WO1994001536A1 - Cancer immunotherapy with antibodies to cancer procoagulant - Google Patents

Abstract

This invention describes a method of tumor prevention using cancer procoagulant (CP) antigen and antibodies in immunization procedures. CP is also used in a method to specifically destroy malignant cells. In addition, a method is described herein for producing a stable hybridoma for the production of monoclonal antibodies to CP.

Description

CANCER IMMUNOTHERAPY WITH ANTIBODIES TO CANCER PROCOAGU ANT

TECHNICAL FIELD

This invention relates to antibodies specific to cancer procoagulant (CP) , a protein elaborated by malignant, but not normal, animal and human cells and to the use of these antibodies to CP in the immunotherapeutic treatment of cancer.

BACKGROUND ART

For years investigators have looked for substances from malignant cells that may modulate or directly affect the growth and/or the metastasis of malignant cells. An increased incidence of vascular thrombosis in patients with cancer, first described by Trousseau in the late 1800's, led to the identification of abnormal fibrin metabolism with malignant disease. Fibrin was deposited on the advancing margin of solid tumors and also on blood borne, potentially metastatic, malignant cells. Administration of anticoagulants and fibrinolysins decreased tumor growth and metastasis (Wood et al. (1955) in The Pathogenesis of Cancer, J.E. Gregory (ed.), Fremont Foundation, Pasadena, CA, pp. 140-151) . Also, thromboplastic and fibrinolytic activities associated with malignant tissue were increased (Svanberg (1975) Thromb. Res. .6:307; Unkeless et a!. (1974) J. Biol. Chem. 249:4295) . Thus, altered fibrin metabolism appeared to be associated with malignant tissue, but it was not clear what caused the altered activity of the coagulation cascade that resulted in abnormal fibrin deposition.

To explain the hypercoagulable state associated with neoplasia, the concept emerged that malignant tissue produced a substance capable of initiating coagulation. Various factors, including fatty acids, tissue factor and a mucus-derived component, were tested as possible candidates responsible for the increased coagulation activity of malignant cells. In 1975, Gordon et al. Thromb. Res. ,6:127 reported the isolation of a protein called cancer procoagulant (CP) , from rabbit V2 carcinoma cells that would initiate coagulation by a mechanism that was distinguishable from that of the intrinsic and extrinsic pathways. CP was shown to be different from tissue factor, which is normally released from damaged cells and participates via the extrinsic pathway in the activation of the coagulation system. It was shown that CP initiated coagulation in the absence of factor VII and was inhibited by diisopropylfluorophosphate (DFP) , two characteristics that distinguish it from tissue factor. CP has now been characterized as a cysteine proteinase having a molecular weight of 68,000 and capable of initiating coagulation by directly activating factor X in the coagulation cascade (Gordon, U.S. Patent number 4,461,833; Falanga et al. (1985) Biochem. 2£:5558-5567, and Biochim. Biophys. Acta 831:161-165. Most importantly, although CP could be extracted from human tumor cells, no CP activity or antigen could be detected in extracts from normal cells or from benign melanocytic lesions (Donati et al. (1986) Cancer Res. 46.:6471-6474) . The presence of CP was clearly associated with the malignant phenotype and its activity appears to be particularly high in metastatic cells. For years investigators have sought to identify diagnostic markers of cancerous cells. In 1970, Bubenek et al. (Int. J. Cancer 5_:310) demonstrated that serum from cancer patients contained antibodies that bound to tumor cell surface antigens. Subsequently, many reports were published on the presence of antigens on the surface of human melanoma and on other neoplastic cells. The ultimate goal in these investigations was to utilize the corresponding specific antibodies in the immunotherapy of cancer.

Of the many antigens characterizing tumor cells, some of the more notable are carcinoembryonic antigen (CEA) , alpha-fetoprotein (AFP) and acute lymphoblastic leukemia associated antigen (cALLA) . CEA was first detected in colon carcinomas and fetal gastrointestinal tract tissue (Gold and Friedman (1965) J. Exp. Med. 121:439) . It was later shown to be produced by a variety of mucin producing normal epithelial tissues. In addition, a variety of nonmalignant disorders, for example, peptic ulcers, pancreatitis, inflammatory bowel diseases, hepatitis, jaundice, biliary tract disease and cirrhosis, were found to be associated with elevated plasma levels of CEA. Although attempts were made to use CEA levels as predictive of various types of malignant diseases including tumors of the gastrointestinal tract, gastric cancer, pancreatic cancer, breast cancer, lung and respiratory tract tumors and gynecological tumors (Beatty et aJL. (1982) Cancer 8>:9), there appeared to be no reliable close correlation between the type and size of the tumor and the plasma CEA level.

AFP, normally produced by fetal liver, has also been found in hepatocellular carcinoma. The level of AFP is elevated to varying degrees in the serum of patients with liver tumors, teratocarcinomas, gastric carcinomas, colorectal carcinomas, hepatic carcinomas and biliary tract carcinomas (Ruddon (1982) Semin. Oncol. 9_:416). In spite of the small number of noncancer diseases that appear to produce elevated AFP levels, the overall value of AFP as a selective marker for tumors was found to be unreliable.

cALLA is under evaluation as a potential determinant of acute leukemic cells (Ritz et al. (1980) Nature 283:583) . However, the antigen has recently been found on normal cells, for example, on normal kidney epithelium as well as melanomas - a finding that could compromise its role as a possible tumor marker.

A number of other tumor associated antigens have been studied as possible tumor detectors (Hellstro et al. (1982) Springer Semin. Immunopathol. 5_:127). In most cases there is little evidence to indicate that any of these antigens have value as a potential tumor marker.

It is still believed that the finding of a selective marker diagnostic of tumorigenic cells would enable subsequent immunological use of specific antibodies not only to detect and/or to modify malignant cells, but also to allow treatment of neoplasia. At the present time there are three general immunopharmacological approaches that are used in treatment of neoplasia: (1) a nonspecific stimulation of the immune system, (2) active and passive immunization using antibodies specific for tumor cell components, and (3) conjugation of specific antibodies with pharmacological agents for selective transport of drugs via antibody carriers.

The use of nonspecific adaptive stimulation of the patient's immunologic system has focused on the use of Bacille Calmette-Guerin (BCG) and BCG cell wall preparations to nonspecifically stimulate the patient's immune system as an adjunct to other types of therapy, such as chemotherapy. Objective benefit from this treatment was not observed in tests on breast cancer (Giuliano et al. (1984) Proc. Am. Soc. Clin. Oncol. 3_:120), melanoma (Paterson et al. (1984) Can. Med. Assoc. J. 131:744) and colon cancer (Gray (1984) Proc. of 4th Intl. Conf. on the Adjuvant Therapy of Cancer, Tucson, Arizona, Toronto, p. 69) . However, in gastric cancer and lung cancer such passive adoptive treatment proved beneficial (Eriguchi et al. (1984) J. Surg. Oncol. 2_6:100). Active nonspecific adoptive therapy using intermediate cell products such as interleukin-II, interferon, thymosin and tuftsin have also been used to stimulate the patient's immune system with better results (Smalley et al. (1986) Springer Semin. Immunopathol. 9.:73- 83; Ettinghausen et al. (1986) Springer Semin. Immunopathol. .9:51-53).

The use of specific active and passive immunotherapy finds many applications in cancer treatment. There are many studies of immunotherapeutic applications of tumor associated antigens in neoplasia. For example, immunotherapy with an antigen from lung cancer improved the five year survival of patients from 46% to 78% (Hollinshead et al- (1981) Yale J. Biol. Med. 54.-367). Similar results were obtained with squamous cell carcinoma and adenocarcinoma of the lung (Takita et a_l. , Cancer Immunol. Immunother. , in press). In studies where patients with Duke's B, C and D adenocarcinoma of the colon were treated with specific active immunotherapy utilizing antigens from human colon carcinoma cells, 82% of the patients were alive after 21 months and 59% of the patients had no evidence of disease (Hollinshead et al. (1985) Cancer 5_6:60-69). In animal model studies, monoclonal antibodies against mouse B16 melanoma cells have been shown to inhibit metastases in the murine animal model by passive immunization (Vollmers et ajL. (1983) Proc. Natl. Acad. Sci. 80:3729-3733) .

Pharmacological agents carried by specific antibodies constitute the basis of an immunopharmacologic approach used to treat neoplasia. Agents such as cytotoxic drugs and radioisotopes are conjugated to antibodies that recognize malignant cell surface proteins (Baldwin et al. (1986) Springer Semin. Immunopathol. 9_:39-50). When the antibody is injected into the patient with a tumor, the antibody will seek out and recognize the tumor cell surface; in so doing, the antibody will bring the pharmacological agent to the cells and, thereby, effect the destruction of the malignant cell. A variety of the systems utilizing antibodies such as those to CEA and AFP as antibodies to which pharmacological agents are conjugated, have been developed and tested in animal and human cancers (Baldwin (1983) Pharmacy Int. 4_.:137; Vitetta et al. (1983) Science 219:644) .

DISCLOSURE OF THE INVENTION

This invention provides for a method of tumor prevention wherein live subjects, including humans and animals, are actively immunized with cancer procoagulant (CP) such that the host develops antibodies to CP. Since malignant cells require the presence of CP for viability, the presence of anti-CP antibodies prevents the formation of all types of tumors in an immunized host, that includes but is not limited to, any mammal, such as humans, primates, rodents (i.e., mice, rats, rabbits), bovines, ovines and canines, but the present invention will be described in connection with mice. Active immunization for tumor prevention is effective with CP in all forms, for example, CP at different levels of purification and CP that is conjugated to different ligands. Usually, CP is extracted from animal, preferably from rabbit, mouse, rat or human, tumors or human amnion- chorion tissue and subsequently purified to desired levels, preferably to greater than 2,500 fold purity, by established methodology. CP can be used as an immunotherapeutic antigen at levels of purity which yield antibodies that have a cross reactivity to nontumor associated antigens of about 15% or less. Preferably the level of purity is such that undesired side immune reactions are not produced, as may be readily ascertained by those skilled in the art. Also, chemically modified CP structures, including addition of ligands to the CP structure and the substitution, addition or deletion of a ino acids within the CP sequences, are considered to be equivalent to unmodified CP whenever such modifications do not prevent detection of CP enzymatic and immunologic activity in the final product and do not prevent the final product from being therapeutically effective. CP is deemed to be therapeutically effective in active immunization when it can be shown to produce antibodies which retard or prevent tumor formation, decrease tumor metastasis, reduce the size of a tumor or kill malignant cells.

The present invention also provides a method of tumor immunotherapy wherein animals and humans having a diagnosed tumor are injected parenterally with an immunotherapeutically effective amount of an antibody which is specific to CP, henceforth referred to as "passive immunization." An immunotherapeutically effective amount of anti-CP antibody is an amount that can be shown to retard or prevent tumor formation, decrease tumor metastasis, reduce the size of a tumor, or kill tumor cells, e.g., by causing lysis. Both polyclonal and monoclonal antibodies having specific immunoreactivity to CP are effective in tumor therapy. It is preferred that if other antibodies are present, the mixture should have an immunoreactivity of at least about 70%.

For tumor immunotherapy, anti-CP antibodies may be delivered by intravenous infusion or other means known to the art. Antibodies to CP may also be injected directly into tumors. In addition, tumor therapy using anti-CP antibodies can be used adjunctly to other types of cancer therapy, for example, in conjunction with radiation therapy or chemotherapy. Also, it can be used in patients having a diagnosed tumor as a preventive measure against tumor metastasis in conjunction with surgery for tumor or nontumor conditions when the probability for increased incidence of tumor metastasis is enhanced.

Additionally, this invention provides a method for killing malignant cells. Anti-CP antibodies cause the death of malignant cells within body fluids or tissues or on established tumors. Thus, in cancer patients the cytotoxic property of anti-CP antibodies destroys malignant cells .in situ and also those which may be in the migratory or invasive phase of metastasis. Therefore, immunotherapy with anti-CP antibodies provides a method for killing malignant cells and, thus, for preventing the establishment of secondary tumors.

Both monoclonal and polyclonal antibodies are useful in the methods of this invention. The preparation of monoclonal antibodies to cancer procoagulant presents certain unique problems in that the hybridoma produces the CP antigen as a result of its derivation from a malignant cell. Applicants have developed a novel method of preparing stable antibody-producing hybridoma cell lines. This method comprises maintaining the hybridomas on a conditioned standard medium, e.g., RPMI, containing sufficient CP antigen to prevent destruction of productivity and harm to the hybridoma cell caused by antibody-antigen binding by providing CP antigen outside the hybridoma in addition to that produced by the hybridoma. Preferably the medium contains about one part filtered medium previously used to grow CP-producing cells and three parts unused medium.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 displays the effect of anti-cancer procoagulant IgM antibody on the viability (cell number) of B16-F10 murine melanoma cells. Each point is the average of six cell counts on three different flasks at each experimental point (the dots and the dashed regression line) ; data was edited by eliminating one data point each at 6 and 602 ug of IgM (triangles and the solid regression line) .

Figure 2 displays the total cell count and cell viability as a function of concentration of anti-cancer procoagulant IgM antibodies incubated with B16-F10 murine melanoma cells.

Figure 3 displays the effect of complement on the viability of cultured small cell lung carcinoma cells (SCLC) in the presence and absence of anti-cancer procoagulant IgM (anti-CP IgM) . Cell viability was determined using the MTT assay.

Figure 4 displays the effect of different amounts of anti-cancer procoagulant IgM (anti-CP IgM) and nonsense IgM, with and without complement, on SCLC cell viability as assayed using the MTT method. Figure 5 displays the effect of different amounts of anti-cancer procoagulant IgM (anti-CP IgM) and nonsense IgM, with and without complement, on the % viability of SCLC cells in vitro using the MTT method.

Figure 6 displays the binding of different amounts of anti-cancer procoagulant IgM (anti-CP IgM) to SCLC cells in vitro. The saline control contained anti-CP IgM in the absence of SCLC cells.

Figure 7 displays the mean ± standard error of the mean of the binding of anti-cancer procoagulant IgM (anti- CP IgM) and nonsense IgM on the SCLC cells in vitro. The saline control contained anti-CP IgM in the absence of SCLC cells. The individual data of these binding studies are shown in Figure 6.

Figure 8 displays the effect of different amounts of anti-cancer procoagulant IgG on the viability of normal human skin fibroblasts ( ) and on human SCLC (H345) cells ( ) as determined by the (³H)-thymidine incorporation method (³HT) . Results are expressed as a percent of the untreated control (without IgG) .

Figure 9 displays the relationship between Absorbance (optical density) at 540 nm and cell number in the 4hr or 24hr MTT assay using human SCLC (H345) cells, mouse melanoma (B16-F10) cells and normal human skin fibroblast cells. The correlation coefficient, R, for each "best fit" standard curve was calculated using linear regression analysis.

Figure 10 displays the effect of different amounts of anti-cancer procoagulant IgG on the viability of normal human skin fibroblasts ( ) and on human SCLC (H345) cells ( ) as determined by the MTT assay after cell incubation with IgG for 24hr. Absorbance (optical density) was measured at 540 nm against a reference wavelength of 690 nm.

Figure 11 compares the effect of different amounts of anti-cancer procoagulant IgG on the viability of human

SCLC (H345) cells as determined by the (³H)-thymidine incorporation assay ( ) and the MTT assay ( ) .

Results are expressed as a percent of the untreated control (without IgG) .

BEST MODES FOR CARRYING OUT THE INVENTION

Cancer procoagulant (CP) is a proteolytic enzyme that directly activates factor X, bypassing both the extrinsic and intrinsic pathways of the coagulation cascade. Thus far, CP has been described in neoplastic and amnion- chorion cells of fetal origin and is believed to be an antigenic marker for malignancy. CP, isolated from rabbit V2 carcinoma and purified approximately 2,664-fold (Gordon, U.S. Patent No. 4,461,833), is a 68 kDa protein having an isoelectric point of 4.8. CP is a cysteine proteinase; it is sensitive to cysteine proteinase inhibitors, such as iodoacetamide and mercury, and activators, such as dithiothreitol and KCN; and it binds to p-chloromercuribenzoate agarose (Donati et aJL. (1986) supra) . The presence of CP can be detected enzymatically using a one-stage plasma recalcification assay or immunologically using antibodies developed to pure CP. CP that is chemically modified, for example by addition of chemical or biological ligands or by substitution, addition or deletion of amino acids, is considered to be equivalent to unmodified CP whenever such modifications do not prevent detection of CP enzymatic and immunologic activity in the final product and do not prevent the final product from being therapeutically effective. Anti-CP antibodies refer to either polyclonal or monoclonal antibodies developed to purified CP. Methods used to prepare and purify polyclonal and monoclonal antibodies are those commonly used by those skilled in the art. Anti-CP antibodies used for tumor prevention and tumor immunotherapy are preferably monospecific having an immunoreactivity only to CP. As is know to the art, cross reactivity of monospecific antibodies is minimal. Also, as will be appreciated by those skilled in the art, cross- reactivity of anti-CP antibodies to antigens critical to the functioning of normal cells should be minimized. Preferably cross-reactivity to nontumor-associated antigens is less than or equal to about 15%.

The term therapeutically effective as used herein is defined by the situation wherein tumor formation is retarded or prevented, tumor metastasis is decreased, tumor size is reduced or malignant cells are destroyed.

As demonstrated herein, CP antibodies cause death of cancer cells. The desired degree of therapeutic effectiveness to be achieved by administration of CP antigen or antibody depends on numerous factors known to the art, such as tolerance of the patient to the dosage, presence or absence of known metastatic cells, tumor size and location, etc. Dosages of antigens and antibodies having the desired degree of effectiveness are readily ascertained by art-known methods.

Antigenic composition refers to a composition comprising cancer procoagulant in an amount that is therapeutically effective or that is effective for the destruction of malignant cells and a pharmaceutically acceptable carrier. Immunogenic composition refers to a composition comprising an antibody specific to cancer procoagulant in an effective amount as defined above.. A typical pharmaceutically acceptable carrier according to this invention contains in 1.0 ml of composition 10 mg human serum albumin, 0.9% NaCl and 0.01M phosphate buffer at pH7.5. Other conventional pharmaceutically acceptable carriers may be used where indicated, such as for oral administration, topical application, parenteral injection, subdural injection, intravenous administration, organ injection, etc.

Malignant disease is associated with disorders of the hemostatic system, particularly with high incidence of thromboembolis . The production of a procoagulant substance by cancer cells is believed to provide a possible mechanism for initiating blood coagulation in malignancy. CP is a proteolytic procoagulant whose presence is associated with the malignant phenotype and whose activity is particularly high in metastatic cells. To date, CP has not been detected in normal tissue or in benign tumors.

The presence of CP activity associated with the malignant state suggests that neoplastic cells produce a protein that is not synthesized by normal cells. This unique CP protein can be isolated from tumors of human or animal origin for use as antigen for the production of antibodies specific to CP. Both polyclonal or monoclonal antibodies can be produced using established methodology known to those of ordinary skill in the art. The resultant purified antibodies are highly monospecific to CP. When such anti-CP antibodies are mixed with either normal or malignant cells, they react specifically with a component of malignant cells, but not with normal cells, as visualized by immunohistochemical techniques routinely used by those of ordinary skill in the art. Thus, the antigenic nature of CP allows production of anti-CP antibodies which are able to target malignant cells and to distinguish between normal and malignant cells. In accordance with one aspect of this invention, a property of antibodies to CP has been discovered which, when anti-CP antibodies are exposed to malignant cells, allows anti-CP antibodies to kill malignant cells. As described in the specific embodiments of Example 8 and 11, malignant cells that are exposed to anti-CP antibodies exhibit decreased viability, most probably, through specific binding and neutralization of CP which is necessary for viability of malignant cells. Example 11 and Figure 7 describe specific binding of anti-CP antibodies to tumor cells. It is generally believed that an agent capable of destroying malignant cells is potentially capable of preventing the establishment of secondary tumors and, therefore, of preventing metastasis and, therefore, of preventing cancer.

Immunization, active or passive, provides a method universally used to prevent development of specific diseases. An antigen, when injected into a host, evokes a response to produce antibodies specific to the antigen so that the antigen is neutralized. A background titer of such antibodies readies the host for any subsequent appearance of the same antigen.

In a specific embodiment of this invention immunization with CP antigen provides a method for cancer prevention in human or animal hosts. As exemplified in Example 5, mice, preim unized with purified CP isolated from rabbit tumors, were injected with a variant of mouse melanoma cells. A significant reduction in metastatic capacity of the melanoma cells in CP-immunized animals was observed. Control mice contained from 25 to 30 lung colonies per mouse, whereas CP-immunized mice contained from zero to three lung colonies per mouse. This invention also affords a similar protection against tumor formation in humans actively immunized with CP. In further studies concerning metastasis in which a primary tumor is allowed to metastasize spontaneously to a secondary site (see Example 6) , preimmunization with CP resulted in a reduction in metastatic capacity and apparent enhanced necrosis of primary tumor tissue. Thus, the presence of antibodies specific to CP in a preimmunized host reduces the size of existing tumors and prevents tumor metastasis.

Another aspect of this invention is to provide a method of tumor immunotherapy for patients having a diagnosed tumor. Patients with tumor desire regression of known tumors and prevention of new tumor formation. For such purposes, immunotherapy, as a primary or adjunct therapy, is employed preferentially. In this invention, antibodies specific to CP are used to target and destroy malignant cells, in situ or invasive, in body tissues and fluids.

A further aspect of this invention is to provide a method for neutralizing the effect of the presence of cancer procoagulant antigen on malignant cells. Cells that are malignant exhibit cancer procoagulant activity and require the presence of cancer procoagulant for cell viability. This invention indicates that polyclonal and monoclonal antibodies specific to cancer procoagulant, for example, IgM, IgG, etc. , bind specifically to cancer procoagulant antigen on the cell surface of malignant cells, for example, melanomas, carcinomas, etc. The neutralization of cancer procoagulant on malignant cells results in the destruction (cytoxicity) of these cells.

In a specific embodiment, this invention provides a method for parenterally injecting a patient having a diagnosed tumor with anti-CP immunoglobulins in order to reduce tumor metastasis. Such tumor treatment with anti- CP antibodies can constitute a primary therapy or an adjunct therapy in addition to, for example, chemical or radiation therapy. Immunotherapy with anti-CP antibodies can also be used as a preventive measure against tumor metastasis in cancer patients, and even in patients not previously diagnosed as having tumors, in conjunction with, e.g., before or during surgery when the incidence of tumor perturbation leading to malignant cell shedding and metastasis can occur with increased probability.

In a more preferred embodiment of this invention, anti-CP immunoglobulins can be delivered to a patient through intravenous injection or injection directly into the tumor site, prelocalized using established medical diagnostic procedures. For example, radiolabelled antibodies to specific tumor antigens are given to patients and the tumors are subsequently detected and localized by scintillation scanning. If desired, similar technology can be used to monitor the path and destination of the antibodies administered during the immunotherapy.

Alternative methods exist for preparation of injectable compositions of antibodies and for administration of antibodies to patients. Where alternative methods exist, the procedures used in the specific embodiments of this invention may be substituted by those skilled in the art.

Preparation of monoclonal antibodies to cancer procoagulant presents unique problems. Cancer procoagulant is believed to be an oncofetal antigen, and the hybrid cells used to produce monoclonal antibodies are developed from a malignant cell line (the myeloma variant) . The hybridoma cells derived from mouse myeloma cells thus produce CP antigen. The CP antibodies produced by the hybridomas, in binding to the antigen, exert negative selection pressure on the hybridomas such that good antibody producers become non-producers in a relatively short period of time. To solve this problem, applicants use a special conditioning medium containing CP antigen as described in the Examples.

It is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the disclosure.

EXAMPLES

Example 1: Preparation and Purification of CP Antigen

Purified CP antigen was obtained from rabbit V2 carcinoma (Gordon, U.S. Patent No. 4,461,833), human amnion-chorion tissue (Falanga et al. (1985) Biochim. Biophys. Acta 831:161-165) , human tumor cells (Donati et al. (1986) Cancer Res. 4j5:6471-6474) or other cellular sources. Briefly, surgically removed tissue was extracted in three changes of vernal buffer, the extracts were pooled and concentrated 10-fold and used as a source of CP antigen.

In the purification of CP extracts, the five step chromatographic procedure described in patent no.

4,461,833 was followed. The five chromatographic steps involved benza idine-Sepharose affinity chromatography

(step one) , 1.5 M agarose gel filtration column chromatography (step two) , a second benzamidine-Sepharose affinity chromatography column (step three) , a p- chloromercurial benzoate-Sepharose column chromatography

(step four) , and a phenyl-Sepharose hydrophobic affinity chromatography column step (step five) . The purified protein exhibited all of the proper enzymatic and chemical characteristics of cancer procoagulant. For example, CP is a proteolytic enzyme that directly activates factor X, bypassing both the extrinsic and intrinsic pathways of the coagulation cascade. It has been chemically characterized as a 68 kDa protein without measurable carbohydrate, and with an isoelectric point of 4.8. Enzymatically, CP behaves like a cysteine proteinase; it is sensitive to cysteine proteinase inhibitors (i.e., iodoacetamide and mercury) and activators (i.e., dithiothreitol, KCN) and it binds to p-chloromercuri-benzoate agarose) .

Example 2: Preparation of Polyclonal Antibodies to CP

One hundred micrograms of purified CP were emulsified in an equal volume of complete Freund's adjuvant and injected subcutaneously in multiple sites along a host animal's, for example, a goat's mid-back or other sites near lymph nodes. Booster immunizations were made at three week intervals by suspending 30-50 μg of purified CP in equal volume of incomplete Freund's adjuvant and injecting the goat in the same way. Blood samples were obtained by jugular vein venipuncture at monthly intervals and tested for antibody by crossed immunodiffusion. After four months, an antibody titer of 1:16 was reached and sustained for about 12 months. The goat antibody (a polyclonal IgG immunoglobulin) was partially purified from goat serum by ammonium sulfate precipitation and DEAE- cellulose ion exchange chromatography by standard techniques.

The partially purified antibody was found to contain antibodies to rabbit serum proteins, probably minor contaminants from the purified CP preparations of rabbit V2 carcinoma. To remove these contaminating antibodies, rabbit serum was coupled to cyanogen bromide activated Sepharose to form a normal rabbit serum protein affinity column, and the partially purified goat antibody preparation was passed over the normal rabbit serum column to remove the contaminating antibodies. A human serum protein affinity column was also prepared and used to remove further non-CP antibodies from the partially purified goat antibody preparation. In both cases, the column was washed to remove unbound antibodies, eluted with NaSCN (3M Guanidine) , reequilibrated and used again.

Example 3: Further Purification of Cancer Procoagulant

The goat antibody preparation was used to prepare an immunoaffinity column by coupling with cyanogen bromide activated sepharose. Partially purified cancer procoagulant of Example 2 was applied to the column; it was then placed on a rotating wheel and allowed to rotate overnight so that the sample and resin were thoroughly mixed. The next morning the column was allowed to settle and the column was washed with 20 mM veronal buffer until all unbound protein was washed off the column (the absorption at 280 nm is the same as that of the buffer) ; this required from.250-350 ml of buffer. The column was washed with 100 ml of 5% deoxycholate dissolved in 20 mM veronal buffer [deoxycholate should be recrystallized from acetone:water (3:1)] followed by 3-4 column volumes of 20 mM veronal buffer.. This removed all adsorbed proteins from the column. The column was eluted with 100 ml of 3 M NaSCN followed by 50-100 ml of veronal buffer. The eluate was dialyzed immediately against 20 mM Bis-Tris propane buffer (pH 6.5) at 5° overnight. The dialyzed eluate was concentrated on an Amicon PM10 ultrafiltration membrane and assayed for activity as described below. Every third or fourth run the column was cleaned with 5 M NaSCN and reequilibrated with veronal buffer. This immunoaffinity procedure removed the majority of contaminating proteins from the cancer procoagulant sample. An additional purification step was used. A p- chloromercurial benzoate (PCMB) organomercurial Agarose column (Affi-gel 501) was purchased from Bio-Rad. The column was prepared according to the Bio-Rad technical information. The column was equilibrated in 20 mM Bis- Tris propane buffer (pH 6.5). The sample was applied to the column and washed slowly onto the column with 20 mM Bis-Tris propane buffer. The column was allowed to stand for 1 hr at 4°C and washed slowly overnight with 20 mM Bis-Tris propane buffer. When the absorption at 280 nm was the same as that of the Bis-Tris propane buffer, the column was washed with about 50 ml of 1 M urea and 1% Tween in water and followed by enough 20 mM Bis-Tris propane buffer to completely remove all residual Tween- urea from the column. The column was eluted with HgCl₂ or glutathione, and each elution was dialyzed immediately in 20 mM Bis-Tris propane buffer at 4°C overnight with several changes of buffer. The samples were concentrated on a PM-10 ultrafiltration membrane and checked for activity as described above. The purified samples from the immunoaffinity column and the PCMB affinity column were evaluated by SDS-polyacrylamide gel electrophoresis and the protein content of each sample was determined with the Lowry protein determination. The activity in the samples was preserved by making them 1 mM with HgCl₂ which will inhibit and preserve the activity for later use.

Example 4: Preparation of Monoclonal Antibodies

Standard techniques were used to prepare the hybridomas. Using the second purification technique described above, mice were immunized with purified CP to raise B cell antibodies as described by Yelton et al. (1980) in Monoclonal Antibodies. Kennett et al. (eds.) Plenum Press, New York, pp. 3-17, although other means of raising hybridoma antibodies may also by employed. Briefly, 40 μg of purified antigen were suspended in an equal volume of complete Freund's adjuvant and injected subcutaneously into Balb/C mice. This was followed by 2 injections of 35 and 10 μg amounts of antigen suspended in incomplete Freund's adjuvant and injected subcutaneously at monthly intervals. Three weeks after the last subcutaneous immunization, 3 intraperitoneal immunizations of 10 μg, 70 μg, and 70 μg of antigen in saline were administered intraperitoneally at 3-day intervals, 2 weeks later a blood sample was obtained by retroorbital bleeding and tested for serum antibody by crossed immunodiffusion. Once the presence of an antibody was confirmed, a last intraperitoneal immunization (40 μg) was administered, and 3 days later the animals were sacrificed. The spleen lymphocytes were removed and hybridized with P3/X 63AG8.653 variant of the mouse myeloma cell line with 50% polyethylene glycol. Hybrid cells were plated in a 96 well microtiter plate with 2 X 10° normal murine spleen cells as a feeder layer, and unhybridized myeloma cells were eliminated by growing the cultures in hypoxanthine- aminopterin-thymidine (HAT) medium for 4 weeks. The standard medium used to grow the P3/X 63 AG8.653 cells was RPMI 1640 medium (Gibro, Grand Island, New York) .

An ELISA was used to screen the medium from the microtiter wells for antibody-producing cells. In this assay purified antigen was adsorbed to the surface of the microtiter wells, the wells were blocked with 2% BSA, and media was incubated in the wells for 1 hr at 37°C, and an alkaline phosphatase labelled rabbit antimouse immunoglobulin preparation was added to identify the antibodies that had attached to the antigen.

Positive wells were expanded in the presence of 2 X 10⁶ normal spleen cells using a conditioned RPMI medium to prevent negative selection pressure on the hybridomas as a result of their production of CP antibody. The medium was prepared by adding one part of filtered RPMI medium that was used to grow the mouse melanoma cells and which contains excess CP synthesized by these cells to three parts unmodified RPMI medium. Expanded wells were retested and positive wells were cloned 2 more times at low density to obtain clean and stable populations of hybrid cells for use in the experiments. Three clones were identified, each clone produced immunoglobulin antibodies, e.g., IgM and IgG, to cancer procoagulant antigen. In the specific embodiment, IgM antibodies to CP antigen were used. Antibody-producing hybridomas were maintained on conditioned medium and became stable after a period of about a year.

The IgM samples obtained from the hybrid cells

(either as medium from tissue cultured cells or ascites fluid) contained procoagulant activity. In a representative experiment, Balb/C mice were injected with 0.5 ml of pristane to desensitize their immune system. Three weeks later, the mice received 2 X 10⁶ hybridoma cells intraperitoneally, and ascites fluid was drained 3 or 4 times at 2-day intervals from the mice by intraperitoneal needle stick until the mice died. Ascites fluid was assayed for procoagulant activity. The procoagulant activity was tentatively characterized as that of cancer procoagulant.

A further unique problem presented by cancer procoagulant monoclonal antibodies is that because of production of CP by the hybridomas, the immunoglobulin antibody is likely to be bound to the antigen in the ascites fluid, rendering it i munologically unreactive in the assay system. Therefore, it was necessary to separate the antigen from the antibody so that the antibody was rendered immunologically reactive to antigen in other samples. The ascites fluid was made 3 M with urea and applied to a 1 X 90 cm 1.5 M agarose gel filtration column that was preequilibrated in 3 M urea. The sample was eluted from the column in 3 M urea and the first peak (void volume) was assayed for IgM and procoagulant activity; it was free of procoagulant activity and contained all of the IgM. A second peak from the column contained procoagulant activity and no IgM. Fractions from the first peak were pooled, dialyzed against at least 3 changes of 5 mM Tris-HCl buffer (pH 7.5) , the sample was concentrated over an Amicon XM50 ultrafiltration membrane, and refrigerated overnight in a centrifuge tube. The next morning, a precipitate embodying IgM had formed under conditions of low ionic strength; it was removed by centrifugation and resuspended in PBS. The resuspended precipitate sample was found to contain the immunoreactive IgM fraction, and a small amount had remained behind in the supernatant. This purified IgM was assayed against purified antigen, using 2% normal human serum as a control blank and gave a sample to blank ratio of from 10 to 20.

The unpurified ascites gave a sample to background ratio of from 2 to 4, the supernatant gave a sample to background ration of 6 to 10. This purified IgM was then used in the immunoassay. There are other methods for dissociating antigen-antibody complexes so they can be separated. Such methods may include higher concentrations of urea, low pH (pH 2-3.5), 5M guanidine-HCl, high pH (pH 10.5-12) and combinations of dissociating agents and pH adjustment. However, a problem sometimes encountered by applicants was the irreversible denaturation of IgM under these dissociating conditions. A preferred method employed by applicants involved use of 50% ethylene glycol in PBS, used in conjunction with gel filtration on the HPLC. All such methods for separating antibody-cancer procoagulant antigen complexes are included within the purview of this application. Example 5: Assays for Detection of CP

Enzyme assays as well as immunoassays were used to detect CP. CP activity of isolated cells and of tissue extracts was measured visually by a one-stage plasma recalcification assay using a test system containing 0.1 ml of test material or buffer, 0.1 ml of human platelet poor plasma, and 0.1 ml of 0.025 M CaCl₂. The standards for the coagulation assay were a 1:10 dilution of rabbit brain thromboplastin (tissue factor) , giving a clotting time of 39.7 s, or RW (Russell's viper venom), (0.5 μg/ l; Wellcome Research Laboratories, Beckenham, England), giving a clotting time of 39.1 s; the procoagulant activity of these concentrations was arbitrarily considered to represent 100 units. Procoagulant activity in the tissue extracts or in the cell preparations were expressed in seconds or as units of either RW or tissue factor per mg protein. There was a linear relationship from 0.2 to 100 units of either thromboplastin or RW and clotting time. The slopes of the curves obtained with thromboplastin and RW were similar and the curves obtained with RW in normal and factor VII deficient plasmas coincided.

CP activity was first identified using human plasmas selectively deficient in factor II, VII, IX, or X (Merz- Dade, Duding, Switzerland) . For further characterization, known inhibitors of cell procoagulants were used, namely the cysteine proteinase inhibitors HgCl₂ and iodoacetamide (Sigma Chemical Co., St. Louis, MO) (Barret (ed.) (1977) Proteinases in Mammalian Cells and Tissues. Amsterdam: Elsevier/North-Holland, Biomedical Press) and the tissue factor inhibitor. Con A, (Sigma) (Pitlick (1975) J. Clin. Invest. 5_5:175-179) . Samples of the tissue extracts or of the cells were incubated with HgCl₂ (0.1 mM, final concentration) or iodoacetamide (2 mM) at 37°C for 30 min 26 containing 0.1 mg MgCl₂.6H₂0/ml and 0.2% NaN₃ and incubated at 37°C for from 45 to 90 min (until color intensity is adequate to read) , and then the plate was read on a Dynatech microtiter plate reader which measured absorbance at 405 nm.

In a second ELISA procedure (a sandwich or double antibody ELISA) , the I munolon I microtiter plate was coated with 1 to 40,000 dilution of partially purified goat IgG and incubated for 2 hrs at 25°C, the wells were washed once with phosphate buffered saline (PBS) and open sites in the wells were blocked with 2% human serum in phosphate buffer. The wells were washed 3 times with PTB. 50 μl of the antigen sample (usually diluted 1:2 with PTB + 0.15 M NaCl) was added to each well and incubated at 25°C for 2 hrs, the wells were washed again with PTB, 50 μl of 1 to 200 dilution of IgM in PBT was added and incubated at 25°C for 2 hrs, and the amount of IgM was measured as described above.

Both of these assays were used to measure purified antigen, purified antigen added to normal human serum, serum from cancer patients, extracts of tumors and other biological samples. The first assay worked better for more purified samples, the second assay worked better for samples like serum and other samples that contain a large number of other proteins that competed with the antigen for binding to the surface of the well because the antigen was absorbed out of the biological sample onto the goat antibody, and the monoclonal antibody was used to quantitate the amount of antigen. Both ELISA procedures were able to detect 10 ng of purified antigen.

The ELISA method is one of a variety of immunoassay techniques that could be employed to assay for cancer procoagulant antigen in biological samples. Other methods 25

(Gordon et a_l. (1981) J. Clin. Invest. .62:1665-1671) and the procoagulant activity was then measured. Control enzymes for procoagulant activity were the cysteine proteinase papain and the serine proteinase RW. Reactivation of PCA after HgCl₂ treatment was obtained by incubation of the cells with 5 mM DL-dithiothreitol (Sigma) , 2 mM EDTA (Merck, Darmstadt, West Germany) , and 10 mM KCN (Sigma) at 5°C for 30 min, followed by overnight dialysis against 20 mM veronal buffer, pH 8.0 (Falanga et al. (1985) Biochem. 24.:5558-5567) . To assess the effect of Con A, portions of tumor extracts were incubated with the inhibitor (100 μg/ml, final concentration) for 1 h at 37°C, and then PCA was determined. To verify the specificity of the effect of Con A, the sample to be tested in the presence of Con A was preincubated for 15 min at 37°C with α-methyl-D-glucoside (Sigma) (Zacharski et al. (1974) Blood 44:783-787.

Two separate immunoassays for the quantification of cancer procoagulant were developed. The first immunoassay system was a direct ELISA in which antigen was adsorbed to the surface of the wells in a 96 well Immulon I microtiter plate at room temperature for 2 hrs, the well was rinsed with phosphate buffered Tween-20, the open sites on the wells were blocked with 2% normal human serum in phosphate buffer at 37°C for 1 hr, and the wells were washed 3 times with 20 mM phosphate buffer (pH 7.5) containing 0.05% Tween-20. Purified immunoglobulin antibody, e.g., IgM antibody, was diluted 1:200 in phosphate buffer and 50 μl was added to each well and incubated at 37°C for l hr. The wells were washed 3 times with phosphate buffer containing 0.05% Tween (PTB). One to 1000 dilution of alkaline phosphatase labeled rabbit antimouse IgM antibody was added to each well, incubated for 1 hr at 37°C, the wells were washed with PTB and 100 μl of p-nitrophenyl phosphate (5 mg/ml) in 10% diethanoloamine buffer (pH 9.8) include radioimmunoassay, immunoinhibition assay, immunofluorescent assay and immunoinhibition assay, immunofluorescent assay and immunoprecipitation assay; all such assays that include the use of an antibody to quantitate the cancer procoagulant antigen should be construed to be included under the description of the assay.

Example 6: Active Immunization with CP to Reduce Metastatic Capacity of Malignant Cells

Thirty Balb/C mice were arbitrarily divided into three groups of ten mice each. Group 1 was a non- immunized control group. Group 2 was immunized with bovine serum albumin (BSA) . Group 3 was immunized with purified cancer procoagulant protein. The immunization protocol followed standard, classic methods in which pure cancer procoagulant (10 micrograms per mouse) was suspended in an equal volume of complete Freund's adjuvant and injected in four to six sites along the mid-back region of each mouse. At 21 day intervals, mice were boosted by injecting from five to ten micrograms of CP suspended in incomplete Freund's adjuvant in the same sites. In the mice immunized with BSA, commercial, grade 4 bovine serum albumin (Sigma, St. Louis, Missouri) was used as the immunogen instead of CP. After 17 weeks, blood was obtained from each animal by retro-orbital bleed and the serum was checked for presence of antibody to the appropriate antigen by crossed immunodiffusion. The presence of the appropriate antibody in Groups 2 and 3 was necessary to proceed with the next step of the experiment.

B16-F10 variants of the mouse melanoma cells were grown in tissue culture as described by Gordon et al. (1982) Thrombos. Res. 16.:379-387. Briefly, cells were grown in early passage, harvested and cell suspensions were prepared for injection into the mice. Fifty-thousand cells per 0.1 ml of serum-free tissue culture medium were prepared and injected into the tail vein of each mouse according to procedures described by Gilbert et al. (1983) Cancer Res. 4J3:536-540. Twenty-one days after inoculation of the B16 melanoma cells, the mice were sacrificed and the number of lung colonies were counted. The control mice contained from 25 to 30 lung colonies per mouse, the BSA-immunized mice contained the same number of colonies per lung. The CP-immunized mice contained from zero to three lung colonies per mouse; this is a significant reduction in metastatic capacity of B16 cells in CP- immunized animals.

Example 7: Active Immunization with CP to reduce

Metastatic Capacity of Primary Tumors

The hematologic phase metastatic murine model is a widely accepted animal model for the study of metastatic capacity of malignant cells. However, since malignant cells are introduced directly into the blood stream rather than shed spontaneously from a primary tumor, it is thought to be a somewhat artificial model. More appropriate is the spontaneous metastases model in which a primary tumor is allowed to metastasize spontaneously to a secondary site.

In this example, the same immunization protocol was followed as that described in Example 6; a controlled group of non-immunized mice, a BSA-immunized control group of mice and a group of CP experimental mice were prepared for the study and their antibody titer was checked as described in Example 6. B16-F10 variants of B16 melanoma were grown in tissue culture and a cell suspension was prepared in serum-free medium. A suspension of 50,000 cells was injected into the footpad of the mice and tumors were allowed to grow for 21 days. At three weeks, the foot was amputated such that the primary tumor was removed and the mice were maintained for an additional four weeks, at which time they were sacrificed and lung colonies were counted. Two of the five mice from the BSA-immunized group had extensive tumor colonies and one other had limited metastatic tumors. In the eight lungs from the untreated controls, seven had metastatic tumor colonies, and one was tumor free. Three of the CP-immunized mice were tumor free, one had limited and one had extensive metastatic tumors. Thus, 77% of the control animals had metastatic tumors and 40% of the CP-immunized animals had tumors. This reduction in metastatic capacity in this animal model confirms the data on metastatic capacity of malignant cells documented in Example 1.

In addition to the decrease in metastatic tumors, it appeared that there was more necrotic primary tumor tissue in the CP-immunized mice than in the unimmunized or BSA- im unized controls. However, it was impossible to evaluate the primary tumors histologically because the amount of melanin (black pigment) in the tumors was excessive, preventing visual or histologic evaluation.

Example 8: Cvtotoxicity of anti-CP Antibodies

Monoclonal antibodies to cancer procoagulant were prepared as described in Example 3. Briefly, Charles Rivers mice were immunized with pure cancer procoagulant as described above. A final injection of antigen was administered in saline intraperitoneally and one week later the mice were sacrificed, their spleens were removed and spleen lymphocytes were hybridized with the P3X variant of the mouse myeloma cell in 15% polyethylene glycol. Hybrid cells were subjected to the standard HAT suicide procedure to eliminate unwanted cell populations. The surviving cells were screened for anti-CP antibody production, cloned and rescreened two more times before cell populations were grown into flasks for storage and antibody production. Hybrid cells were injected into the peritoneal cavity of pristane treated Charles Rivers mice and IgM was purified from the ascites drainage from these mice. IgM was mixed with 50,000 B16-F10 mouse melanoma cells and incubated for 30 minutes at 25°C. The cells were examined by phase contrast microscopy and appeared to be lysed. To check for viability, cells were replated back in tissue culture medium under the standard growing conditions for the B16 melanoma cells and no cells grew out of the replating experiment. These results suggest that the monoclonal antibody to CP may have a direct effect or act indirectly through complement in the fetal bovine serum on cell viability of tumor cells.

Example 9: Patient Immunization with Cancer Procoagulant Antigen

Preventive medicine is generally considered to embody the preferred form of therapy for disease control in the future. The discovery that antibodies to cancer procoagulant are cytotoxic to malignant cells (see Example 7) , reduce the metastatic capacity of malignant cells (Example 5) and decrease metastases of tumor cells (Example 6) has allowed a novel immunization to be formulated and used for the prevention of cancer.

Active immunization of the population, preferably in adolescent years, substantially reduces the incidence of cancer establishment, since anti-cancer procoagulant antibody is cytotoxic to malignant cells. Briefly, purified animal cancer procoagulant is used as active antigen to elicit the production of human antibodies specific to cancer procoagulant. Standard immunological techniques used routinely in the art are used to obtain immunization against cancer procoagulant antigen. The titer of cancer procoagulant antibodies is easily monitored qualitatively using conventional skin patch tests or quantitatively by standard laboratory measurements of blood serum samples.

Example 10: Patient Immunotherapy to Reduce Tumor Metastases

(i) Intravenous administration of anti-human CP immunoglobulins

Intravenous injection of anti-CP immunoglobulins is utilized in immunotherapy directed toward the reduction of tumor metastasis. It may also be used as adjuvant therapy in conjunction with, for example, chemical or radiation therapy; or as a preventive measure in cancer patients before or during surgery when the incidence of tumor perturbation leading to malignant cell shedding and metastasis may occur with increased probability.

A patient having cancerous growth, ascertained by usual methods of tumor detection, is injected by intravenous infusion of 50-100 ml of sterile physiological saline containing 10-1000 μg of anti-human CP immunoglobulin per kg weight of patient and delivered over a period of 15-60 min. Antibodies to purified human CP may be prepared by conventional methods used for the preparation of polyclonal antibodies as, for example, goat anti-human CP IgG or by established protocols for the preparation of monoclonal antibodies, for example, goat anti-human CP IgM as described by Gordon in continuation- in-part application serial no. 069,454 incorporated herein by reference. Patients are pretested for anaphylactic hypersensitivity to the foreign immunoglobulin fraction. Dose levels of foreign antibody proteins ranging from 15 to 1000 mg are well tolerated in humans (Sears et al. (1984) J. Biol. Response Modifiers 2:138-145). After intravenous immunotherapy, tumor size and extent of metastasis are monitored at regular intervals. The dose is repeated as necessary at intervals adjusted on an individual basis.

(ii) Direct injection of anti-human CP immunoglobulin into tumors

The precise localization of a tumor is carried out using established medical diagnostic procedures. For example, radiolabelled antibodies to specific tumor antigens are given to patients and the tumors are subsequently detected and localized by scintillation scanning (Hansen, U.S. patent no. 3,927,193; Goldberg, U.S. patent no. 4,331,647, no. 4,348,376 and no. 4,361,544) .

An injectable composition of anti-human CP immunoglobulins is prepared in a sterile solution comprising per ml:

(a) 10 mg human serum albumin

(b) 0.01 M phosphate buffer, pH 7.5

(c) 0.9% NaCl

(d) 10-1000 μg anti-human CP immunoglobulins prepared as discussed above in Example 9(i).

From 1 to 10 ml of injectable composition of anti- human CP globulins is injected into predefined tumor locations. The tumor size is monitored at regular intervals. Injections are repeated as necessary at intervals adjusted on an individual basis.

Example 11: In Vitro Cytotoxic Effect of Anti-Cancer

Procoagulant Antibody on Malignant Cells

(i) Effect of anti-cancer procoagulant IgM antibody on viability of B16-F10 murine melanoma cells

Monoclonal antibodies to cancer procoagulant were prepared as described in Example 3. Briefly, Charles Rivers mice were immunized with pure cancer procoagulant as described above. A final injection of antigen was administered in saline intraperitoneally and one week later the mice were sacrificed, their spleens were removed and spleen lymphocytes were hybridized with the P3X variant of the mouse myeloma cell in 15% polyethylene glycol. Hybrid cells were subjected to the standard HAT suicide procedure to eliminate unwanted cell populations. The surviving hybrid cells were grown in the presence of CP to stabilize the clones and were screened for anti-CP antibody production, cloned and rescreened two more times before cell populations were grown into flasks for storage and--antibody production. Hybrid cells were injected into the peritoneal cavity of pristane treated Charles Rivers mice and IgM was purified from the ascites drainage from these mice. IgM was mixed with 50,000 B16-F10 mouse melanoma cells in T-25 culture flasks and incubated overnight at 37°C. Incubations were routinely started between 3pm and 4pm of one day and stopped at approximately 9am the next morning. In all cases experimentals and appropriate controls were incubated for exactly the same time intervals. At the end of the incubation period, the cells were counted in a hemocytometer. Cell viability was determined by Trypan Blue exclusion as described in Antibodies. A Laboratory Manual (1988), (Harlow and Lane eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 256. Figure 1 documents the cytotoxic effect of anti-cancer procoagulant IgM antibody on B16-F10 murine melanoma cells. No decrease in cell number was observed in the absence of anti-cancer procoagulant antibody. Figure 2 displays the decrease in total cell number and cell viability as a function of concentration of anti-cancer procoagulant IgM antibodies. These results indicate that the monoclonal antibody to cancer procoagulant has a cytotoxic effect on B16-F10 murine melanoma cells.

(ii) Effect of anti-cancer procoagulant IgM antibody on the destruction of small cell lung carcinoma (SCLC) cells in vitro

It is generally recognized in the art that the binding of antigens by antibodies and T cells triggers either a cell-mediated or a complement-mediated cascade reaction that leads to destruction of the antigens and lysis of antigen-bearing cells. The effect of complement was tested on the viability of small cell lung carcinoma

(SCLC ) cells in the presence and absence of anti-cancer procoagulant antibodies. The MTT method, (Carmichael et al. (1987) Cancer Res. £7:936; Denizot et al. (1986) J. Immunol. Method £9.:271; Mossman, T. (1983) J. Immunol. Method (55_:55) was used to determine the viability of the cells. In microtiter plates 10,000 cells per well of cultured SCLC cells (obtained from The Department of Oncology, University of Colorado Health Sciences Center, Denver, CO) were incubated overnight (from between 3pm and 4pm one day to about 9am the next morning) at 37°C with complement (Sigma Chemical Co., St. Louis, MO) at different dilutions from 1:2 to 1:20. As shown in Figure 3, there was essentially no effect of complement on the viability of SCLC cells except at the highest concentration of complement. Addition of anti-cancer procoagulant IgM resulted in the destruction of SCLC cells at a level that was sustained at different dilutions of complement. Similar results (to the curve of Figure 3) were obtained with heat inactivated complement and anti-CP IgM (not shown) .

Figure 4 shows the effect of different amounts of anti-cancer procoagulant IgM and nonsense IgM (Kirkagaard

Co., Inc.) with and without complement, on SCLC cells in microtiter plate culture incubated overnight at 25°C. The nonsense IgM had no effect, with or without complement.

The anti-cancer procoagulant IgM had a definite cytotoxic effect above 4 ug/well (which translates to about 40 ug/ml) . The same data is combined in Figure 5 and presented in terms of % viability. These results indicate that anti-cancer procoagulant antibody kills SCLC cells and that neither complement nor immune cell are required for anti-cancer procoagulant antibody induced destruction of tumor cells in vitro. According to the teachings of the prior art, the IgM must bind to the cell membrane in order to kill the cells directly. To show binding of anti-cancer procoagulant antibodies to SCLC cells in culture, different concentrations of anti-cancer procoagulant IgM or nonsense IgM from 1 to 8 ug per well were reacted with the SCLC cells (10,000 cells per well) overnight at 37°C. After incubation, alkaline phosphate conjugated anti-IgM antibody was added to the incubated cells. The color development represented a measure of the IgM bound to the cells. Figure 6 documents the individual data from five binding curves and a saline, IgM, no cell control. Figure 7 displays the mean and standard error of the mean for the five sets of data. The difference between the anti-cancer procoagulant IgM and the nonsense IgM is the specific binding of anti-cancer procoagulant IgM to cancer procoagulant on the cell surface of SCLC cells.

(iϋ) Effect of anti-cancer procoagulant IgG antibody on viability of human SCLC cells

fa) with (³H)-thymidine incorporation assay

Incorporation of(³H)-thymidine was utilized as a measure of replication or division of human SCLC (H345) cells and normal human skin fibroblasts in the presence of anti-cancer procoagulant IgG. Polyclonal IgG was prepared as described in Example 2. Normal skin fibroblasts were obtained from biopsy specimens and grown out as primary explants in RPMI 1640 medium supplemented with 10% fetal calf serum.

Cell were plated in serum-free medium at a concentration of 5000 viable cells/200 μl/well in 96 well, flat bottom plates (Corning 25860, or Falcon 3075) . Control wells contained RPMI 1640 medium supplemented with HITES (hydrocortisone, insulin, transferrin, estradiol and selenium; Sim s et al. (1980) Cancer Res. 40:4356-4363) . Cells were allowed to recover and settle for three hours at 37°C in a 5% incubator (100% humidity) before adding antibodies. Following a 24 hour incubation in the presence of antibodies, 0.4 μCi [methyl-³H] thymidine, 6.7 Ci/mmol (ICN Biomedicals) , was added in a volume of 10 μl RPMI with HITES medium. Quantitation of incorporation of (³H)-thymidine into DNA was carried out by routine procedures. Briefly, cells were incubated for an additional 24 hours before harvesting using a Titerek® Cell Harvestor (Flow Laboratories, Rockville, MD) . Glass Fiber Filter paper (Gelman Sciences, #61638) , Type A/E, 8 in X 10 in was used. In harvesting cells, the paper was first saturated with distilled water using a blank plate, then the line was cleared. The cells in the wells were then lysed using distilled water and washed onto the paper. The wells were then washed using 80% ethanol which fixes the DNA on the paper. A final rinse was done using distilled water before clearing the lines. This procedure was repeated for each row of wells. The paper was then allowed to dry before counting. One minute sample counts were done using a Beckman LS1801 scintillation counter (Beckman Scientific) . Counts were performed in sets of 4 and mean+/-SE values were compared to control values of counts in medium without peptide agonist.

As illustrated in Figure 8, there was no decrease in incorporation of (³H)-thymidine by normal skin fibroblasts in the presence of anti-cancer procoagulant IgG. Instead, a slight stimulation of thymidine incorporation was observed in the presence of IgG as might be expected due to the normal process of cell division. In contrast, the SCLC (H345) cells were noticeably stimulated to incorporate (³H)-thymidine in the presence of about 20 μg IgG, but exhibited a progressive decrease in amount of (³H)-thymidine incorporation with increasing concentration of IgG. The results graphed in Figure 8 show that addition of anti-cancer procoagulant IgG has no effect on normal cells but results in a substantial suppression of cell proliferation, most likely, due to destruction of the malignant SCLC cells. (h) with MTT assay

The MTT method (Carmichael et al. (1987) Cancer Res. 47:936; Denizot et al. (1986) J. Immunol. Method j}£:271; Mossman (1983) J. Immunol. Method j55_:55) was used to determine cell viability. The MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method is a colorimetric assay based on the ability of live (but not dead) cells to reduce a pale yellow tetrazolium-based compound (MTT) to a blue for azan product by the mitochondrial enzyme succinate dehydrogenase. The amount of formazan product generated and then measured at 540 nm against a reference wavelength of 690 nm is proportional to cell number. Standard curves for 4h and 24h MTT assays using human SCLC(H345) cells, mouse melanoma (B16-F10) cells and normal human skin fibroblast cells are presented in Figure 9. A straight- line relationship is indicated for all cases (Correlation coefficients varied from 0.96 to 0.98).

SCLC (H345) cells or normal skin fibroblasts were incubated 24h at 37°C essentially as described in the method above, in the presence of different concentrations of anti-cancer procoagulant IgG (prepared as described above) or normal goat IgG as a control. 25 μl of a 2 mg/ml solution of MTT (Sigma No. M2128) was dissolved in RPMI 1640 with HITES medium and added to each well. After incubation for 4h at 37°C in a 5% C0₂ incubator, the plate was centrifuged at 1600 rpm for 10 minutes, and the supernatant was carefully aspirated off leaving the dark blue formazan product in the bottom of the wells. The reduced MTT product was solubilized by adding 100 μl of a solution containing 2% concentrated HC1, 75% isopropanal and 23% distilled water to each well. Thorough mixing was carried out using a Titerek® multichannel pipettor. The absorbance (optical density) of each well was measured using an automatic plate reader (Titerek® Multiscan MCC) with a 540 nm test wavelength and a 690 nm reference wavelength) .

As shown in Figure 10, normal skin fibroblasts showed no decrease in Absorbance (optical activity) with increasing levels of IgG, and instead, a stimulatory effect was observed at higher levels of IgG. In contrast, SCLC cells showed a rapid decrease in Absorbance (optical density) at low levels of IgG, while at higher levels of IgG, the rate of MTT metabolism continued to decrease. Thus, the data presented in Figure 10 show that increasing amounts of anti-cancer procoagulant IgG has no effect on the viability of normal cells, e.g. human fibroblasts, but results in the substantial loss of viability of malignant SCLC cells, due to loss of viability and increased destruction of the malignant cells. Based on the MTT assays, the number of living cells decreases in the presence of increasing concentrations of anti-cancer procoagulant antibodies. Thus, anti-cancer procoagulant antibodies appear to have specific cytotoxicity, killing or destroying, specifically, cancer cells but not normal cells. In fact, there do not appear to be any long-term effects of the presence of anti-cancer procoagulant antibodies in animals. For example, three rabbits were actively immunized with cancer procoagulant and were determined to produce antibodies to cancer procoagulant in chronic toxicity studies. They showed no detrimental effects due to the presence of anti-cancer procoagulant antibodies for a period of over 4 years. When these rabbits finally died, necropsy results indicated that death was associated with the common causes of old age.

Figure 11 restates data from Figures 8 and 10 to show the consistent effect of anti-cancer procoagulant IgG on SCLC (H345) cells as determined with the (³H)-thymidine incorporation assay which reflects cell division or proliferation and the MTT assay which reflects cell viability. At low concentrations of IgG, there appears to be a slight stimulation in rate of (³H)-thymidine incorporation, whereas a rapid decrease in Absorbance (optical density) is observed with the MTT assay. However, in both assays, it is clear that incubation of SCLC cells in the presence of anti-cancer procoagulant IgG results in a loss of cell viability.

INDUSTRIAL APPLICABILITY

This invention may be used for active and passive immunization for the prevention of tumors in an immunized host. Also, this invention provides a method of tumor immunotherapy for diagnosed tumors and a method for destroying malignant cells. Further, a method is provided for preparing a stable hybridoma cell line for producing the antibody if the invention.

Claims

1. Use of an effective amount of antibody specific to cancer procoagulant for the preparation of an immunogenic composition for application to the destruction of in situ or invasive malignant cells.

2. Use of cancer procoagulant for the preparation of an antigenic composition for application to tumor prevention which comprises active immunization of a live subject with an effective amount of said antigenic composition such that the host develops antibodies to said cancer procoagulant.

3. The method of claim 2 wherein said cancer procoagulant is conjugated to a chemical or biological ligand, such that the presence of cancer procoagulant enzymatic and immunologic activities remain detectable and therapeutically effective.

4. Use of an antibody specific to cancer procoagulant for the preparation of an immunogenic composition for application to tumor immunotherapy which comprises injecting into a live subject having a tumor which produces or is associated with cancer procoagulant an immunotherapeutically effective amount of said immunogenic composition such that tumor formation is retarded or prevented, tumor metastasis is decreased, tumor size is reduced or malignant cells are destroyed.

5. The method of claim 4 wherein said antibody is a monoclonal antibody.

6. The method of claim 4 wherein said parenterally injecting refers to injection directly into a prelocalized tumor.

7. The method of claim 4 wherein said method is used adjunct to other types of cancer therapy.

8. The method of claim 4 wherein said method is used in conjunction with surgery as a preventive measure against tumor metastasis.

9. A method for producing a stable hybridoma producing monoclonal antibodies to CP comprising maintaining said hybridoma on a conditioned medium comprising CP antigen.

10. A stable hybridoma cell line producing monoclonal antibodies to CP prepared by the method of claim 9.