WO1994001536A1 - Immunotherapie contre le cancer realisee avec des anticorps diriges contre le profacteur de coagulation du cancer (pc) - Google Patents

Immunotherapie contre le cancer realisee avec des anticorps diriges contre le profacteur de coagulation du cancer (pc) Download PDF

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Publication number
WO1994001536A1
WO1994001536A1 PCT/US1992/005726 US9205726W WO9401536A1 WO 1994001536 A1 WO1994001536 A1 WO 1994001536A1 US 9205726 W US9205726 W US 9205726W WO 9401536 A1 WO9401536 A1 WO 9401536A1
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cells
cancer
tumor
antibodies
procoagulant
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PCT/US1992/005726
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Stuart G. Gordon
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University Research Corporation
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Priority to AU23193/92A priority patent/AU2319392A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • 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.
  • CP cancer procoagulant
  • CP initiated coagulation in the absence of factor VII and was inhibited by diisopropylfluorophosphate (DFP) , two characteristics that distinguish it from tissue factor.
  • DFP diisopropylfluorophosphate
  • 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.
  • investigators have sought to identify diagnostic markers of cancerous cells.
  • Bubenek et al. Int. J. Cancer 5_:310
  • serum from cancer patients contained antibodies that bound to tumor cell surface antigens.
  • 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.
  • CEA carcinoembryonic antigen
  • AFP alpha-fetoprotein
  • cALLA acute lymphoblastic leukemia associated antigen
  • 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).
  • 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) .
  • 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.
  • BCG Bacille Calmette-Guerin
  • 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, Arlington, Arizona, Toronto, p. 69) .
  • 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).
  • the antibody 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.
  • 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.
  • CP cancer procoagulant
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • the medium contains about one part filtered medium previously used to grow CP-producing cells and three parts unused medium.
  • 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 ( 3 H)-thymidine incorporation method ( 3 HT) . 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 ( 3 H)-thymidine incorporation assay ( ) and the MTT assay ( ) .
  • Results are expressed as a percent of the untreated control (without IgG) .
  • CP Cancer procoagulant
  • CP is a proteolytic enzyme that directly activates factor X, bypassing both the extrinsic and intrinsic pathways of the coagulation cascade.
  • 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.
  • 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.
  • cross-reactivity of anti-CP antibodies to antigens critical to the functioning of normal cells should be minimized.
  • cross-reactivity to nontumor-associated antigens is less than or equal to about 15%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • immunization with CP antigen provides a method for cancer prevention in human or animal hosts.
  • 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.
  • 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.
  • immunotherapy as a primary or adjunct therapy, is employed preferentially.
  • 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.
  • this invention provides a method for parenterally injecting a patient having a diagnosed tumor with anti-CP immunoglobulins in order to reduce tumor metastasis.
  • 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.
  • 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.
  • Cancer procoagulant is believed to be an oncofetal antigen
  • 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.
  • applicants use a special conditioning medium containing CP antigen as described in the Examples.
  • 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.
  • step one 1.5 M agarose gel filtration column chromatography
  • step two 1.5 M agarose gel filtration column chromatography
  • step three second benzamidine-Sepharose affinity chromatography column
  • step three a p- chloromercurial benzoate-Sepharose column chromatography
  • 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.
  • 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) .
  • cysteine proteinase inhibitors i.e., iodoacetamide and mercury
  • activators i.e., dithiothreitol, KCN
  • 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 ⁇ g 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.
  • 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.
  • 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.
  • PCMB p- chloromercurial benzoate
  • the column was allowed to stand for 1 hr at 4°C and washed slowly overnight with 20 mM 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 HgCl 2 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 HgCl 2 which will inhibit and preserve the activity for later use.
  • 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 ⁇ g 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 ⁇ g amounts of antigen suspended in incomplete Freund's adjuvant and injected subcutaneously at monthly intervals.
  • 3 intraperitoneal immunizations of 10 ⁇ g, 70 ⁇ g, and 70 ⁇ g 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 ⁇ g) 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.
  • HAT hypoxanthine- aminopterin-thymidine
  • 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.
  • 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 10 6 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.
  • procoagulant activity contained procoagulant activity.
  • Balb/C mice were injected with 0.5 ml of pristane to desensitize their immune system. Three weeks later, the mice received 2 X 10 6 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.
  • 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 CaCl 2 .
  • 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 ⁇ g/ 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) .
  • known inhibitors of cell procoagulants were used, namely the cysteine proteinase inhibitors HgCl 2 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 HgCl 2 (0.1 mM, final concentration) or iodoacetamide (2 mM) at 37°C for 30 min 26 containing 0.1 mg MgCl 2 .6H 2 0/ml and 0.2% NaN 3 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.
  • 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.
  • PBS phosphate buffered saline
  • 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
  • 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
  • phosphate buffer 50 ⁇ l 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).
  • mice 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.
  • BSA bovine serum albumin
  • 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.
  • 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.
  • Example 6 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.
  • 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.
  • 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 substantially reduces the incidence of cancer establishment, since anti-cancer procoagulant antibody is cytotoxic to malignant cells.
  • 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.
  • 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 ⁇ g 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.
  • 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:
  • 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.
  • 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.).
  • 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.
  • SCLC 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.
  • 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.
  • Figure 4 shows the effect of different amounts of anti-cancer procoagulant IgM and nonsense IgM (Kirkagaard
  • 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.
  • HITES hydrocortisone, insulin, transferrin, estradiol and selenium
  • 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.
  • 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.
  • 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.
  • the reduced MTT product was solubilized by adding 100 ⁇ l 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) .
  • 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 ( 3 H)-thymidine incorporation assay which reflects cell division or proliferation and the MTT assay which reflects cell viability.
  • SCLC SCLC
  • 3 H-thymidine incorporation assay which reflects cell division or proliferation
  • MTT MTT assay
  • 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.

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Abstract

Procédé de prévention des tumeurs dans lequel on utilise l'antigène et des anticorps dirigés contre le profacteur de coagulation du cancer (PC) dans des procédures d'immunisation. On utilise également le PC dans un procédé permettant de détruire de manière spécifique des cellules malignes. Cette invention concerne également un procédé de production d'un hybridome stable utile pour produire des anticorps monoclonaux dirigés contre le profacteur de coagulation du cancer.
PCT/US1992/005726 1992-07-07 1992-07-07 Immunotherapie contre le cancer realisee avec des anticorps diriges contre le profacteur de coagulation du cancer (pc) WO1994001536A1 (fr)

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PCT/US1992/005726 WO1994001536A1 (fr) 1992-07-07 1992-07-07 Immunotherapie contre le cancer realisee avec des anticorps diriges contre le profacteur de coagulation du cancer (pc)
AU23193/92A AU2319392A (en) 1992-07-07 1992-07-07 Cancer immunotherapy with antibodies to cancer procoagulant

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6344026B1 (en) 1998-04-08 2002-02-05 Senorx, Inc. Tissue specimen encapsulation device and method thereof
US6540693B2 (en) 1998-03-03 2003-04-01 Senorx, Inc. Methods and apparatus for securing medical instruments to desired locations in a patients body
US8137346B2 (en) 1998-09-01 2012-03-20 Senorx, Inc. Electrosurgical lesion location device
US9408592B2 (en) 2003-12-23 2016-08-09 Senorx, Inc. Biopsy device with aperture orientation and improved tip
CN114062564A (zh) * 2022-01-12 2022-02-18 北京第一生物化学药业有限公司 一种塔夫素及其变异体的检测方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4461833A (en) * 1982-06-23 1984-07-24 University Patents, Inc. Chromatographically purifying proteolytic procoagulant enzyme from animal tissue extract
EP0162977A2 (fr) * 1984-05-02 1985-12-04 University Research Corporation Essai immunologique d'une enzyme procoagulante, indicatrice du cancer
EP0314496A2 (fr) * 1987-10-29 1989-05-03 E.I. Du Pont De Nemours And Company Production d'hybridomes
WO1989005307A1 (fr) * 1987-12-09 1989-06-15 The General Hospital Corporation Antigene sf-25 de l'adenocarcinome du colon et anticorps reconnaissant cet antigene

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4461833A (en) * 1982-06-23 1984-07-24 University Patents, Inc. Chromatographically purifying proteolytic procoagulant enzyme from animal tissue extract
EP0162977A2 (fr) * 1984-05-02 1985-12-04 University Research Corporation Essai immunologique d'une enzyme procoagulante, indicatrice du cancer
EP0314496A2 (fr) * 1987-10-29 1989-05-03 E.I. Du Pont De Nemours And Company Production d'hybridomes
WO1989005307A1 (fr) * 1987-12-09 1989-06-15 The General Hospital Corporation Antigene sf-25 de l'adenocarcinome du colon et anticorps reconnaissant cet antigene

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6540693B2 (en) 1998-03-03 2003-04-01 Senorx, Inc. Methods and apparatus for securing medical instruments to desired locations in a patients body
US6344026B1 (en) 1998-04-08 2002-02-05 Senorx, Inc. Tissue specimen encapsulation device and method thereof
US6508773B2 (en) 1998-04-08 2003-01-21 Senorx, Inc. Tissue specimen encapsulation device and method thereof
US8137346B2 (en) 1998-09-01 2012-03-20 Senorx, Inc. Electrosurgical lesion location device
US9408592B2 (en) 2003-12-23 2016-08-09 Senorx, Inc. Biopsy device with aperture orientation and improved tip
CN114062564A (zh) * 2022-01-12 2022-02-18 北京第一生物化学药业有限公司 一种塔夫素及其变异体的检测方法

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