WO1990011765A1

WO1990011765A1 - Antitumor preparation obtained following oncolysate treatment

Info

Publication number: WO1990011765A1
Application number: PCT/US1990/000787
Authority: WO
Inventors: Eva Lotzova; Jim Klostergaard; Ralph S. Freedman; James M. Bowen
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1989-04-11
Filing date: 1990-02-12
Publication date: 1990-10-18
Also published as: AU5275290A

Abstract

Disclosed is the use of viral oncolysates in the preparation of a complement-dependent, soluble antitumor factor having particular antitumor activity against ovarian carcinomas. The viral oncolysates employed in the preparation of the antitumor factor of the present invention is derived through the use of an influenza type. A strain PR8/34 virus, which is employed to modify the membranes of ovarian carcinoma cells in vitro without virally lysing these cells. The antitumor factor itself is prepared by administration of an effective amount of the viral oncolysate to an ovarian carcinoma patient bearing an ascites-producing ovarian carcinoma. Following treatment, the presence of the antitumor factor is elicited in the ascites of the individual. Ascites fluids bearing the antitumor factor are collected, and the antitumor factor is obtained therefrom. Also disclosed are particularly useful ovarian carcinoma cell lines for the preparation of oncolysates, the MDAH 2774 and the CaOV3 cell lines.

Description

ANTITUMOR PREPARATION OBTAINED

FOLLOWING ONCOLYSATE TREATMENT

The present invention relates to novel antitumor preparations, including processes for their preparation and use. In particular aspects, the invention concerns antitumor preparations obtained from viral oncolysate treated individuals bearing ascites-producing ovarian tumors.

Viral oncolysates have been the subject of studies in cancer research for a number of years. Virus augmentation is a biologic response modifier approach to antitumor therapy by which the immunogenicity of tumor cell extracts is enhanced by infecting the tumor cells with a selected virus such as influenza virus, vesicular stomatitis virus (VSV), Newcastle disease virus (NCDV) and vaccinia (1-5).

The virus-augmentation effect was first demonstrated in animals. Researchers compared the reactions of animals protection from transplanted tumors by prior immunization with viral oncolysates to those of animals receiving non- viral modified tumor cell extract, virus alone, or an admixture of virus and extract. Intensified delayed-type hypersensitivity reactions were observed in animals injected with viral oncolysates . Some animals with established tumors survived unexpectedly longer when injected with viral oncolysates (2,4) Humans receiving allogeneic extracts of cultured tumor cells similar to their own tumors also showed intensified delayed-type hypersensitivity reactions (1) and potentiation of natural immunity (22). Studies have shown that delayed-type hypersensitivity reactions are augmented in ovarian cancer patients injected with viral oncolysate derived from influenza virus-modified ovarian carcinoma cultures (6). Furthermore, possible therapeutic applications have been investigated in various tumors, including the treatment of malignant melanoma using

Newcastle disease virus oncolysate (7), the treatment of vulvar carcinoma (8) squamous carcinoma (9) and others (10-15). Various novel oncolysate preparations have been described in patent disclosures for use in the treatment of cancer. For example, Wallack (16) describes a viral oncolysate vaccine for stimulating the immune mechanism of mammals to species-specific tumors. The vaccine was prepared by infecting monolayers of tumor cells with live vaccinia virus, incubating for three to five days and collecting the lysate in the form of a supernatant

obtained after centrifugation of the lysed cellular debris. The vaccine prepared in this manner was employed to treat species-specific tumors, apparently through stimulating the immune mechanism of the tumor burdened animal.

Unfortunately, while these preparations have shown both utility and promise in antitumor therapy, they are clearly not ideal. Novel treatment preparations and modalities are needed in order to press forward in our search for effective approaches to antitumor therapy. The present invention addresses shortcomings in the prior art, including deficiencies in the treatment of cancer, by providing a novel antitumor preparation

prepared through the use of viral oncolysates. The invention concerns generally a soluble, complement- dependent antitumor factor which is derived from ascites fluids of viral oncolysate-treated ovarian cancer

patients. In addition to being soluble and complement- dependent, the antitumor factor itself is characterized as having the molecular weight of approximately 90-120 kilodaltons when subjected to exclusion chromatography, such as chromatography on a gel exclusion matrix.

In certain aspects, the invention concerns a process for preparing an antitumor preparation which comprises treating an individual bearing an ascites-producing ovarian tumor with a viral oncolysate to stimulate the appearance of a soluble, antitumor factor into the ascites fluid of the treated individual. This is followed by collecting the ascites fluid, and fractionating it in a manner to provide a preparation enriched for the antitumor factor. As used herein, the term "fractionating" is meant to refer broadly to any procedure wherein a biologically active portion or fraction of the ascites containing the antitumor factor is obtained. Therefore, fractionating includes concentrating, dialyzing, molecular weight fractionation, chromatography and the like.

The particular oncolysate which has been found to work well in connection with the present invention has been termed an influenza virus viral oncolysate in that influenza virus, preferably a type A influenza virus, is employed to infect cell lines used in a preparation of the oncolysate. A particularly useful type A influenza virus is provided by strain PR8/34, such as described by

Sinkovics et al. in reference 17. This virus may be obtained generally from the CDC or Flow Laboratories. In the preparation of suitable viral oncolysates for use in connection with the present invention, one will desire to use ovarian tumor cells for viral infection. The ovarian tumor cells employed, typically an ovarian tumor cell line such as OV-2774 and CaOV3, are characterized in that upon infection with the influenza virus, the infected cells will not be lysed prior to extract preparation. For practical purposes, the cell line will be allogeneic. However, autologous tumor or established cell line may be used. The selected ovarian tumor or cell line will be characterized by phenotypic cell surface features and chromosomal analysis. In particular, one can identify suitable allogeneic ovarian tumor cells for use in connection with the present invention by their

sensitivity to PR8/34 virus infection.

Two ovarian carcinoma cell lines, designated MDAH 2774 and CaOV3, have been found by the inventors to work well in connection with the practice of the present invention. The MDAH 2774 cell line has been described in some detail in previous publications (18, 19). Moreover, the CaOV3 cell line, has been characterized in various publications (6). The CaOV3 cell line has been deposited in the ATCC, and accorded serial number ATCC HTB75.

In the practice of the invention, one therefore seeks to modify the surface membranes of the selected allogeneic cultured ovarian tumor cells through the use of the influenza virus, without lysing the cells. Surface membrane modification is achieved by infecting the selected ovarian carcinoma cells with the virus until virus modification of the membrane occurs, usually on the order of approximately 20 hours. The cells are then washed and an extract prepared. The virus-infected ovarian tumor cell extract is then administered, preferably intraperitoneally, to an individual bearing an ascitic ovarian tumor in order to elicit the appearance of the soluble antitumor factor into the ascites fluid. The ascites fluid bearing the antitumor factor is then

fractionated by one of various methods in order to provide a more purified or enriched preparation of the antitumor factor hereof.

In certain embodiments of the invention, the ascites fluid is fractionated by means of dialysis. Dialysis serves to remove small molecules from the preparation in order to purify the preparation, and render it more acceptable pharmacologically. Thus, dialysis is employed to remove molecules such as low molecular weight hormones and polypeptide growth factors. Although virtually any suitable dialysis membrane can be employed for dialysis of the ascites fluid, a preferred dialysis membrane is one having relatively large pore sizes, allowing the passage of molecules of on the order of 8 or even 12-14 kilodaltons in size. Such large pore dialysis membranes are employed in order to assist in removing relatively large molecular weight contaminations. However, since the size of the antitumor factor has been estimated to be on the order of 90 to 120 kilodaltons, a large pore dialysis membrane will have little or no effect in removing the antitumor factor from the ascites fluid preparation upon dialysis.

Accordingly, in certain aspects, the invention is directed to a process which includes dialyzing the ascite fluid, or a biologically active fraction thereof, to provide a preparation enriched for the antitumor factor.

As noted, the antitumor factor of the present invention has been characterized as having a molecular weight of on the order of about 90 to about 120 kilodaltons, when the ascites fluid is subjected to exclusion chromatography, and fractions therefrom are assayed for antitumor activity. However, it will be appreciated by those of skill in the art that exclusion chromatography will not generally give a precise measurement of size.

Therefore, the size range of 90 to 120 kilodaltons refers only to the apparent molecular weight obtained for this factor when ascites fluid is subjected to exclusion chromatography in the manner detailed herein. Therefore, other molecular sizing techniques may reflect an apparent molecular weight for the factor which is different than that observed upon exclusion chromatography as detailed herein. In any event, molecular exclusion chromatography can be employed to fractionate the antitumor "activated" ascites fluid and thereby provide the antitumor factor of the present invention in a more highly purified form.

Accordingly, in certain aspects of the present invention, the process for preparing the antitumor

preparation will include subjecting the ascites fluid, or a fraction thereof, to exclusion chromatography to provide a fraction enriched for the antitumor factor.

In further aspects of the invention, it will be generally desirable to render the entire tumor factor- enriched ascites fluid extract or fraction pharma- chrologically acceptable so that it can be used directly in the treatment of humans bearing cancer. This can be achieved by techniques generally known to those who have skill in the art in light of the disclosure herein. For example, the ascites fluid-derived material can be sterilized through the use of sterilization filters such as nitrocellulose membranes and the like.

In certain aspects, therefore, the invention is concerned with the use of one of the foregoing antitumor treatment of ovarian cancer. It has generally been found that the antitumor factor is highly active against several types of ovarian cell lines. The antitumor preparation of the present invention has not shown activity against nonovarian derived cells, such as the leukemia cell lines K-562 or the murine tumor YAC-1. Accordingly, the antitumor factor of the invention can be classified in particular as an anti ovarian tumor factor. However, since limited panel of tumors has been analyzed, this antitumor factor might not be limited to ovarian tumors.

Figure 1 illustrates the cytotoxic effect of the soluble antitumor factor against ovarian carcinoma cell line OV-2774, induced by intraperitoneal administration of viral oncolysates. Viral oncolysates were administered to patients with ovarian carcinoma in two injections, three weeks apart and ascitic fluids were drawn before or 24 hours after each injection. The cytotoxic effect of the soluble antitumor factor was assayed at 40 percent concentration in a three hour Cr-release assay.

Figure 2 illustrates a dose-response curve of the cytotoxic activity of the soluble antitumor factor against the ovarian carcinoma cell line OV-2774. Ascitic fluids collected from peritoneum of patients with ovarian

carcinoma treated with viral oncolysates were tested for cytotoxic effect against OV-2774 in a three hour ⁵¹Cr- release assay. The fluids were collected three weeks after a single injection of oncolysates. The figure shows the results (mean plus/minus s.d.) of the effect of ascitic fluids from three different patients.

The present invention is based on the surprising discovery that the ascitic fluids of patients with ovarian carcinoma who have been treated with viral oncolysates exhibit the appearance of a soluble antitumor factor highly cytotoxic for human ovarian tumor cells in vitro. This soluble antitumor factor, in studies to date, have displayed a certain degree of selectivity. Its cytotoxic activity has been exhibited most profoundly in connection with the treatment of ovarian tumors. While ovarian tumor cell lines have been found to be sensitive to the antitumor factor, non-ovarian cell lines have generally not been found to be sensitive. This selectivity may be related to the antigenic profile of viral oncolysates used for patients' treatment. Initial purification of the factor has demonstrated it to have an apparent molecular weight of on the order of 90,000 to 120,000 daltons as measured by gel exclusion chromatography on Ultrogel AcA44 (a gel exclusion chromatography matrix obtained from IBF Biotechnics, France).

In a general sense, preparation of the compositions containing the antitumor factor of the present invention is achieved in the following manner:

1. The preparation of an influenza virus-generated viral oncolysate through the non-lytic infection of ovarian carcinoma cell lines with an influenza type A virus in a manner sufficient to modify the membranes thereof, followed by the preparation of an extract from the virus-modified tumor cells.

2. The intraperitoneal administration of the oncolysate to an individual bearing an ascites producing ovarian tumor so as to elicit the appearance of the antitumor factor in ascites fluid of the individual.

3. Collection of the ascites fluid and fractionation in a manner so as to provide a preparation enriched for the antitumor factor or, additionally, rendered pharmacologically acceptable. I. Preparation of Viral Oncolysates

The preparation of viral oncolysates suitable for use in connection with the present invention has been

described previously (6,8,14). As described in the foregoing references, two cultured and characterized ovarian carcinoma cell lines, MDAH 2774 and CaOV3, were used in preparing the viral oncolysates. CaOV3 cell line has been deposited with the American Type Culture

Collection, and accorded accession number ATCC HTB75. The cells have undergone isozyme analysis. Both lines were found to be independent of HeLa cell contamination in that they express the B phenotype for G6PV.

A. History and Geneaology of

the MDAA 2774 Cell Line

The MDAH 2774 cell line was developed from ascites borne cells obtained from a 40 year old female (0 Rh+ blood group) bearing an ovarian tumor. Tissue diagnosis indicated a mixed mucinous and serous adenocarcinoma of the ovary. The tumor was untreated. Ascites fluid collected from the patient was allowed to sediment in a vacuum collecting bottle. The sedimented cells were collected, resuspended and plated. The cells were cultured in L-15medium+10% Fetal Bovine Serum (FBS) and a split ratio of about 1:40, weekly, was employed. The cells were characterized on passage 132 (18). The following represents various results obtained in characterizing MDAH 2774 cells: 1. Cytogenetics

The modal number of chromosomes in G banded meta- phases prepared from MDAH 2774 was 66-68 with a general trend toward triploidy which was constantly found in chromosomes 1, 2, 3, 6, 11, 12, 16, X, whereas monosomy was found in 17 and 21. Three marker chromosomes were identified. 2. Transmission Electron Microscopy (TEM)

TEM confirmed a poorly differentiated epithelial tumor with interdigitation of cells, surface microvilli, desmosomes, large indented nuclei with prominent

fenestrated nucleoli, granular endoplasmic reticulum, numerous mitochondria and annular lamellae.

3. Isoenzyme patterns Isoenzyme determinations have been routinely

performed. The last result provided the following

analysis for MDAH 2774:

PGM1 PGM3 ESD AK1 ADA ACP1 PGD G6PD

1 1 1,2 1 NE a,b a b

ME2 GL01 PGP Frequency

1 1,2 1,2 9.15 X 10^-4 Analyses confirm the stable phenotypic characteristics of this cell line and the absence of HeLa contamination. MDAH 2774 is heterozygous 1,2 for ESK whereas CaOV³ (the other ovarian cell line used in

preparation of oncolysates) is only type 1 for that locus, indicating independent origins for these cell lines. 4 . HLA typing:

Locus A Locus B Locus C DR Others

A3,24 Bw 45,57 Cw2,X W2 , 5 TD11,

MT1,2,4

le5

5. Monoclonal antibody phenotyping cell surface antigen analysis of blood group and tumor associated antigens was performed by fluorescent activated cell sorter analysis:

MoAb Code# OC125 29-1 Co44.1 GA73.3

% cells bound 89 26 15 20

MoAb Code# 33/25/17 E283-52 E283-52 C051-4 CO

% cells bound 20 0 1 0 0 Control preparations incubated with goat antimouse FITC conjugated antibody labelled 2% of MDAH 2774 cells.

Antigen defined by monoclonal antibodies in phenotyping analysis of MDAH 2774 mAb ANTIGEN DEFINED Code Name

OC-125 Glycoprotein*

29-1 LNFIII

CO 44-1 Protein

GA73-3 35kd protein 180-3-44 Not identified

WG-HS-NS-29-1

33/25/17 Blood group A

E2 83-52 Blood group B, 1+2

CO 51-4 Lewis A

CO 43-1 Lewis B

GI 3174 Glycoprotein

*Identified with ovarian and some other carcinomas

Common blood group isoantigens were absent.

The cell culture was obtained by the inventors at about passage 132 and has been cultured in L-15 (Gibco #320-1415AJ) and 10% FBS (Hazelton #12-10378). The cells were grown as thick, multilayered colonies, with an epithelial-like morphology. The cells exhibited a lack of contact inhibition, numerous mitotic figures, irregular hyperchromatic nuclei, high nuclear cytoplasmic ratios and multiple nucleoli.

B. The CaOV₃ Cell Line

The CaOV₃ cell line is an ovarian carcinoma cell line which was derived and characterized as described in reference 6,20. Whereas, the cell line MDAH 2774 was heterozygous for EsD, and CaOV3 was homozygous type 1 for that locus, which indicates that the origins of the lines were independent of each other. Genotypes at certain other loci may be informative for identifying these cell lines in the future: MDAH 2774 was heterozygous (2,2) for PGP and had the less common form (type 1) for GLO1; CaOV3 had the less common forms, 2 and 1, for ME2 and GLO1, respectively.

Human leukocyte antigen analysis of CaOV3 was performed by a standardized two-stage NIH microcytotoxicity assay, and revealed the following additional characteristics:

HLA-A HCA-B HLACW

28,29 Y9,W61 4,X

HLA-DR MT

5,W8 MT1,4 Surface-antigen characterization of tumor cells used in preparing ovarian VO was performed with murine monoclonal antibodies (MoAb) to blood group and tumor- associated antigens by fluorescence-activated cell sorter analysis. The Ca-125 antigen, commonly associated with nonmucinous ovarian carcinoma tissues, was detected on the surface membrane of 89% of MDAH 2774 cells and 33% of CaOV3 cells, whereas blood groups A, B, and Lewis a and b were notably absent. Control antibody stained 2% of MDAH 2774 cells and 3% of the CaOV3 cells.

C. Virus

Type A influenza virus, PR8/34, was used to infect the ovarian carcinoma cells. The virus was egg adapted and originally obtained from Flow Laboratories, Rockville passages in leukosis-free embryonated hens' eggs.

Limiting dilutions of the virus in phosphate-buffered saline (PBS) were injected into the allantoic cavities of 10-day-old embryonated chicken eggs.

To prepare the virus, eggs were incubated at 35°C for 48 hours. Allantoic fluids were harvested and pooled.

Pooled allantoic fluids, checked for sterility, titrated for viral hemagglutin in hemagglutination assay (HA), were dispensed into small ampoules, flash-frozen, and stored at -70°C. Presence of virus of all preparations were

routinely monitored by titrating HA using a standard tube dilution technique and read by the pattern method after the virus dilutions had been incubated with 0.4% washed, pooled chicken erythrocytes.

1. Virus infection of Cultured Human

Tumor cell Line MDAH 2774 The kinetics of PR8 influenza A virus infection were studied in the MDAH 2774 ovarian cell line. Viral

replication was detected by titrating culture fluid for hemagglutination of chicken red blood cells. Peak antigen modification of the cell membrane was measured by cell membrane immunofluorescence developed by polyvalent rabbit anti-PR8 serum. Peak virus modification for MDAH 2774 was observed at 12-24 hours.

2. Preparation of MDAH 2774 Lysates

Cells were seeded into T-150 plastic tissue culture flasks in L-15 medium supplemented with 10% fetal bovine serum. When the monolayers were just subcontiguous, they were washed free of growth medium and incubated for a further 72 hours in L-15 without serum. No antibiotics were used in these cultures at any time. After incubation in serum free medium, the monolayers were washed again in sterile pH 7.3 phosphate-buffered saline (PBS) and

infected with 5 ml/flask of a dilution of frozen stock virus in PBS sufficient to give approximately 10⁵ egg infection units per flask. The infected cultures were incubated at 35°C. After a 20-hour incubation, cells were scraped from the flask with a sterile rubber policeman. The resulting suspensions were aseptically pooled, centrifuged at 300xg, and the supernatants discarded. The cell pellets were resuspended in PBS and washed by a further centrifugation.

The washed cell pellets were then resuspended in an equal volume of 1 mM MgCl₂ and DNAse. A preferred source for DNAse has been Sigma Chemical Company Type II-S D4513 (endotoxin-free and chromatographically purified).

The resulting suspension was sonicated for 3 minutes in a Raytheon cup sonicator with an output of 310 KHz.

Sonications were carried out in small plastic tubes packed in ice in the sonicator cup. Ultraviolet irradiation to reduce viral infection of the sonicates was carried out as follows: A homogenate layer of less than 4 mm in a sterile 50 mm Petri dish was irradiated in a hood with a shortwave UV lamp, set to deliver 40 erg sec^-1M₂. The irradiated lysates were then pooled and adjusted to a uniform protein concentration. Each was placed in labeled precapped serum vials and stored at -70°C. Lysates of the virus infected cell line MDAH 2774 have been designated OVO1.

The CaOV3 cell line is infected, and oncolysate prepared, in essentially the same manner as the foregoing MDAH 2774 description. II. Viral Oncolysate Administration,

Dose, and Schedule

In general, oncolysates are administered intra- peritoneally (i.p.) to patients in aliquots containing protein contents ranging from about 1.5 to about 9.0 mg, with about 9 mg being preferred. Such aliquots are typically administered i.p. by a schedule such as bimonthly or monthly, with monthly administration being found optimal in many cases. However, this schedule can be varied, depending on the patient or response. Most preferably, about 9 mg of the oncolysate is administered by a schedule such as monthly or bi-monthly. It has been found that mixtures of oncolysates obtained from different cell sources may be particularly useful in eliciting the appearance of the antitumor factor. The advantage of the mixture of ovarian cell lines is to broaden the immunogenicity of the preparation. Of course, other reasons for the improved performance of oncolysate mixtures may exist.

The present inventors have discovered that mixtures of oncolysates derived through the use of MDAH 2774 and CaOV₃ ovarian carcinoma cell lines are effective in eliciting the antitumor factor. The following methods represent protocols which have been employed in

administering oncolysates of these cell lines to ovarian cancer patients:

For development of the antitumor factor preparation of the invention, epithelia ovarian cancer patients are selected on the basis of ascites production and failure to respond to conventional therapy. Ascites production is determined both through physical examination of the patient and by cytology. The particular type of ascites producing ovarian tumor has not been found to be important in obtaining a highly active antitumor preparation.

Lysates of the two virus-infected ovarian carcinoma cell lines, MDAH 2774 and CaOV3, prepared as above were designated OVO1 and OVO2, respectively. An aliquot of each extract was adjusted to a protein content of 4.5 mg and suspended in 5 ml N saline for i.p. injections. OVO1 and OVO2 aliquots were mixed and suspended in 10 ml nonpyrogenic saline for i.p. injection, usually through a No. 15-gauge angiocatheter. Each i.p. dose was

equivalent to 9.0 mg. No concomitant chemotherapy was administered.

Patients received 9.0 mg doses of a 1:1 combination of OVO1 and OVO2 by i.p. injection. A few patients received either 9.0 mg of OVO1 or OVO2 alone (when the accompanying extract was unavailable at that time).

During the initial treatment the patients received a single injection, injections 2 weeks apart, or weekly injections. Three i.p. injection schedules, weekly, biweekly, or monthly were used. Subsequent injections were given at monthly intervals. A few patients received additional i.p. injections of a 1:1 mixture of OV1 and OV2 equivalent to 3.0 mg. i.p. injections were administered simultaneously with initial i.p. injections in patients who had presented with pleural effusions and ascites, and at later dates to 2 patients who developed pleural effusions while undergoing i.p. OVO injections.

In terms of antitumor factor production, the

preferred dosage and schedule involved the i.p. administration of 9 mg of a 1:1 mixture of OVO1 and OVO2, at bi- monthly to monthly intervals, administered over several minutes. III. ASCITES COLLECTION AND

ANTITUMOR FACTOR PREPARATION Following oncolysate treatment, the antitumor factor- bearing ascites fluid is removed from the patient by paracenthesis (removal of fluid from abdomen). It has generally been found that 2 injections of oncolysate are needed in order to obtain a high titer of antitumor

activity in the resultant ascites fluid.

Once collected, the ascites fluid is centrifuged at 1500 rpm to remove particulate matter, cells, and cellular debris and either tested for its effect on tumor cells in cytotoxicity assay or stored at -20° C and tested later.

Following clarification of the collected ascites, one will typically desire to subject it to dialysis to remove undesirable salts, contaminating peptides and other small molecular "weight components unrelated to the antitumor factor. A preferred dialysis treatment employs a large pore membrane, such as one having a pore size allowing the passage of molecules on the order of 8 to 14 kilodaltons. This facilitates the removal of contaminating low molecular weight material. A preferred membrane for dialysis is one such as Spectra/Por, obtained from Spectrum Medical Industries, Inc., Los Angeles, CA.

It has generally been found desirable to dialyze the ascites fluid with a buffer such as Dulbeccos' phosphate buffered saline. This is because the preparation is stable at the pH and ionic strength of this buffer and the biological assay is not influenced by this buffer.

However, other buffers which allow retention of biological activity and which do not perturb the biological assay should also be suitable. Following dialysis, the ascites fluid will typically be subjected to further fractionation, for example, on the basis of molecular weight or size. A preferred technique employs gel exclusion chromatography of the ascites fluid on a gel exclusion matrix such as Ultrogel AcA44 (IBF Biotechnics, France). Ultrogel AcA44 is an acrylamide cross-linked agarose matrix with an effective range of 10,000-130,000 daltons, which has been found to work particularly well in the practice of the invention.

However, where molecular weight fractionation of the ascites fluid by exclusion chromatography is employed, it should be appreciated that numerous other gel matrices can be employed, including, for example, Sepharose G-200, and the like.

Alternatively, or in addition to molecular weight fractionation, one may desire to concentrate the ascites fluid or fractions thereof. Suitable concentration can be achieved by a variety of means known in the art,

including, for example, by vacuum concentrators, lyophilization ultrafiltration, ammonium sulphate fractionation, or even precipitation by alcohol or a mild acid. Of course, upon concentration it will be appreciated that one will typically desire to further dialyze the concentrated material.

The antitumor activity of the ascites preparation containing the antitumor factor can be monitored during fractionation by a variety of techniques, such as through the use of in vitro cytotoxicity assays employing ovarian carcinoma cell lines, or using cell lines developed from the patient to be subsequently treated. A preferred assay for screening the antitumor activity is the ⁵¹Cr-release assay. This assay measures the release of radioactive chromium by dead tumor "target" cells in vitro, upon incubation with agents which lyse these cells.

To determine the therapeutic efficacy of the factor, it will generally be the case that one will desire to administer the antitumor preparation parenterally and, in particular, by a i.p. route. Therefore, one will

generally desire to render the preparation suitable for parenteral administration. This involves rendering the material sterile and may include solubilization of

precipitated powdered or lyophilized samples in a sterile aqueous solution. Aqueous solutions should be suitably buffered if necessary and the liquid diluent first

rendered isotonic with sufficient saline or glucose.

These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intra- peritoneal administration. In this connection, the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art. Moreover, the novel factor of the invention may be

administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. The dosage, of course, will be

determined by the physician who administers the antitumor preparation, and will be determined on the basis of both the antitumor effect observed as well as the appearance of undue toxic or immunologic reaction in the particular patient.

ANTITUMOR FACTOR CHARACTERIZATION

Turning now to Figure 1 is demonstrated the cytotoxic effect of the soluble antitumor factor elicited in the ascites fluids of three separate individuals. The cytotoxic activity of the soluble factor was measured in an in vitro ⁵¹Cr-release assay using the ovarian carcinoma OV-2774 as a target. Three separate patients were treated with the viral oncolysate injection in the manner

described above in section II. Ascitic fluid obtained from these patients at various intervals was tested for its ability to lyse the target ovarian carcinoma cells (see Ref. 21). Therefore, the data is representative of the kinetics of appearance of the antitumor factor in the ascitic preparations.

In the Figure 1 studies, viral oncolysates were administered to patients with ovarian carcinoma in two injections, three weeks apart, and ascitic fluids were drawn before or 24 hours after each injection. The cytotoxic effect of the soluble antitumor factor was assayed at 40% final concentration.

Figure 2 represents a dose response curve of the cytotoxic activity of the soluble antitumor factor against the OV-2774 cell line, again in a three hour ⁵¹Cr-release assay. Ascitic fluids were collected from the peritoneum of patients with ovarian carcinoma who had been treated with viral oncolysates prepared in the manner detailed above in section II. The fluids were collected three weeks after a single injection of viral oncolysate. Shown in the figure are the results of the effect of ascitic fluids obtained from three different patients and tested at various volume percent concentrations (mean plus/minus s.d.).

A variety of different studies were performed in order to further characterize the antitumor factor. In one series of experiments, the cytotoxicity of ascitic fluids from viral oncolysate-treated ovarian cancer patients was tested against various tumor cells. Tumor cells employed (see Table 1 below) consisted of a series of target cells including various ovarian carcinomas as well as a CML tumor cell line target (K-562). In the Table 1 series of experiments, ascitic fluids were collected from patients both before and after viral oncolysate treatment and these materials were tested in the in vitro assay. As can be seen, viral oncolysate treatment elicited the appearance of an antitumor factor in the ascitic fluid of patients following a single injection of oncolysate. The antitumor factor exhibited a fair degree of specificity for ovarian carcinoma tumor targets. A high degree of antitumor activity was exerted against both the OV-2774 and OV-F1 tumor targets. No activity was observed against the CML tumor target.

Further studies using the patient's ascitic fluid consisted of testing for the abrogation of cytotoxicity by specific adsorption of the ascitic fluid targets in vitro. These studies were performed by collecting ascitic fluid from oncolysate-treated patients approximately three weeks after a single injection. The ascitic fluid

(approximately 0.5 ml) was then incubated with one of various cell lines (5 x 10⁵ or 4 x 10⁶ cells) in order to determine whether the cells could, through absorption, remove the antitumor activity from the ascitic

preparation. Following incubation of the ascitic fluid with the indicated cell line (see Table 2 below) for 45 minutes at room temperature, the supernatant was collected and tested for cytotoxicity in the ⁵¹Cr-release assay.

The results, shown in Table 2 below, demonstrated that, in general, the OV-2774 cell line was best able to remove cytotoxicity observed against the OV-2774 cell line.

Further studies were performed in order to determine the adsorption of cytotoxic activity of ascitic fluids against the OV-2774 target, using OV-2774 cells for adsorption. Again, the ascitic fluids (0.5ml) were obtained approximately three weeks after a single viral oncolysate treatment. The ascitic fluids were then incubated with various numbers of OV-2774 cells for 45 minutes at 4°C. The supernatant was then collected and added to 5 x 10³ of OV-2774 cells targets and cytotoxicity was measured in the ⁵¹Cr-release assay. The results of this experiment, shown below in Table 3, demonstrated that the OV-2774 adsorption effectively removed the antitumor activity in a dose responsive manner. That is, the larger the number of cells used for adsorption, the greater the amount of antitumor factor removed from the ascitic fuuids tested.

Other experiments were performed which were directed at testing the effect of dialysis on the cytotoxicity of ascitic fluid against the OV-2774 target. In this study, ascitic fluids were obtained three weeks after first viral oncolysate injection, and the fluid was subjected to dialysis treatment. Two types of membranes were employed, an 8 KD dialysis membrane as well as a 12-14 KD dialysis membrane (Spectrum Medical Industries). Dialysis was conducted at 4° overnight using a Dulbecco's phosphate buffered saline dialysis buffer. Both the dialysate as well as the retentate was then tested for cytotoxicity against the OV-2774 target in the 3 hr ⁵¹Cr-release assay.

The results, shown in Table 4 below, demonstrated that dialysis using either an 8 KD or 12-14 KD dialysis membrane did not result in the removal of appreciable antitumor activity from the ascitic fluid. This therefore demonstrated that the factor could be successfully

dialysized without it being removed from the ascitic fluid.

Fractionation of the ascitic fluid was achieved on Ultrogel AcA44 (IBF Biotechnics) as follows:

A 0.5-1.0 ml sample of patient's ascites fluid obtained immediately prior to the second administration of viral oncolysate was loaded on an AcA44 column of 30 ml volume, equilibrated in Dulbecco's phosphate buffered saline. Fractions of 0.25-0.5 ml volume were collected. Fractions from the molecular sieving of the ascitic fluid on Ultrogel AcA44 were then tested in the 3 hr

5¹Cr-release assay. Furthermore, the effect of rabbit complement was determined in a side-by-side analysis.

From the results as shown in Table 5 below, it was found that the antitumor activity was complement-dependent, and was exhibited against the OV-2774 cell line but not against the K-562 CML cell line. By comparison against the Molecular sieving of molecular weight markers, it was observed that the antitumor activity migrated broadly from a size equivalent approximately to BSA (68 KD) up to just less than size of IgG (150 KD). However, the bulk of the activity was centered in a size range of approximately 90 to 120 kilodaltons.

Additionally, the effect of heat treatment and complement were tested independently on the cytotoxicity of ascitic fluid using the OV-2774 target. In particular, ascitic fluid samples were tested for activity with or without heat treatment (30 min at 56' C), and with or without the addition of rabbit complement (1:10).

Complement alone was not found to be cytotoxic against the OV-2774 cell line. The results, as shown in Table 6 below, demonstrated that the activity of the factor was sensitive to heat treatment at 56' for 30 minutes. In all cases, heat- sensitivity appeared to be related to complement

inactivation, since the samples to which complement was added tended to demonstrate high percent lysis of the target cell in the 3 hr ⁵¹Cr-release assay.

The foregoing description has been directed to particular embodiments of the invention in accordance with the requirements of the patent statutes for the purposes of illustration and explanation. It will be apparent, however, to those skilled in the art, that many modifications and changes in the procedure set forth will be possible without departing from the scope and spirit of the invention. For example, techniques are known for clarification and/or fractionation of the ascitic fluid in light of the present disclosure in order to obtain a fraction enriched for the soluble antitumor factor described herein. These, and other modifications of the invention, will be apparent to those skilled in the art with the aid Applicants disclosure. It is applicant's intention in the following claims to cover all such equivalent modification and variation which fall within the true spirit and scope of the invention.

REFERENCES

The following references are hereby incorporated by reference.

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9. Freedman, R.S., and Rutledege, F.N., Adjunctive immunotherapy with VMTCE in patients with high risk squamous carcinoma of the uterine cervix, Amer. J. Clin. Oncol., 6:155-156 (1983). 10. Green, A.A., Pratt, C., Webster, R.G., and Smith, K., Immunotherapy of osteosarcoma patients with virus-modified tumor cells. Ann. N.Y. Acad. Sci., 277:396-411 (1976). 11. Murray, D.R., Cassel, W.A., Torbin, A.H., Olkowski, Z.L., and Moore, M.E., Viral oncolysate in the management of malignant melanoma II. Clinical studies. Cancer,

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Claims

CLAIMS:

1. A process for preparing an antitumor preparation comprising treating an individual bearing an ascites- producing ovarian tumor with a viral oncolysate to

stimulate the appearance of a soluble, complement- dependent antitumor factor into ascites fluid of the treated individual, collecting the ascites fluid, and fractionating the ascites fluid to provide a preparation enriched for the antitumor factor.

2. The process of claims 1 wherein the antitumor factor is defined as capable of being retained by a 12-14 KD dialysis membrane.

3. The process of claim 2 wherein the ascites fluid or a fraction thereof, is dialyzed to provide a preparation enriched for the antitumor factor.

4. The process of claim 1 wherein the antitumor factor comprises a protein exhibiting a molecular weight of about 90 to about 120 kilodaltons when subjected to exclusion chromatography.

5. The process of claim 4 wherein the ascites fluid, or a fraction thereof, is subjected to exclusion chromatography to provide a fraction enriched for the antitumor factor.

6. The process of claim 1 further comprising the step of rendering the ascites fluid, or a fraction thereof, pharmacologically acceptable.

7. The process of claim 1 wherein the viral oncolysate is prepared by a process which includes infecting ovarian carcinoma cells with influenza virus to modify the

membranes of the carcinoma cells without virus-induced lysing of the cells.

8. The process of claim 7 wherein the influenza virus comprises Type A, strain PR8/34, influenza virus.

9. The process of claim 7 wherein the ovarian carcinoma cells comprise allogeneic cultured ovarian tumor cells.

10. The process of claim 9 wherein the allogeneic cultured ovarian tumor cells comprise MDAH 2774 or CaOV3 cells.

11. The process of claim 7 wherein the viral oncolysate is prepared by a process which includes virally infecting two separate ovarian carcinoma cell lines.

12. The process of claim 9 wherein the allogeneic cultured ovarian tumor cells comprise MDAH 2774 and CaOV3 cells.

13. The process of claim 1 wherein the individual bearing the ascites producing ovarian tumor is treated with the viral oncolysate by a process which includes intraperitoneal administration of an effective dose of the viral oncolysate.

14. An antitumor preparation made by a process as defined by any one of claims 1-13.

15. A method of treating ovarian tumor cells to induce cytotoxicity therein comprising subjecting said cells to an effective amount of an antitumor preparation made in accordance with claim 1.