induced tumors based on the number and size of foci observed in the lungs of inoculated BALB/c mice is accomplished herein using video assisted computer imaging. Use of experimental tumor models to evaluate effectiveness of prophylactic and therapeutic treatment modalities require quantitative analysis to establish levels of tumor burden. Manual foci analysis is tedious and presents great variability due to its subjective nature. It also requires multiple iterations to obtain consistent and satisfactory results. In addition, counting error due to indistinguishable overlap of foci in late stages of disease is difficult to characterize. Other investigators have utilized manual enumeration to count the number of tumor foci. The size of the foci were not considered. Thus, the means to quantitate lung tumor foci size and number is novel and has not been reported previously for a tumor model . The present invention comprises using a video camera with a charged-coupled device (CCD) and associated video imaging software to obtain more reliable counts and measurements of tumor. This method is both quantitative and reproducible and therefore offers advantages over other protocols. Namely, it decreases variability, allows long-term storage of original data (i.e., stained lungs), lends itself to computer assisted data analysis, and removes the subjective nature of manual analysis.

The demonstration that the viral encoded tumor antigen SV40 T-ag is expressed on the surface of tumor foci in the lungs clearly indicates the potential of this model for evaluating viral encoded tumor antigen specific and non-specific strategies to treat the tumor. Further, the ability to quantitate the tumor burden using a computer analysis modality allows one to evaluate and compare cancer therapies that may afford only partial tumor immunity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C are photos of indirect i munofluorescence (IF) of lung tissue (Figure 1A) and spleen tissue (Figure IB) recovered from BALB/c mice at 14 days post-injection with SV40- transformed mKSA cells. Cells were stained using Pab 405 anti- T-ag monoclonal antibody (mAb) as primary antibody and a FITC- conjugated goat anti-mouse Ig label. Figure 1C is a photo of a representative control of uninoculated BALB/c lung tissue.

Figures 2A-2D are photos showing tumor formation in lungs from BALB/c mice injected i.v. with 5xl0⁵ mKSA cells at (Figure 2A) 5 days post injection (right) compared to naive lung (left) , (Figure 2B) 10 days post injection (right) compared to naive lung (left) , and (Figure 2C) 14 days post injection (right) compared to naive lung (left) . Figure 2D represents tumor formation in lungs taken from BALB/c mice injected intravenously with mKSA cells (day 21) (right) compared to naive lung (left) . Photographs are of whole lungs stained with India ink and destained with Fekete's solution for foci visualization.

Figures 3A-3B are graphs representing data obtained from groups of five mice which were inoculated with 5xl0⁵ mKSA cells and whose lungs were removed, stained with India ink, then destained and stored in Fekete's solution. Tumor foci were quantitated on the ventral surface using image analysis. Figure 3A represents foci number. Figure 3B represents foci diameter in pixels. The standard error of the mean is less than 5.0% of the mean value. At later time points, some mice succumbed to the lethal effects of the tumor. The data presented represent only those mice that survived to that time point.

Figures 4A-4B show representative video image prints of lungs taken from (left) an alum injected BALB/c mouse (Figure 4A) and a SV40 T-ag immunized BALB/c mouse (Figure 4B) at 25 days post-i.v. inoculation with 5xl0⁵ mKSA cells.

DETAILED DESCRIPTION OF THE INVENTION As explained in more detail below, the present invention comprises a method of stimulating or increasing the immune response against SV40 T-ag-type metastatic cancer in a mammal. The method comprises administering to the mammal a vaccine composition comprising an intact recombinant SV40 tumor antigen or an immunogen comprising one or more immunogenic epitopes of SV40 tumor antigen or a combination thereof. The vaccine composition may comprise an adjuvant. The immunogen may be defined as comprising a Pab 405-binding epitope of SV40 T-ag, a Pab 101-binding epitope of SV40 T-ag, SEQ ID NO:l or SEQ ID NO: 2 or immunogenic variants thereof, or combinations of these immunogens or epitopes of other antibodies which bind to SV40 T-ag.

The present invention may also be defined as comprising a method of treating a metastatic tumor or cells thereof in a mammal, wherein the cells express SV40 large tumor antigen, or an antigenic portion thereof. The method comprises administering to said mammal having said metastatic tumor or cells thereof an effective amount of an anti-SV40 T-ag antibody or a vaccine comprising recombinant SV40 tumor antigen or an immunogenic portion thereof to kill, inhibit, or induce regression of said metastatic tumor or cells thereof.

The antibody may be Pab 101, Pab 108, Pab 419, or Pab 405, or another antibody having binding specific for SV40 T-ag. The immunogenic portion may comprise SEQ ID NO:l or SEQ ID NO: 2, or immunogenic variants thereof . The vaccine may be monovalent or polyvalent comprising combinations of the various vaccine components described herein. The mammal may be a human, or other mammal such as a rodent, including a mouse.

The cancer vaccination method may be further defined as comprising administering to a mammal subject a vaccine comprising an amount of SV40 large T-ag or an immunogenic portion thereof, and a pharmaceutically acceptable carrier, diluent or excipient wherein the vaccine is capable of inducing an immune response comprising production of anti-SV40 T-ag antibodies effective against metastatic cancer cells which express SV40 T-ag. The mammal subject may be human, or a rodent, for example, including a mouse. The vaccine may comprise an adjuvant. The large SV40 T-ag or immunogenic portion may be a recombinant polypeptide or peptide, or SEQ ID NO:l or SEQ ID NO : 2 , or immunogenic variants thereof. The immunogenic portion may further comprise the binding epitopes of Pab 108 and Pab 419. The present invention further comprises a method of modelling metastatic SV40 T-ag type cancer by intravenously administering SV40 T-ag-transformed tumorigenic cells into a test animal. The test animal may be a mammal, such as a rodent, for example, a mouse. In a preferred embodiment the SV40 T-ag- transformed tumorigenic cells are mKSA cells.

The present invention further comprises a method of screening for compounds which inhibit, reduce or prevent SV40- type metastatic cancer, by providing a test animal treated to induce an SV40 T-ag-type metastatic cancer, treating the test animal with a test compound, and assessing the metastatic tumor foci in the test animal after a predetermined period of time. The test compound may be a vaccine, for example. Alternatively, the test compound may be an antibody against SV40 T-ag or against a portion thereof .

In the method the test animal may be treated with the test compound before or after the test animal is treated to induce SV40 T-ag metastatic cancer. The metastatic cancer is induced in the test animal preferably by intravenously administering the test animal with SV40 T-ag transformed tumorigenic cells.

In a further embodiment, the present invention comprises a method of quantifying tumor burden in a test animal. The method comprises providing an organ of a test animal treated in a manner known to induce a metastatic tumor response in the organ in the test animal, treating the organ with a stain for distinguishing between tumor foci and normal tissue, obtaining a photographic image of the treated organ, and automatically analyzing the photographic image using computer image analysis software to determine the number and size of tumor foci in the organ by selecting object images which exceed a predetermined minimum size threshold and which exceed a predetermined minimum density value. The photographic image may be obtained using a CCD camera. The organ used in the analysis method may be a lung, or other organs which can be analyzed in accordance with the present invention. The organ may be stained with India ink and bleached to enhance tumor foci visualization. Preferably the organ is placed and flattened between two transparent plates before the step of obtaining the photographic image.

This invention will be more fully understood by reference to the following description and examples. However, the examples are merely intended to illustrate embodiments of the invention and are not to be construed to limit the scope of the invention.

Materials and Methods

Mice. Cells, and Media Six to eight-week old female BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained under standard conditions. Treatment and care of the animals were in accordance with institutional guidelines and the Animal Welfare Assurance Act. The SV40-transformed BALB/c mouse kidney fibroblast cell line, designated mKSA (27) , is tumorigenic in BALB/c mice and was used for in vivo tumor induction. mKSA cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with L-glutamine (Gibco BRL) supplemented with 0.1 mM non- essential amino acids, 500 units/mL penicillin, 500 μg/mL streptomycin (Mediatech, Washington, DC) and 10% heat inactivated fetal bovine serum (BioWhittaker, Walkersville, MD) . Flasks were incubated in a humidified, 5% C0₂ atmosphere at 37°C. Prior to injection, cells were detached from flasks by 5 minute exposure to phosphate buffered saline (PBS) and ImM EDTA (pH 7.5). The tumor cells were washed once, resuspended in PBS, counted, and adjusted to the appropriate density with additional PBS prior to injection. In Vivo Determination of mKSA Cell DORP

Various doses of mKSA cells were i.v. injected into groups of naive BALB/c mice (five mice per group) in order to determine a dose capable of inducing detectable tumors . Each set of experimental inoculations with the different doses of mKSA cells was performed at least two times. A 50 μl volume of tumor cell dose was injected into the dorsal tail vein. All mice were bled on specified days prior to euthanasia. Animals from each tumor cell titration group were euthanized at either 5, 10, 14, 21, 28 or 35 days at which time a post-mortem examination was conducted on animals and tissues (unless the dose proved lethal) . Spleen, lymph nodes, liver, lungs, kidneys, and brain were collected for further analysis. Survival Experiments In order to assess the long-term outcome of the various doses of mKSA cells in mice inoculated i.v., several mice from each group were allowed to progress in illness and observed daily to evaluate mean survival time. For comparative purposes, groups of mice were also inoculated with 5 x 10⁵ mKSA cells via the i.p. route by methods described (5, 7, 54) .

In Vitro Tumorigenic Cell Recovery and Identification

In order to determine whether mKSA cells were detectable at sites distal to the inoculation site, spleen, lymph nodes, liver, lung, kidney, and brain tissues were cultured in vitro . Organs were removed from animals immediately after euthanization at days 5, 10, 14 and 21 post-inoculation and disaggregated in 10 ml of supplemented DMEM. Medium containing tissue was transferred to 25 cm² culture flasks and incubated at 37°C. After 3-4 days, all non-adherent cellular matter was removed and replaced with fresh medium. All cell cultures were maintained for 21 days and observed for the presence of mKSA cells by microscopic examination. IF staining (described below) was carried out on remaining adherent cells within 10-21 days of culture.

Indirect Inrniunofluorescenr.fi

In order to identify the presence of mKSA cells in the organs and tissues of inoculated mice, indirect IF for SV40 T-ag surface expression was performed. Cells cultured as described above were detached and resuspended in PBS before fixing onto poly-L-lysine coated slides with acetone at -20°C for 5 min. A murine monoclonal antibody specific for the carboxy terminus of SV40 T-ag, designated Pab 405 (IgGl) (76) , served as the primary antibody. Fixed cells were incubated with 30 μl solution of 4 μg/ml Pab 405 in 10% normal goat serum (NGS)/borate buffered saline (BBS) for 1 h at 37°C, washed in 0.02% (w/v) Tween-20/BBS, and incubated with FITC-conjugated goat anti-mouse immunoglobulin (Ig) (1:10 dilution in 10% NGS/BBS, Becton Dickinson Immunocytometry Systems, San Jose, CA) for an additional hour. After a final wash, cells were analyzed at 500 x using an Olympus BH2-RFC reflected light fluorescence microscope. For negative controls, tumorigenic cells were stained using a control murine monoclonal antibody (specific for hepatitis B surface antigen) of the same isotype as Pab 405 and goat anti-mouse Ig-FITC. Uninoculated mice and their respective tissues also served as controls. rSV40 T-aσ Production and Purification

Recombinant SV40 T-ag (rSV40 T-ag) was generated in the Sf9 insect cell line using the baculovirus Autographa californica nuclear polyhedrosis virus expression vector system (31) . Sf9 cells were incubated at 27°C and then washed three times in phosphate-buffered saline (PBS) . Cellular proteins were solubilized in detergent extraction buffer (50mM Tris-HCl, pH 9.0, lOOmM NaCI, 1% NP-40, and lOOuM leupeptin) . Cellular extract was clarified at 15,000 g for 15 min. The immunoaffinity resin was prepared by covalently coupling a mouse monoclonal anti-SV40 T-ag antibody, designated Pab 419, to Affi- gel 10 (Bio-Rad Laboratories, Richmond, CA) or cyanogen bromide activated Sepharose according to the manufacturer's specifications. The Affi-gel 10 containing the monoclonal anti- SV40 T-ag preparation was mixed with SV40 T-ag detergent extracts and incubated overnight at 4°C. Immune complexes were washed three times with extraction buffer supplemented with 1% deoxycholate and 0.1% SDS . Bound proteins were eluted with 2% SDS and 2% 2-mercaptoethanol . Proteins were separated by SDS PAGE and detected with Coomassie blue or Silver staining. (53, 55) . Protein concentration was determined using an extinction coefficient of 1.14 for a 1% solution at 280 nm prior to precipitation in alum.

Spodoptera fugi forma (Sf9) cells are propagated in Graces insect cell medium supplemented with lactalbumin hydrolysate, yeastolate and 10% fetal bovine sera (FBS) . The cells are maintained in T-75 flasks at a density of 2 x 10⁶ cells/mL with a 50:50 split every 24 hours. After several days and when enough cells are propagated the cultures are pooled and transferred to a 1 liter spinner flask. When the cells have reached log phase and are approximately 100% viable they are suitable for infection with the recombinant baculovirus expressing the SV40 T-ag gene. Pellet the cells at 200 x g for 10 minutes . Remove the supernatant fraction and resuspend the cell pellet in 50 mL of phosphate buffered saline (PBS) . Pellet the cells again and repeat the washing process once more. To the washed pellet of cells and 10 mL of virus stock and allow adsorption for 1 hour at room temperature. After the adsorption period bring the culture volume back up to one liter with fresh complete Graces medium. Incubate the infected cells for 48 hours at room temperature. Harvest and pellet the cells at 200 x g for 10 minutes. Remove the supernatant fraction and save as virus stock. Place the cell pellet on ice. Lyse the cells with 10 mL of ice cold NP40 lysis buffer (Buffer A: 150 mM NaCI, 1.0% NP-40, 50 mM Tris (pH 8.0)). Hold lysate on ice for 30 minutes vortexing every 5 minutes. Clarify the lysate by centrifugation at 10,000 x g for 30 minutes (S10) . Remove the supernatant S10 fraction and store frozen at -80°C or immediately purify the SV40 T-ag.

Purify the SV40 T-ag from the S10 NP40 lysate of infected cells over a monoclonal antibody affinity column. The antibody designated Pab 419 was immobilized on Sepharose prior to purification. The column is prepared by mock elution with triethylamine (TEA) buffer (Buffer B: 20 mM triethylamine (pH 11) ) and equilibration in ice cold buffer A. Pass the infected cell lysate over the column 3 times at a rate of one drop per five seconds. Wash away and unbound material with 100 mL ice cold buffer A followed by 100 mL of ice cold borate buffered saline (Buffer C: 100 mM NaCI, 50 mM KC1, 50 mM boric acid (pH 8.0)). Monitor the wash spectrophotometrically at an optical density (OD) of 280 nm. When the OD of the wash falls below a value of 0.1 elute the bound T-ag with buffer B stepwise collecting one mL fractious . Monitor the fraction spectrophotometrically for the T-ag. Pool the positive fractions and dialyze with four changes of buffer C. Measure of Quality and Variation in rSV40 T-ag Lots

Each lot of rSV40T-ag produced was examined for quality by ELISA using a panel of mAb specific for SV40 T-ag. Specifically, a 96-well plate is coated overnight at 4°C with 200ng/well rSV40 T-ag diluted in borate buffered saline (BBS) , pH 8.2. Non-specific binding sites are then blocked by incubation with 200uL of 5% normal goat serum (NGS) diluted in BBS (NGS-BBS) at 37°C for 30 minutes. The wells are washed 3-5 times with BBS containing 0.02% (w/v) Tween-20. The panel of primary antibodies include: mAb Pab 405 and Pab 101 (recognize the C-terminus of SV40 T-ag) , and Pab 419 and Pab 108 (recognize the N-terminus of SV40 T-ag) (73) . Polyclonal BALB/c serum reactive to rSV40 T-ag is used as primary antibody as a positive control. Naive BALB/c sera, 5% NGS-BBS, and irrelevant antibody Al .2 (recognizing hepatitis B surface antigen) are used as negative controls. Primary antibodies undergo a two-fold serial dilution in 5% NGS-BBS beginning with a 1:20 dilution. mAb starting at a concentration of 100 μg/ml are similarly serially diluted. Each dilution is then added in triplicate to the wells (50ul/well) and the plate is incubated at 37°C for 1 hour. The plate is washed (as described above) . 50uL of horseradish peroxidase-conjugated gost-anti-mouse IgG Fc (1:1000) diluted in 5% NGS-BBS is then added to each well and the plate is incubated 1 hour at 37°C. The plate is washed (as described above) and lOOuL of ABTS containing 0.01% (v/v) H₂0₂ is added to each well and the plate is incubated at room temperature for 5 minutes. The reaction is stopped with lOOuL of 5% (w/v) SDS. Color intensity is measured at an OD of 410nm with a reference of 490nm on an automated plate reader. Minimum positive value is calculated as three times the OD 410nm obtained for a 1:50 dilution of pre-immune sera. Binding of all four monoclonal antibodies to the rSV40 T-ag preparation indicates a pure lot with no leaching of Pab 419 from the affinity column and/or no conformational damage to the rSV40 T-ag and the epitopes recognized by the monoclonal anti-SV40 T-ag preparation.

ELISA for the Detection of Antibodies to Recombinant .qV40

T-ag

A 96-well plate is coated overnight at 4°C with 200 ng/well rSV40 T-ag diluted in borate buffered saline (BBS), pH 8.2. Non-specific binding sites are then blocked by incubation with 200uL of 5% normal goat serum (NGS) diluted in BBS (NGS-BBS) at 37°C for 30 minutes. The wells are washed 3-5 times with BBS containing 0.02% (w/v) Tween-20. Monoclonal antibody Pab 405 and polyclonal BALB/c serum reactive to rSV40 T-ag are used as primary antibody as a positive control. Naive BALB/c sera, 5% NGS-BBS, and irrelevant antibody A1.2 (recognizing hepatitis B surface antigen) are used as negative controls. Primary antibodies undergo a two-fold serial dilution in 5% NGS-BBS beginning with a 1:20 dilution. Each dilution is then added in triplicate to the wells (50uL/well) and the plate is incubated at 37°C for 1 hour. The plate is washed (described above) . 50uL of horseradish peroxidase-conjugated gost-anti-mouse IgG Fc (1:1000) diluted in 5% NGS-BBS is then added to each well and the plate is incubated 1 hour at 37°C. The plate is washed (as described above) and lOOuL of ABTS containing 0.01% (v/v) H₂0₂ is added to each well and the plate is incubated at room temperature for 5 minutes. The reaction is stopped with lOOuL of 5% (w/v) SDS. Color change is read at an OD of 410nm with a reference of 490nm on an automated plate reader. Minimum positive value is calculated as three times the OD 410nm obtained for a 1:50 dilution of pre- immune sera (5) . Cy otoxic T Lymphocyte Assay

To measure SV40 T-ag cytotoxic T lymphocyte (CTL) activity, a 4 hour ⁵¹Cr release assay was performed as previously described (7) . In 96 well plates, target cells (mKSA) were grown to subconfluency in supplemented RPMI and harvested by incubating the monolayer in PBS containing ImM EDTA for 3-5 min. Detached cells were washed twice with medium and screened for viability by vital dye exclusion. Viable mKSA cells were then incubated with lOOuCi of ⁵¹Cr for 1 hour at 37°C. Radiolabeled mKSA were washed with medium and resuspended at a dilution of 5 x 10⁴ cells/mL. Effector cells were splenic lymphocytes from recombinant SV40 T-ag immunized mice. Effectors were cultured with 20 units/mL recombinant IL-2 and 10⁴ inactivated mKSA cells for 6 days in vi tro at 37°C. Samples of 100 uL of effector cells in medium were added to lOOuL of radiolabelled targets at E:T ratios of 10:1, 20:1, 50:1, 100:1, and 200:1. Microtiter plates were then centrifuged at 300 x g for 5 minutes, followed by incubation for 4 hours at 37°C in 5% C0₂. Following incubation, lOOuL aliquots were collected in separate tubes and radioactivity (cpm) was determined by gamma scintillation. Percentage lysis was calculated as % specific lysis 1- [ (E-S) / (M-S) ] x 100. E represents radioactivity in the presence of effector cells . S represents spontaneous radioactivity released in medium alone . M represents maximum radioactivity released in the presence of 2N NaOH. Immunization and Tumor Challenge

In one set of experiments, mice were immunized with rSV40 T-ag or plasmid DNA encoding rSV40 T-ag before challenge with mKSA cells. Groups of BALB/c mice were immunized via intraperitoneal injection with either an alum precipitate of immunoaffinity purified recombinant SV40 T-ag, or alum alone. In previous studies, we demonstrated that injection of alum alone gave comparable results to injection with alum precipitated control antigens with regards to the lack of protective tumor immunity against SV40 tumorigenic cells (5, 54) . Recombinant SV40 T-ag immunized mice were primed with 5μg of recombinant SV40 T-ag as an alum precipitate. Three subsequent injections of 2 μg recombinant SV40 T-ag as an alum precipitate were given at approximately 14 day intervals or in intervals known to induce amnestic immunologic responses .

The route of immunization is any route that induces an immune response (e.g., intramuscular intradermal, subcutaneously) . The immunogenic range of SV40 T-ag is 200 ng or greater in mice administered as single or multiple injections. A dose of 200ng provides tumor immunity in mice while a dose of 20ng fails to provide tumor immunity. A mouse equivalent immunogenic dose can be used to vaccinate humans to induce an SV40 T-ag specific immune response. Approximately 30 days after the final immunizations, groups of mice were injected with a 50 μL volume of either 5 x 10 (initial experiments) or 1 x 10⁶ (confirmatory experiments) mKSA cells in PBS into the dorsal tail vein. For the lower dose, the majority of the animals were used for tumor challenge survival experiments . Four of the ten animals from each group given the higher dose (10⁶ mKSA cells) were euthanized at 10 days post- challenge at which time a post-mortem examination was conducted. The remaining six animals were used in tumor challenge survival experiments .

To evaluate the effects of genetic immunization, groups of mice were immunized intramuscularly with plasmid DNA encoding the SV40 T-ag gene or with a control plasmid not containing the T-ag gene. These plasmids have been described in detail elsewhere (7) . Plasmid DNA immunized mice were primed with lOOμg of DNA in saline and subsequently received three additional injections of lOOμg of DNA per injection at approximately 14 day intervals. Mice were challenged 30 days following the fourth injection with 5 x 10⁵ mKSA cells and experiments were performed as described above . The tumorigenic dose that represented a 100% lethal dose in inoculated mice was approximately 1 x 10⁵ mKSA cells.

In another set of experiments, mice were immunized with rSV40 T-ag after challenge with mKSA cells. Groups of mice were injected with a 50μL volume ranging from 1 x 10⁵ to 1 x 10⁶ mKSA cells in PBS into the dorsal tail vein. Groups of mice were used to monitor survival time or were euthanized for post-mortem measure of foci number and size. Beginning two days after challenge, BALB/c mice were immunized via intraperitoneal injection with either an alum precipitate of immunoaffinity purified recombinant SV40 T-ag, or alum alone. In previous studies, we demonstrated that injection of alum alone gave comparable results to injection with alum precipitated control antigens with regards to the lack of protective tumor immunity against SV40 tumorigenic cells (1, 13) . Recombinant SV40 T-ag immunized mice were primed with an immunogenic dose (e.g., 5 μg) of recombinant SV40 T-ag as an alum precipitate. Injections continued at 5-10 day intervals for a total of up to four injections post challenge. Those mice that survived were immunized with an immunogenic dose of rSV40 T-ag at monthly intervals for maintenance. Pulmonary Tumor Foci Quantification

The left lung of each animal was removed after euthanasia and stained by injecting lobes with 10% India ink. Lungs were then suspended in Fekete's destaining solution (67) using a technique described previously (65) . The staining procedure results in a clear distinction between tumor foci (white) and normal lung tissue (black) on visual analysis. The lung is placed in a layer of water between two plates of glass in order to create a flattened surface for optimum image acquisition and analysis. Flattening of the lung sample between the two glass plates also alleviates some of the foci quantification problems associated with tumor foci overlap. After destaining 15 minutes, the number of foci visible on the ventral surface of the lung was quantified using an IS-1000 Digital Imaging System

(Alpha Innotech Corporation, San Leandro, CA) . The Alpha Innotech IS-1000 Digital Imaging System is a computer controlled CCD video camera with a 12.5mm wide angle lens and interference filter under control of the IS-1000 DOS-based image analysis software and is available from Alpha Innotech Corporation (San Leandro, CA) . This system allows images to be optimized, captured as quantitative data, and stored for later analysis.

Plates are centered under a mounted CCD camera. A 75-watt light source is placed approximately 6" above the plates. A photograph stored within the system that represents a naive lung stained similarly is then brought onto the screen. The image of the lung between the plates is superimposed on the stored image to obtain a set distance from the camera lens to allow consistent measurement of foci diameter in image pixels. The image is optimized by adjusting contrast, gray scale, and exposure time and saved in a picture file format before being analyzed using the IS-1000 image analysis software. The lung parameter is outlined on the computer to select the object to be analyzed. The same plane of focus was selected for comparison purposes .

Density threshold parameters were defined to ensure that the foci counted consistently fell above a gray scale value of 25 (compared to black lung background) . Size threshold parameters were set to count only those foci exceeding 4 pixels in diameter on the computer image (66) . Both a foci quantity range and estimate of foci diameter were defined and employed as an indicator of disease progression. The foci are then counted using the "object count" option in the program. For size quantification, individual spots were measured by the program and a mean of the tumor foci and standard error of the mean was calculated for the entire lung to demonstrate variability within each group . In instances where tumor foci were deemed too numerous to count and the individual foci became indistinguishable due to overlap (tumor foci number>60) , visible from an individual lung are shown and not quantitated. Although the software particularly described here for use is available commercially from Alpha Innotech Corporation, it will be understood by a person of ordinary skill in the art that any quantitative analysis software which functions similarly may be used herein.

Survival Experiments

In order to assess the long-term outcome of the various doses of mKSA cells in inoculated mice, animals from each group were allowed to progress in illness and observed daily for up to 120 days post-challenge to evaluate mean survival time.

Statistical Analysis

Data were analyzed using the mean collected for animals in each group and standard error of the mean to demonstrate the amount of variability within each group.

RESULTS

Example 1

Correlation between mKSA dose and survival rate. A dose of 1 x 10⁵ mKSA cells has been used in studies incorporating the i.p. route of inoculation (7) . In preliminary studies, we examined the effects of various doses of mKSA cells in order to determine the i.v. inoculum to be used in the detailed characterization of this model . We examined the effects of doses ranging from 1 x 10⁴ to 1 x 10⁶. Doses of mKSA cells lower than 5 x 10⁵ produced a slow rate of disease progression as assessed by visible foci in the lung. Doses greater than 5 x 10⁵ exhibited rapid disease progression, not allowing sufficient time to measure the various parameters employed to characterize the consequences of tumor cell inoculation. Therefore, a dose of 5 x 10⁵ cells was chosen as optimal for the characterization of this model. Post-mortem examinations were carried out on experimental groups and indicators of disease progression following tumor inoculation included foci development in the lungs, mKSA recoverability from various organs, and mortality. In survival experiments, we examined the mortality rate among mice that were inoculated with an i.v. injection of mKSA cells. For comparison, we also inoculated mice with a previously defined lethal dose of mKSA via the i.p. route. This data is presented in Table 1. The mean survival time for 12 mice inoculated intravenously with 5 x 10⁵ mKSA cells was 20.4 days. Five mice were also inoculated intravenously with 1 x 10⁶ mKSA cells to determine any change in mortality at this higher dose. The mean survival time for these mice was 18 days. Mice that received 5 x 10⁵ mKSA cells intraperitoneally survived a mean of 21.4 days that is comparable with previously published studies using this route of inoculation (5, 7, 54) .

TABLE 1. Mean Survival Time Observed In BALB/c Mice After Inoculation With Live mKSA Cells³

^aVarious doses of mKSA cells were injected i.v. or i.p. into BALB/c mice and animals were monitored until death due to tumor burden.

In vivo Tumorigenic Cell Localization

Disaggregated tissue from organs selected for post-mortem examination was cultured in order to determine whether mKSA cells were invading sites distal to the inoculation site. Spleen, kidneys, liver, lungs, lymph nodes, and brain were selected for culture as these are among the most frequent sites of metastatic spread (77) . Detection of tumor cell localization was first determined by morphological identification of mKSA cells that normally exhibit a distinctive fibroblast morphology and visible nucleus. By light microscopy, mKSA cells tend to be less elongated than normal kidney fibroblasts, grow in a random fashion, and exhibit two to three cytoplasmic extensions. Both spleen and lung cell cultures possessed mKSA cells which reached confluency within approximately 7-10 days of culture, exhibiting no difficulty attaching and establishing in primary culture. The mKSA cells were detected in 100% of lung tissue cultures at every post-inoculation day examined (Table 2) . Eighty percent of the spleen cultures obtained 5 days after tumor cell inoculation contained mKSA cells in (Table 2) . Spleen tissues contained mKSA cells in 100% of the cultures obtained on days 10, 14 and 21 post-inoculation (Table 2) . The detection of mKSA cells in kidney tissue cultures were delayed until approximately 12-15 days of culture, a later timepoint compared to lung and spleen cultures. Only three out of the five kidney tissue cultures examined at day 5 post-inoculation (60%) exhibited mKSA cells. The number of positive kidney samples increased as days 10 and 14 post-inoculation when 80% of the kidney cell cultures contained mKSA cells . One hundred percent of the day 21 post-inoculation kidney cultures contained mKSA cells. Only two mice were examined for this experimental group since three mice from this group did not survive to day 21. The cultures of brain tissue exhibited mKSA cell presence at approximately the same time as the kidney cell cultures (within 12-15 days of culture) . Sixty and 80% of the brain cell cultures obtained from the day 5 and 10 post-inoculation groups, respectively, exhibited mKSA cells. In addition, 100% of the brain tissue cultures taken from both day 14 and 21 post- inoculation groups demonstrated mKSA cell presence (Table 2) . Unlike the spleen, lung, kidney, and brain, the liver and lymph node cell cultures failed to exhibit the presence of mKSA cells by morphologic analysis. However, visable tumor foci have been observed in the liver taken from mice after i.v. administration of mKSA cells (data not shown) . Therefore, mKSA cells may be establishing themselves in the liver but were not detected in culture. To confirm the morphological identification of mKSA cells in tissues from inoculated mice, indirect IF for SV40 T-ag was performed on cells grown in primary culture. Representative IF employing Pab 405 anti-SV40 T-ag antibody shows SV40 T-ag staining of cells obtained from the lungs (Figure 1A) and spleen cultures (Figure IB) at 14 days post inoculation. In addition, SV40 T-ag expressing cells were detectable by this method in kidney and brain cultures . No staining was observed when an isotype matched control mAb was employed rather than Pab 405 (data not shown) . Pab 405 failed to stain tissues obtained from untreated control groups of mice (Figure 1C) . By utilization of two different methods, we therefore demonstrate the ability to easily identify the presence of virally transformed tumorigenic cells in this murine model.

TABLE 2. SV40 Transformed Cell (mKSA) Recovery From Various Organs of BALB/c Mice After Intravenous Injection With Live mKSA Cells³

^a5xl0⁵ cells/dose i.v. injected into dorsal ta l vein. ^bmKSA presence determined by morphological identification of transformed fibroblasts in primary cell culture. ^cNot determined. ^dThree mice from this group died before day 21 examination. Tumor Development in the Lungs

Tumor foci formation in the lungs was one of the most prominent features in this SV40 murine tumor model. Therefore, presence of foci in the lungs was used as a main indicator of disease progression. Lungs were obtained from mice in each group and bleached with Fekete's solution which results in a clear distinction between foci (white) and normal lung tissue (black) on visual analysis. Both the size and number of foci increased with the number of days post-inoculation, and this increase was consistent for each mouse in each group. Representative examples of the presence of lung foci at various times post-inoculation are shown in Figure 2. Upon gross examination of lungs at day 5 post-inoculation, a limited number of small foci were apparent around the periphery (Figure 2A) . By day 10, the foci were small in size, however, they had increased in number and were scattered evenly over the surface of the lung (Figure 2B) . At day 14 post-inoculation, the lungs exhibited a slightly higher number of foci when compared to the lungs at day 10, and the size of the foci also increased (Figure 2C) . The diameter of these foci at 14 days post-inoculation was calculated to be two times larger than the foci at 10 days post- inoculation. A noticeable difference in the gross morphology was seen in the 21 day lung when compared with lungs taken from earlier time points (Figures 2D) . In the early post-inoculation groups, the lung periphery was smooth and even and the only indication of. foci was the color difference. In contrast, the day 21 post-inoculation lungs had extremely uneven surfaces and large nodules of foci (Figure 2D) , indicating extensive invasion and establishment in the lungs.

To quantify these visible changes in the lung foci, computer assisted image analysis of the ventral surface of the lungs was employed as described elsewhere herein. Density and size threshold parameters were defined in order to keep counts consistent from experiment to experiment. For the lungs obtained from the day 5 post-inoculation group, a mean number of 1.8 foci were observed and the diameter of these foci measured an average of five computer image pixels (Table 3) . The day 10 pos -inoculation group exhibited a mean foci number of 26.2 and average foci diameter of 14 image pixels. As time increased post-injection, foci enumeration and measurement became more difficult because the individual foci became indistinguishable. Lungs that contained greater than 60 foci on the ventral surface were deemed too numerous to count . These lungs were assigned an arbitrary value of 60 because we were able to reliably count only numbers approaching 60 per lung. Day 14 post-inoculation lungs had a mean foci number of 42 and average diameter of 30 pixels. The two lungs examined from the surviving day 21 post-inoculation group had a foci number mean of 43 and a diameter of 36 pixels (Table 3) . The foci diameter measurements of these two groups could only be estimated due to the foci overlap. Counting error due to indistinguishable overlap is referred to as "masking' and this phenomena has been described in detail elsewhere (75) . This method of tumor foci enumeration provides a unique approach for accurate measurement of tumor progression in this model . TABLE 3 . Tumor Formation Measured As Number Of Visible Foci^a On Lungs Of BALB/c Mice After Intravenous Inj ection With

Live mKSA Cells^b

^aLungs were removed, stained with india ink, then stained and suspended in Fekete ' s solution (EtOH, formalin, glacial acetic acid) . Foci were quantitated on the ventral surface using image analysis with defined parameters . ^b5xl0⁵ cells/dose i .v. injected into dorsal tail vein. Represents range of foci determined for group (S .E .M. ) ^dRemaining mice succumbed to the lethal tumor dose prior to examination. "In these instances, 60 foci were used to calculate the mean (represents greater than 60 foci per lung) .

Studies using the i.p. route of tumorigenic cell inoculation in the SV40 tumor model have employed survival time as the only defined parameter for tumor progression (5, 7, 54, 74, 25) . Alternatively, the i.d. route of inoculation of mice with mKSA cells results in a visible tumor cell mass that is quantitated by measurement and/or weight (25) . However, quantitation of the tumor mass is extremely variable from mouse to mouse after intradermal inoculation of tumor. The advantage of i.v. inoculation of tumorigenic cells for the induction of SV40 tumors, used in the model described herein, is that multiple endpoints, such as lung foci number and size and survival time, can be employed to address issues related to tumor development and evaluation of treatment modalities. This murine model can be used in exploring questions related to tissue-specific tropism, immunologic based therapies, tumor cell evasion of host immune response, and a number of other clinically relevant questions.

Example 2

Imaging Systxem . The results obtained for the metastatic tumor model using the imaging system described above are shown in Figures 3A-B. Groups of 5 mice were inoculated with 5 x 10⁵ mKSA cells and lungs were removed, stained with India ink, then destained and stored in Fekete's solution as described above. Tumor foci were quantitated on the ventral surface using image analysis. Figure 3A represents foci numbers. Figure 3B represents foci diameter in pixels. The standard error of the mean is less than 5.0% of the mean value. At later time points, some mice succumbed to the lethal effects of the tumor. The data presented represents only those mice that survived to that time point.

For the lungs obtained from the day 5 post-inoculation group, a mean number of 1.8 foci was observed and the diameter of these foci measured an average of 5 computer image pixels. The day 10 post-inoculation group exhibited a mean foci number of 26.2 and average foci diameter of 14 image pixels. As time increased post-injection, foci enumeration and measurement became more difficult because the individual foci became indistinguishable. Day 14 post-inoculation lungs had a mean foci number of 42 and average diameter of 30 pixels. Lungs examined from the surviving day 21 post-inoculation group had a foci number mean of 43 and a diameter of 36 pixels (Figure 3) . This computer assisted method has been previously employed in the enumeration of viral plaques and bacterial colonies (5, 6) . This program has been modified to quantitate the size and number of individual tumor foci . To our knowledge this is the first report that utilizes a computer assisted method for the enumeration and quantitation of individual tumor foci. By adapting a computer assisted video imaging analysis system to the enumeration and quantification processes, we present here a unique approach for accurate measurement of tumor progression in our model that is convenient, objective, and reproducible.

The method has been described herein for measurement of tumor foci in lungs, but it will be understood by a person of ordinary skill in the art that other organs may be quantitatively analyzed in a similar manner. Where used herein, the term organ may refer to portions of organs .

Example 3

Comparison of the immune response induced by recombinant

SV40 T-ag versus DNA immunization .

To examine the humoral immune responses generated to SV40 T-ag, anti-SV40 T-ag responses were measured by ELISA following each immunization with recombinant SV40 T-ag (Table 4) . The antibody responses were specific for SV40 T-ag. The pre-immune sera as well as sera specific for HBsAg failed to recognize SV40 T-ag (data not shown) . The reciprocal anti-SV40 T-ag antibody endpoint titers in BALB/c mice ranged from 50 to 3200 and from 12,800 to 1,638,400 following two and four injections with recombinant SV40 T-ag, respectively. To evaluate the CTL responses, splenocytes were obtained from recombinant SV40 T-ag immunized mice after the fourth injection. At various effector to target cell ratios, the immune splenocytes fail to lyse syngeneic tumor cells in the CTL assay (data not shown) . Less than 5% specific lysis was observed with effector cells obtained from rSV40 T-ag immunized mice (E:T cell ratios ranged from 6:1 to 200:1) . This was comparable to the levels of specific lysis obtained with effector cells from control immunized mice. These results indicate that recombinant SV40 T-ag immunization does not induce a specific CTL response in the splenocytes of immune mice, however does induce SV40 T-ag-specific antibodies. SV40 T-ag expressing plasmid DNA immunized mice did not generate detectable antibodies to SV40 T-ag following the fourth injection (titers<50) . However, in vitro CTL responses were observed in splenocytes from SV40 T-ag DNA immunized mice when compared to recombinant SV40 T-ag and control plasmid immunized mice (>30% specific lysis at E:T cell ratio of 100:1, data not shown) . These results confirm previous studies regarding the dichotomy of the anti-SV40 T-ag immune response to SV40 T-ag protein versus genetic immunization (5, 7) .

TABLE 4. Anti-SV40 T-ag Endpoint Titers In Sera From Immunized Mice/

^*Titers were determined by an indirect ELISA.

"Groups consisted of 20 female BALB/c mice, immunized and bled biweekly. ^***The values represent the reciprocal of the dilution of sera that was three times the OD value obtained with a 1 : 10 dilution of the preimmune sera . The mean of each group is shown with the range of individuals in parenthesis .

Protection from Lethal Tumor Challenge

In initial experiments , we examined whether immunization with recombinant SV40 T-ag protects mice from lethal tumor challenge and establishment of experimental pulmonary metastasis . In these experiments , we evaluated two parameters indicative of protective tumor immunity, survival post tumor cell challenge and absence of lung tumor foci development . Table 5 shows that the rSV40 T-ag immunized mice were protected from a lethal tumor challenge of 5 x 10⁵ mKSA cells . Specifically, control mice survived a mean of 24 days post challenge (a range of 22 -31 days ) . Mice immunized with rSV40 T-ag survived greater than 120 days , at which time no indication of tumors was evident . It is clear that rSV40 T-ag immunization also prevented establishment of experimental pulmonary metastasis (Figures 4A-B) . One mouse from each group was euthanized at 25 days post tumor cell inoculation and the lungs were evaluated for tumor foci. The lung obtained from a control mouse contained greater than 60 tumor foci (Figure 4A) , while a lung obtained from the SV40 T-ag immunized mouse exhibited no visible tumor foci (Figure 4B) .

To confirm, these initial observations, a higher tumor challenge dose was selected and an additional parameter was added to evaluate tumor immunity, namely quantification of tumor foci in the lungs . We examined mortality among control and immunized mice after challenge with an i.v. injection of mKSA cells (Table 5) . The mean survival time for six control mice inoculated with 1 x 10⁶ mKSA cells was 21 days (range of 19-23 days) . Recombinant SV40 T-ag immunized mice survived greater than 120 days, during which time no sign of illness or tumors was apparent.

TABLE 5 . Effect of SV40 T-ag Immunization On Survival After SV40 Tumor Cell Challenge

"Each group contained BALB/c mice immunized four times and challenged once with mKSA cells .

"Values represent the mean of the individual survival times . Values in parenthesis represent the range . Plasmid DNA immunized mice that had been previously shown to confer complete tumor immunity in a primary solid tumor model (7) demonstrated partial to no protection in the experimental pulmonary metastasis model with regards to survival. SV40 T-ag gene expressing plasmid DNA immunized mice were challenge with 5 x 10⁵ mKSA cells and mean survival time was 31 days (range 27- 41 days). This compared to 23.5 days (range 23-25 days) for control plasmid DNA immunized mice (Table 5) .

Protection from Lunα Tumor Foci Development Presence of foci in the lungs is used as a main indicator of disease progression in this SV40 murine tumor metastasis model . Lungs were obtained from mice in each group and bleached with Fekete's solution which results in a clear distinction between foci (white) and normal lung tissue (black) after staining. Both the size and number of foci were measured at 10 days post-inoculation. To quantify the lung foci, computer assisted image analysis of the lung ventral surface was employed. Density and size threshold parameters were defined in order to keep counts consistent from experiment to experiment as previously described (65) . Table 6 shows data for individual mice and mean values for the groups. Upon examination of lungs taken from control mice at day 10 post-inoculation, a mean of 33 foci were counted (Table 6) . Foci diameter measurements fell between 5 to 8 pixels by computer image analysis . No foci were detected on lungs taken from immunized mice. Together, these data clearly indicate that SV40 T-ag immunization can prevent the establishment of pulmonary tumors in this experimental animal model of pulmonary metastasis. Alternatively, four mice immunized with rSV40 T-ag DNA contained lung foci following challenge with 5 x 10⁵ mKSA cells. One of the four SV40 T-ag DNA-immunized mice had lung foci comparable in number to the control DNA immunized group of mice (Table 6) . The other three mice immunized with DNA encoding SV40 T-ag had a reduced number of lung foci when compared to controls, however, the size of the foci based on pixel units was comparable or larger. These data indicate that immunization with DNA encoding SV40 T-ag fails to provide complete tumor immunity in this experimental pulmonary metastasis model.

TABLE 6 . Tumor Formation Measured As Number Of Visible Foci On Lungs Of BALB/c Mice After Intravenous Inj ection With mKSA Cells

"Foci count mean±standard deviation for control mice=33±9. 9.

'Since no visible foci were detected, the foci size could not be determined. UTILITY

Simian virus 40 large tumor antigen (SV40 T-ag) is known to be associated with a number of lethal human cancers including human choroid plexus tumors, malignant mesotheliomas, osteosarcomas, astrocytomas, gliomas, and oligodendrocytomas . Many of these tumors are very aggressive, often do not respond well to therapy, and are often difficult or impossible to remove .

The present invention involves an immunotherapeutic approach for the prevention and/or treatment of cancer. The administration to a patient of a vaccine in accordance with this invention for the prevention and/or treatment of SV40 T-ag-type cancer can take place before or after a surgical procedure to remove the cancer, before or after a chemotherapeutic procedure for the treatment of cancer, and before or after radiation therapy for the treatment of cancer and any combination of surgery, chemotherapy and radiation treatment. Therefore, the cancer immunotherapy is administered in accordance with this invention is a preferred treatment for the prevention and/or treatment of certain metastatic cancers, particularly since the risk and side effects involved are substantially minimal compared with the above-identified treatments. A unique aspect of this invention is that the vaccines have the potential or capability to prevent SV40 T-ag-type metastatic cancer in individuals without cancer but who are at risk of developing cancer or to cause regression in individuals who are already afflicted with such metastatic cancer. The administration of a cancer vaccine prepared in accordance with this invention, is generally applicable to the prevention or treatment of cancer. Cancers which could be suitably treated in accordance with the practices of this invention include, but are not limited to, human choroid plexus tumors, malignant mesotheliomas, osteosarcomas, astrocytomas, gliomas, and oligodendrocytomas characterized as having cells which express SV40 T-ag.

In the practice of this invention as set forth herein, the vaccine is intradermally or subcutaneously administered to the extremities, arms and legs, of the patients being treated. Although this approach is generally satisfactory, other routes of administration, such as i.m. or into the blood stream may also be used in a manner known to those of ordinary skill in the art. In addition, the vaccine can be given together with adjuvants and/or immuno-modulators to boost the activity of the vaccine and the patient's response.

The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise 0.1-1000 μg of protein, preferably 1-200 μg, and most preferably 10-100 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunization adequately spaced. Accordingly in one aspect, the invention provides a method of treatment comprising administering an effective amount of a vaccine of the present invention to a patient.

The vaccine formulations of the present invention may be used for both prophylactic and therapeutic purposes . The vaccine compositions of the present invention can be formulated according to known methods of preparing pharmaceutically useful compositions, whereby these materials are combined in a mixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remingtons' Pharmaceutical Sr.-i SΠΓ.PR _Γ (Mack Publishing Co., 1980) .

Those of ordinary skill in the art will recognize that the best treatment regimens for using the vaccine of the present invention to suppress SV40T-ag cancers can be straightforwardly determined. This is not a question of experimentation, but rather one of optimization, which is routinely conducted in the medical arts. The in vivo studies in BALB/c mice provide a starting point from which to begin to optimize the dosage and delivery regimes. The frequency of injection will initially be the frequency used herein in the mice studies (i.e., each two weeks) . However, this frequency might be optimally adjusted from one day to every two weeks to monthly, depending upon the results obtained from the initial clinical trials and the needs of a particular patient. Human dosage amounts can initially be determined by extrapolating from the amount of immunogen used in mice, for example, approximately 200 ng of protein per mouse. Based on this, a 50 kg woman would require treatment with about of 50 μg of protein per dose. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient. The antibodies used herein can be, for example, polyclonal or monoclonal antibodies . The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab (F(ab')2 fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments .

Antibodies generated against the SV40 T-ag or peptide portions thereof corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by other appropriate forms of administering the polypeptides to an animal, preferably a nonhuman. In this manner, even a sequence encoding only a fragment of the SV40 T-ag can be used to generate antibodies binding to the whole native SV40 T-ag.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al . , 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al . , 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) . Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. The polyclonal or monoclonal antibodies may be used prophylactically or may be used therapeutically to treat preexisting metastatic tumors and may be conjugated to the active portion of a toxic substance (e.g. ricin « chain; pseudomonas endotoxin) or radioactive materials . Examples of the many suitable radioactive materials known to those of ordinary skill in the art include S³⁵, Cu⁶⁴, Ga⁶⁷, Zr⁸⁹, Ru⁹⁷, Tc^99m, Rh¹⁰⁵, Pd¹⁰⁹, In¹¹¹, I¹²³, I¹²⁵, I¹³¹, Re¹⁸⁶, Au¹⁹⁸ , Au¹⁹⁹ , Pb²⁰³ , At²¹¹, Pb²¹² and Bi²¹² and in particular high gamma radiation emitters. It will be clear to a person of ordinary skill in the art that the radioactive metals and other metals contemplated herein are not limited to those described herein. The antibodies may be administered for example by intravenous or parenteral injection. The antibodies may kill the tumor cells by mediating apoptosis, oncosis, or antibody-dependent cell-mediated immunologic mechanisms. Effector cells of antibody-dependent cell-mediated immunologic mechanisms may require activation via the exogenous addition of activators of these effector cells in a manner known to those of ordinary skill in the art . Examples of antibodies which may be used during prophylactic a therapeutic treatment are Pab 405, Pab 101, Pab 108 and Pab 419.

Methods for conjugating or labelling the antibodies discussed above with the representative labels set forth above may be readily accomplished using conventional techniques such as described in U.S. Pat. No. 4,744,981 (Trichothecene Antibody); U.S. Pat. No., 5,106,951 (Antibody Conjugate); U.S. Pat. No. 4,018,884 (Fluorengenic Materials and Labelling Techniques); U.S. 20 Pat. No. 4,897,255 (Metal Radionucleotide Labeled Proteins for Diagnosis and Therapy); U. S. Pat. No. 4,988,496 (Metal Radionuclide Chelating Compounds for Improved Chelation Kinetics); Inman, Methods in Enzymology, Vol. 34, Affinity Techniques, Enzyme Purification; Part B, Jacoby and Wichek (eds) Academic Press, New York, P. 30, 1974; and Wilcheck and Bayer, The Avidin-Biotin Complex in Bioanalytical Applications Anal. Biochem. 171:1-32, 1988.

The present invention further includes antigens having immunogenic activity which have a primary amino acid sequence comprising all or part of the amino acid sequence of the large SV40 T-ag protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution as long as the antigen has immunogenic activity in accordance with the present invention. Particularly preferred portions of SV40 T-ag which retain immunogenic activity are amino acids 632-652 (SEQ ID NO:l) and 690-708 (SEQ ID NO:2) or portions of SV40 T-ag which are the binding epitopes of Pab 405 and Pab 101.

The invention contemplated herein further comprises a pharmaceutical composition and method for in vivo or in vitro diagnosis of metastatic cancers characterized by the expression of SV40 T-ag. In one embodiment, the pharmaceutical composition comprises an SV40 T-ag-specific antibody and a radioactive metal or paramagnetic, superparamagnetic, or ferromagnetic metal, e.g., Gd or Mn, bound therto. In one diagnostic method, an effective amount of the compound is administered to an individual known to have or suspected of having metastatic cells or tumors. The compound is delivered by vascular tissue to the site of the malignant tissues where the compound binds preferentially to SV40 T-ag expressed on the surface of cells of metastatic cancer tissues . Once the compound has sufficiently bound to the SV40 T-ag-expressing cells, the sites of the affected tissues within the body can be imaged using standard nuclear medicine imaging techniques or standard magnetic resonance imaging techniques well known to those of ordinary skill in the art. For example, the detection imaging may be gamma scintigraphy, specific photon emission computerized tomography, positron emission tomography or magnetic resonance imaging.

Biologically Functional Equivalents

For example, certain amino acids may be substituted for other amino acids in a protein structure or inserted therein without appreciable loss of equivalent antigenic activity. It is thus contemplated by the inventors that various changes may be made in the sequence of the SV40T-ag proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity. It is also well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted. Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.

Amino acid substitutions, such as those which might be employed in modifying either SV40T-ag or antigenic portions thereof are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side- chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

In making such substitutions or insertions, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, J. Mol. Biol. 157:105-132, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity i.e. with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. As detailed in U.S. patent No. 4,554,101, the following hydrophilicity values have been assigned to 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). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those which are within +0.5 are even more particularly preferred. While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid.

The present invention is not to be limited in scope by the specific embodiments described herein, since such embodiments are intended as but single illustrations of one aspect of the invention and any functionally equivalent embodiments are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims .

All references cited herein are hereby incorporated herein in their entirety. References

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Claims

What is claimed is :

1. A composition comprising recombinant SV40 tumor antigen or an immunogenic portion thereof or an anti-SV40 tumor antigen antibody for use in treating or preventing in a mammal a metastatic tumor, or cells thereof, which express SV40 tumor antigen.

2. The composition of claim 1 wherein said antibody is selected from the group consisting of Pab 101, Pab 108, Pab 419, and Pab 405.

3. The composition of claim 1 or 2 wherein said immunogenic portion comprises SEQ ID N0:1 or SEQ ID NO: 2, or immunogenic variants thereof .

4. The composition of any one of claims 1-3 wherein the composition is a vaccine .

5. A medicine for treating or preventing SV40 T-ag-type metastatic cancer in a mammal comprising recombinant SV40 tumor antigen or an immunogenic portion thereof or an anti-SV40 tumor antigen antibody.

6. The medicine of claim 5 wherein said antibody is selected from the group consisting of Pab 101, Pab 108, Pab 419, and Pab 405.

7. The medicine of claim 5 or 6 wherein said immunogenic portion comprises SEQ ID NO:l or SEQ ID NO: 2, or immunogenic variants thereof.

8. The medicine of any one of claims 5-7 wherein the medicine is a vaccine.

9. Use of recombinant SV40 tumor antigen or an immunogenic portion thereof or an anti-SV40 tumor antigen antibody to treat or prevent SV40 T-ag-type metastatic cancer in a mammal .

10. A method of stimulating or increasing the immune response against SV40 T-ag-type metastatic cancer in a mammal, comprising: administering to the mammal a vaccine composition comprising an intact recombinant SV40 tumor antigen or an immunogen comprising one or more immunogenic epitopes of SV40 tumor antigen or a combination thereof.

11. The method of claim 10 wherein the composition further comprises an adjuvant.

12. The method of claim 10 or 11 wherein said immunogen comprises a Pab 405-binding epitope of SV40 T-ag.

13. The method of claim 10 or 11 wherein the immunogen comprises SEQ ID NO: 2 or an immunogenic variant thereof.

14. The method of claim 10 or 11 wherein said immunogen comprises a Pab 101-binding epitope of SV40 T-ag.

15. The method of claim 10 or 11 wherein the immunogen comprises SEQ ID NO:l or an immunogenic variant thereof.

16. The method of claim 10 or 11 wherein said immunogen comprises a Pab 405 -binding epitope and a Pab 101-binding epitope of SV40 tumor antigen.

17. A method of treating a metastatic tumor or cells thereof in a mammal, cells of which tumor express large SV40 tumor antigen, or an antigenic portion thereof, comprising: administering to said mammal having said metastatic tumor or cells thereof an effective amount of an anti-SV40 T-ag antibody or a vaccine comprising recombinant SV40 tumor antigen or an immunogenic portion thereof to kill, inhibit, or induce regression of said metastatic tumor or cells thereof .

18. The method of claim 17 wherein said antibody is selected from the group consisting of Pab 101, Pab 108, Pab 419, and Pab 405.

19. The method of claim 17 or 18 wherein said immunogenic portion comprises SEQ ID NO:l or SEQ ID NO : 2 , or immunogenic variants thereof.

20. The method of any one of claims 17-19 wherein the vaccine is monovalent .

21. The method of any one of claims 17-19 wherein the vaccine is polyvalent .

22. The method of any one of claims 17-21 wherein the mammal is a human.

23. A cancer vaccination method, comprising: administering to a mammal subject a vaccine comprising: an amount of large SV40 T-ag or an immunogenic portion thereof, and a pharmaceutically acceptable carrier, diluent or excipient; and wherein the vaccine is capable of inducing an immune response comprising production of anti-large SV40 T-ag antibodies effective against metastatic cancer cells which express large SV40 T-ag.

24. The method of claim 23 wherein the mammal subject is a human.

25. The method of claim 23 wherein the mammal subject is a rodent .

26. The method of claim 25 wherein the rodent is a mouse.

27. The method of any one of claims 23-26 wherein the vaccine comprises an adjuvant.

28. The method of any one of claims 23-27 wherein the large SV40 T-ag or immunogenic portion thereof is a recombinant polypeptide or peptide.

29. The method of any one of claims 23-28 wherein the vaccine comprises SEQ ID NO:l or SEQ ID NO : 2 , or immunogenic variants thereof.

30. A method of modelling metastatic SV40 T-ag-type cancer, comprising: intravenously administering SV40 T-ag-transformed tumorigenic cells into a test animal .

31. The method of claim 30 wherein the test animal is a mammal .

32. The method of claim 30 or 31 wherein the SV40 T-ag- transformed tumorigenic cells are mKSA cells.

33. A method of screening for compounds which inhibit, reduce or prevent SV40-type metastatic cancer, comprising.- providing a test animal treated to induce an SV40 T- ag-type metastatic cancer; treating the test animal with a test compound; and assessing the metastatic tumor foci in the test animal after a predetermined period of time .

34. The method of claim 33 wherein the test compound is a vaccine.

35. The method of claim 33 or 34 wherein the test compound is an antibody against SV40 T-ag or against a portion thereof.

36. The method of any one of claims 33-35 wherein the test animal is treated with the test compound after the test animal is treated to induce SV40 T-ag metastatic cancer.

37. The method of any one of claims 33-36 wherein the test animal is treated with the test compound before the test animal is treated to induce SV40 T-ag metastatic cancer.

38. The method of any one of claims 33-37 wherein the test animal is a mammal .

39. The method of claim 38 wherein the mammal is a rodent.

40. The method of any one of claims 33-39 wherein metastatic cancer is induced in the test animal by intravenously injecting the test animal with SV40 T-ag transformed tumorigenic cells .

41. A method of quantifying tumor burden in a test animal, comprising: providing an organ of a test animal treated in a manner known to induce a metastatic tumor response in the organ in the test animal; treating the organ with a stain for distinguishing between tumor foci and normal tissue; obtaining a photographic image of the treated organ; and automatically analyzing the photographic image using computer image analysis software to determine the number and size of tumor foci in the organ by selecting object images which exceed a predetermined minimum size threshold and which exceed a predetermined minimum density value.

42. The method of claim 41 wherein the photographic image is obtained using a CCD camera.

43. The method of claim 41 or 42 wherein the organ used in the analysis method is a lung.

44. The method of any one of claims 41-43 wherein the organ is stained with India ink and bleached to enhance tumor foci visualization.

45. The method of any one of claims 41-44 wherein the organ is placed and flattened between two transparent plates before the step of obtaining the photographic image.

46. A composition comprising recombinant SV40 tumor antigen or an immunogenic portion thereof or an anti-SV40 tumor antigen antibody for use in a mammal in treating, preventing, killing, inhibiting, or inducing regression of a metastatic tumor, or cells thereof, which express SV40 tumor antigen.

47. A medicine for treating, preventing, killing, inhibiting, or inducing regression of SV40 T-ag-type metastatic cancer in a mammal comprising recombinant SV40 tumor antigen or an immunogenic portion thereof or an anti-SV40 tumor antigen antibody.

48. Use of recombinant SV40 tumor antigen or an immunogenic portion thereof or an anti-SV40 tumor antigen antibody to treat, prevent, kill, inhibit, or induce regression of SV40 T-ag-type metastatic cancer in a mammal.

49. A method of treating or preventing a metastatic tumor or cells thereof in a mammal, cells of which tumor express large SV40 tumor antigen, or an antigenic portion thereof, comprising: administering to said mammal an effective amount of an anti-SV40 T-ag antibody or a vaccine comprising recombinant SV40 tumor antigen or an immunogenic portion thereof to treat, kill, inhibit, induce regression of, or prevent said metastatic tumor or cells thereof.

50. A method of modelling metastatic SV40 T-ag-type cancer, comprising: administering SV40 T-ag-transformed tumorigenic cells into a test animal.

51. A method of screening for compounds which treat, kill, inhibit, reduce, or prevent SV40-type metastatic cancer, comprising: providing a test animal treated to induce an SV40 T- ag-type metastatic cancer; treating the test animal with a test compound; and assessing the metastatic tumor foci in the test animal after a predetermined period of time.

52. A method of quantifying tumor burden in a test animal, comprising: providing an organ of a test animal treated in a manner known to induce a metastatic tumor response in the organ in the test animal ; treating the organ with a stain for distinguishing between tumor foci and normal tissue; obtaining an image of the treated organ; and automatically analyzing the image using computer image analysis software to determine the number and size of tumor foci in the organ by selecting object images which exceed a predetermined minimum size threshold and which exceed a predetermined minimum density value.

53. A composition as claimed in any one of claims 1-4 and

46, wherein the mammal is a human or a rodent.

54. A medicine as claimed in any one of claims 5-8 and

47, wherein the mammal is a human or a rodent.

55. A use as claimed in claim 9 or 48, wherein the mammal is a human or a rodent .

56. A method as claimed in any one of claims 10-22 and 49, wherein the mammal is a human or a rodent.

57. A method as claimed in any one of claims 30-38, 40- 45, and 50-52, wherein the animal is a human or a rodent .

58. A method as claimed in any one of claims 41-45, wherein the animal is a mammal .

59. A composition as claimed in claim 53, wherein the rodent is a mouse.

60. A medicine as claimed in claim 54, wherein the rodent is a mouse.

61. A use as claimed in claim 55, wherein the rodent is a mouse .

62. A method as claimed in claim 51 or 57, wherein the rodent is a mouse .