CH662573A5 - METHOD FOR THE PRODUCTION OF STRONG ANTIGEN, TUMOR-SPECIFIC SUBSTANCES AND THE USE THEREOF AS A CANCER DIAGNOSIS. - Google Patents

Description

The present invention relates to a method for producing substances with highly tumor-specific antigen activity and their use as cancer diagnostic agents.

It is known that in several human cancer diseases, such as melanomas, lung, breast or colon cancer, weakly antigen-active tumor-specific substances are present on the membranes of the tumor cells which form them. With the usual extraction methods, such tumor-specific antigens, sometimes referred to in the literature as TSTAs and hereinafter "TSAs", prove to be even weaker antigens than when they are naturally associated with malignant tumor cells; see the article by K. Sikora et al. "Partial Purification of Tu-mor-Specific Transplantation Antigens from Methylcholan-trene-Induced Murine Sarcomas by Immobilized Lectins", British J. Cancer 40: 831-838 (1979) ("Partial purification of tumor-specific transplantation antigens from methylcholantrene-induced mouse sarcomas by immobilized Lectine »).

There is a recent report on sensitizing circulating lymphocytes in women with breast cancer to breast cancer TSAs; see the article by Wm. Gm. Ramey et al "Detection of Breast Tumor Antigen-Sensitive Circulating T-Lymphocytes by Antigen-Stimulated Active Rosette Formation", Cancer Research 39: 4796-4801 (1979) ("Detection of breast tumor antigen-sensitive circulating T-lymphocytes by antigen-stimulated active Rosette formation »). This effect is believed to be due to the prolonged release of breast TSAs of weak anti-gene activity into the breast cancer patient's circulation.

Melanoma extracts can react with the lymphocytes of other melanoma patients in vitro, but not with lymphocytes of other tumor types (UW lehn et al “In Vitro Lymphocyte Stimulation By A Soluble Antigen From Malignant Melanoma”, New England J. of Medicine 283: 329-333, 1970) ("In vitro lymphocyte stimulation by a soluble antigen of malignant melanoma"), Similarly, extracts from osteosarcomas in vitro can stimulate lymphocytes of other osteosarcoma patients, but not lymphocytes of other cancer patients. (B.J. Gai-nor et al, "A Method of Immunological Assay in Human Osteosarcoma, Clinical Orthopedics and Related Research (Philadelphia) 111: 83, 1975) (" A method for immunological detection in human osteosarcoma "),

It is known that lymphocytes, e.g. Bacillus Calmette-Guerin (BCG) are sensitized if they come into contact with the strong sensitizing antigen again in vivo or in vitro, merge existing macrophages at the site of the reaction and can form multinucleated giant cells; see the article by

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AH. Warfel "Macrophage Fusion and Multinucleated Giant Cell Formation, Surface Morphology", Expérimental & Molecular Pathology 28: 163-176 (1978) ("Macrophage fusion and formation of multinucleated giant cells, surface morphology"). TSAs previously obtained from cancer cells are, as noted, weak antigens, and when weak TSAs are contacted with lymphocytes in the presence of macrophages, no macrophage fusion reaction occurs either in vivo or in vitro.

Heating to hyperthermal temperatures, e.g. 41.5 to 43.5 ° C, improves the inherently weak immunogenic effect of cancer cells, however, it has been found that the immunological change caused by heat is less than the similar, still ineffective immunogenic changes produced by X-rays; see the article by H.D. Suit et al "Immunogenicity of Tumor Cells Inactivated by Heat", Cancer Research 37: 3836-3837 (1977) ("Immunogenic effects of heat-inactivated tumor cells"). X-rays are also known to have a similar limited effect on embryonic or fetal tissue antigens, according to E. Sega et al, "Specific Blastogenic Response of Peripheral Blood Lymphocytes from Lung Cancer Patients to a Fetal Lung Antigen," Journal of the National Cancer Institute , Vol. 64, No. 5, pages 1001-1006 (1980) (“Specific blastogenic response of peripheral blood lymphocytes from lung cancer patients to a fetal lung antigen”). Reported warming or X-ray exposure may make TSAs somewhat more active than non-warming TSAs, but they are still not activated enough to produce specific histological changes or participate in in vitro responses, such as the macrophage fusion reaction.

US Pat. No. 4,108,849 (A. Thomas) discloses a “process for extracting and processing glycoproteins, mu-copolysaccharides and accompanying substances”, in which such substances are extracted from human tissue, from bacteria and other plant organisms . The substances are stabilized with fatty substances for use in industry, medicine or cosmetics. The products obtained are not suitable for the production of antigens, in particular for diagnostic purposes.

From US Pat. No. 4,150,150 (O. Straub; Bayer AG) a method for obtaining cancerostatic preparations from tumor cells is known, in which mouse tumor cells are treated with alternating current in aqueous suspension to destroy their pathogenic activity. The preparations are used for therapeutic, non-diagnostic purposes.

It has also become known from the British Journal of Cancer, Vol. 41, pages 227-275 (F. Sakai et al.) To extract rat lymphoma cells produced by viruses with saline solutions and to combine the extracts obtained in this way with lipid materials. There is no increase in the antigenic activity of gross virus cell surface anti-gen when introduced into Sakai's liposomes.

It is an object of the invention to produce substances with strong tumor-specific antigenic activity and to use such strongly antigenic TSAs in cancer test methods.

This object is achieved according to the invention by the characterizing features of claims 1 and 8. Refinements and preferred embodiments of the invention are defined in the dependent claims.

The radiation, which causes cyclical, very rapidly changing changes in the intracellular arrangement of the polarity in dipolar molecular components of cancer cells, generates strongly antigenic TSAs.

The applied rays can simultaneously heat the rapidly vibrating cell components if the cells are not cooled during the irradiation. Such irradiations can be followed by elution or extraction of the strong antigenic TSAs produced. During the irradiation, the temperature of the material derived from cancer is preferably kept above 22 ° C. and in particular above approximately 25 ° C., for example between approximately 42 ° C. and approximately 55 ° C. and more, preferably between approximately 42 ° C. and 50 ° C. The duration of the irradiation can vary depending on the energy input per unit of time, for example the irradiation can be carried out for a period of time between about 20 and 180 minutes and more, preferably between about 30 and 90 minutes, cut cancer tissue in the form of tissue slices or aggregates irradiated by tumor cells or cell membranes isolated from cancer cells suspended in aqueous medium, which accumulates in TSAs emerging from cancer cells or cancer cell membranes.

Strongly antigenic TSAs can be extracted from irradiated cancer cell material using various known extraction methods or modifications thereof.

The molecular weight of the TSAs obtained is between about 20,000 and 300,000, preferably between about 25,000 and 150,000 daltons and more, in particular between about 28,000 and 75,000 daltons. Crude TSA extracts also contain histocompatibility antigens (HL-As) which, like the TSAs, (I) come from the membranes of tumor cells and (II) contain beta-2-micro-globulin in a non-covalent bond.

The TSA extracts, which contain substances obtained according to the invention, can preferably be used if HL-As and their bound beta-2-globulin have been removed from the TSA extracts, as described hereinafter.

The strongly antigenic TSA extracts can be encapsulated in lamellar liposome droplets in order to further increase the antigenic effect of the TSAs. Encapsulation can be performed using the method described in the article by G. Poste et al "Lipid Vesicles as a Carrier for Introducing Material into Cultured Cells: Influence of Vesicle Lipid Composition on Mechanisms of Vesicle Incorporation into Cells", Proc. Nat'l. Acad. Sci. 73: 1603 (1976) ("Lipid droplets as carriers for the introduction of materials into cultivated cells: influence of the lipid composition of the droplet on mechanisms of droplet uptake in cells") or by following the procedure of F. Sakai et al. Association of Gross Virus Associated Cell-Surface Antigen with Liposomes ”, British Journal of Cancer 41: 227-235 (1980) (“ Association of Gross Virus Associated Cell Surface Antigen with Liposomes ”).

The strongly antigenic TSAs, which are specific to the type of cancer cells obtained or extracted from them, which are obtained according to the invention, are characterized by their ability to be in the presence of contact with lymphocytes obtained from cancer patients with a similar pathohistological tumor type of neighboring (near) macrophages to cause aggregation and fusion of macrophages.

Strongly antigenic tumor-related TSA generated and contained in or eluted or extracted from malignant tumor cells whose primary location and histological character are known are tested for in vitro induction of lymphokine production by lymphocytes from cancer patients. If a patient's lymphocytes after they have been brought into contact with

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secrete strongly antigenic tumor cells or tumor antigen from histologically known tumor origin, lymphokines, which lead to the aggregation and fusion of added macrophages or to the formation of multinucleated giant cells, then the patient has cancer of a type and / or histological character similar to the known cancer, of which the specific cancer cell material or the TSA extract used in the test.

Strongly antigenic TSAs have other uses than just for use in the macrophage fusion test. They can be used in other tests to detect the presence of related cancer, for example in the platelet aggregation test or in the cytofluorometric test, which are explained below. Strong TSAs also have therapeutic utility, particularly TSAs in isolated cancer cell membranes or encapsulated in liposomes when used for extracorporeal or parenteral immunological activation of mononuclear cells from immunocompetent cancer patients.

The macrophage fusion test is analogous to the test described by B. Galindo et al, "Fusion of Normal Rabbit Alveolar Macrophages Induced by Supernatant Fluids from BCG-Sensitized Lymph Node Cells After Elicitation by Antigen", Infection and Immunity 9: 212- 216 (1974) ("Fusion of normal rabbit alveolar macrophages induced by supernatant fluids from BCG-sensitized lymph node cells after elicitation by antigen") who first used such tests with non-tumor-strongly sensitizing substances such as BCG. The result of this in vitro test is obtained by counting the fused macrophages and multinucleated giant cell formations, which are visible on microscopic examination. Such cell aggregations and fusion are formed by association of single macrophages under the influence of lymphokines, which originate from sensitized lymphocytes, in which the production of lymphokines is triggered by a strong sensitizing specific antigen.

The platelet aggregation test is analogous to the macrophage fusion test. This test was described by K.L. Lavelle et al. "Identification of a New Platelet Aggregating Factor Released by Sensitized Leucocytes", Clinical Immunology and Immunopathology 3: 492-502, 1975) ("Identification of a new platelet aggregating factor that is released by sensitized leukocytes") and uses platelets instead of macrophages as aggregation-indicating cells.

It is known that blastogenic transformation of lymphocytes, which reflects intracellular DNA changes, can occur when reactive lymphocytes from sensitized persons again come into contact with the sensitizing antigen or antigen in vivo or in vitro. Cytofluorometric analysis can detect minor changes in intracellular DNA content. Sensitized lymphocytes in in-vitro culture which come into contact again with the sensitizing TSA antigen can rapidly change in a diagnostically meaningful manner if TSAs to which the lymphocytes are sensitized are added to the in-vitro tissue culture medium. Cytofluorometry can provide accurate quantitative information in a short time (hours) of such changes that are of diagnostic value for cancer. The two proposed tests for cancer diagnosis, i.e. the cell fusion tests and the cytofluorometric test depend on the same test elements, namely TSAs and lymphocytes from cancer patients, and performing both tests can usefully corroborate the result obtained with either test alone.

The molecular composition of the TSAs is currently unknown. The strength of the TSA in extracts can be assessed by their protein content, which is determined according to Lowry's method.

TSAs in aqueous suspension is a useful form of the strongly antigenic tumor-derived substance obtained according to the invention. The TSAs are preferably encapsulated. It is also possible to use irradiated tumor cell material or tumor cell membranes without extracting TSAs from their natural cellular bonds and to use such material as replacement antigens instead of extracted or eluted TSAs as reagents in diagnostic tests or for therapeutic purposes.

Originally, malignant tumor tissue (well differentiated human squamous cell cancer of the lungs) was irradiated to produce substances that are tumor-specific: fish and have strong antigen activity. Such irradiation was carried out for up to 90 minutes using radiation of 13.56 MHz with 0.25 to 1 watt / cm 2 tumor. A second 90 minute treatment was performed under essentially the same conditions. Strongly antigenic TSAs were formed in the treated cancer, which reacted with lung cancer-sensitive lymphocytes, which released lymphokines that reacted with nearby macrophages, thereby causing single macrophage cells to fuse, thereby producing giant cells with multiple nuclei in a single cytoplasm.

The radiation can be carried out by dielectric or inductive electromagnetic radiation. The wavelengths used in the USA are necessarily within the wavelength ranges approved by the US government.

Microwaves can also be used. The frequency of the high-frequency radiation is in the order of a few tens of million hertz. The frequency of microwaves is on the order of several billion Hertz. Since each frequency cycle causes two changes in polarity and affects all dipolar molecular cell components, the subcellular components of cancer cells are continuously subjected to very rapid electromagnetic impulses, which have an intrinsic effect on them. The non-warming but antigenic potentizing intrinsic effect on cells can be separated from the intrinsic and warming effect of the radiation by cooling the vessel which contains the irradiated cancer cells, cell membranes or TSA extracts.

As suggested by J.W. Schereschewsky noted in “Biological Effects of Very High Frequency Electromagnetic Radiation”, Radiolo-gy, Vol. 20, pages 246-253 (1933) (“Biological Effects of High Frequency Electromagnetic Radiation”) can have a different effect on individual tissue components due to the specific frequency occur at which cell components preferentially absorb energy.

The use of radio frequency treatments with a description of the mechanical details of a device for this purpose has been described by J.A. Diskson et al in "Tumor Eradication in the Rabbit by Radiofrequen-cy Hearing", in Cancer Research 37: 2162-2169 (1977) ("Tumor elimination in rabbits by high-frequency heating").

The use of high frequency electromagnetic radiation to treat human tumors has been described in the article by S. Sugaar and H.H. Le-Veen, "A Histopathologic Study of the Effects of Radiofrequency Thermotherapy on Malignant Tumors of the Lung", Cancer, Col. 43, pages 767-783 (1979) ("A histopathological study of the effects of high-frequency Ther -

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motherapy for malignant tumors of the lungs »); and by F.K. Storm et al in "Normal Tissue and Solid Tumor Effects of Hyperthermia in Animal Models and Clinical Trials", Cancer Research 39: 2245 (1979) ("Effect of hypothermia on normal tissue and solid tumor in animal models and clinical studies"). Microwave treatment of tumors has been described in the article by J. Mendecki et al "Microwave-Induced Hyperthermia in Cancer Treatment: Apparatus and Preliminary Results", International Journal of Radiation Oncology Biology and Physics, Vol. 4, pages 1095-1103 (1978) (" Microwave induced hypothermia in cancer treatment: devices and preliminary results »); and in the article by Raymond U et al "Micro-wave-Induced Hyperthermia in Combination with Radio Therapy of Human Malignant Tumors", Cancer, Vol. 45, pages 638-646 (1980) ("Microwave-induced hypothermia in combination with high-frequency therapy by human malignant tumors »), An intrinsic effect of electromagnetic radiation on mouse lymphosarcoma cells is described by V. Riley; Cancer Research, Vol. 20: 132, 1979 (ref. 533).

Ultrasonic waves also produce high-speed subcellular vibrational effects on molecular particles in tumor cells and cell membranes, and their effects are considered useful in the production of antigenic TSAs; see the article by G.C. Li et al “Cellular Inactivation by Ultrasound”, Nature, Vol. 267, pages 163-165 (1977) (“Cellular Inactivation by Ultrasound”), and the article by J.B. Marmor et al “Treatment of Superficial Human Neoplasms by Local Hyperthermia Induced by Ultrasound”, Cancer, Vol. 43, pages 188-197 (1979) (“Treatment of superficial human neoplasms by ultrasound-induced local hypothermia”). X-rays and alpha, beta and gamma radiation cannot be used.

Electromagnetic effects can produce very rapid changes in polarity in dipolar molecules from cancer cells, cancer cell membranes or from the same derived TSAs which produce sufficiently strong antigenic changes therein that when these materials come into contact with lymphocytes caused by cancer of the same type and / or are sensitized to the same histology, aggregation and fusion of macrophages occurs in the presence of macrophages. The amount of electromagnetic energy required in each case depends on the energy source, the device, the geometry and the dielectric properties of the applicator and on the cancer tissue absorbing the energy and is determined empirically in each individual case. (Applicator: an HF heating electrode).

Human cancer-specific antigens can be obtained from surgically removed malignant tumors, tumors recently removed during autopsy or from masses of cancer cells cultured in vitro.

The first two sources may be sufficient for pilot studies; for preparations on a large scale, cancer cell lines cultured in vitro are preferably used.

The following are methods that are best suited to generate strongly antigenic cancer cells and cancer cell membranes that contain antigenic TSAs.

I. (a) With surgically excised, aseptically manipulated, cut cancer tissue or such obtained from organs removed by autopsy, a volume of tumor tissue (e.g. 30 grams) is freed from connective and adipose tissue, visible necrotic tissue and blood into a chemically given an inert plastic bottle containing two volumes (60 ml) of sterile RPMI-1640 culture medium (pH 7.4); or (b) in vitro grown masses (eg 1000 x 106 malignant cells) of cancer cells or (c) 1 volume of leukaemic cells (eg 1000 x 106 malignant cells) in 1 volume of sterile culture medium are placed in 5 the plastic bottle and in each case electromagnetic high frequency or exposed to microwave energy. Dielectric electromagnetic radiation is generated, e.g. at 13.56 MHz or 2450 MHz crystal controlled frequency by a device that can deliver 300 W adjustable output power to direct radiation onto a plastic bottle surrounded by an applicator and useful cancer tissue in an electrolyte, e.g. Tissue culture medium containing protease inhibitor substance (0.1 mm phenyl-methylsulfonyl fluoride or epsilon-aminocaproic acid 0.25 mg / ml. Heat measurement of the enclosed and irradiated cancer tissue is carried out by means of a remote thermometer which is connected to thermistor sensors which are connected to the in The controlled and maintained temperature is measured and displayed during short periods in which the radiation and the mechanical shaking are interrupted for a moment.

A solid, flexible, flexible applicator bag filled with electrolyte is preferably used, which on its working surface encloses the chemically inert plastic bottle which contains the cancer tissue in RPMI-1640 culture medium. The bag type applicator is electrically matched to the tumor-containing plastic bottle used, i.e. it is designed to minimize the reflection of electro-magnetic radiation from the interface between the applicator and the bottle and its contents. It is usually necessary to tune the applicator with a separate coaxial tuner by pressing the bag type applicator against the plastic bottle 35 which contains the tumor material to be heated and vibrated by radio frequency energy. This is done by adjusting the tuner until the energy reflected by the tumor reaches its minimum. Bag type applicators, preferably filled with solid dielectrics, 40 are preferred for this purpose. Electromagnetically generated energy is transmitted from the amplifier to an impedance tuning circuit and from there to two insulated coaxial cables, which are firmly attached to the outside of the applicator bag, which is covered on its surface with absorbent material. The moist tumor tissue enclosed by the bottle, while suspended in the buffered tissue culture medium, is at controlled temperatures of not less than 22 and not more than 55 ° C and for periods of not less than 20 50 and not more than 180 minutes at one Radiation kept. Soluble cancer cell eluate, which enters the buffer solution as a result of the irradiation with electromagnetic waves, is removed and concentrated, because during the irradiation, the aqueous culture medium is gradually enriched with TSAs, which emerge from irradiated cancer cell membranes. Such TSAs can be concentrated using the hollow fiber extraction method.

After exposure to electromagnetic radiation, the moist tumor tissue is cut with a scalpel and scissors 60, and the finely cut tumor particles are sieved through metal sieves of 0.42 mm and then 0.25 mm clear mesh size (40 or 60 mesh). The tumor particles and cells are washed three times with sterile phosphate-buffered saline and then suspended in 40 ml of a hypotonic buffer (10 mM Tris HCl, pH 7.5, the 0.1 mM phenylmethylsulfonyl fluoride (PMSF) to inhibit endogenous protease activity contains). For the chemical extraction and purification of TSAs, the

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method by K. Sikora et al, Br. J. Cancer, Vol. 40, pages 831-838 (1979), which has the following stages:

A. Cancer cell membrane isolation and solubilization

The suspension of tumor particles and cells receives 20

Beat in a dounce homogenizer and centrifuge in a beaker centrifuge at 108,000 xg for 1 hour at 4 ° C on a layer of 45% sucrose. The membrane fraction forms layers on the sucrose and is collected, resuspended in 0.01 M phosphate buffered saline (PBS) and obtained as a pellet by centrifugation at 108,000 xg for 30 minutes. The pellet is broken up by pipetting with a Pasteur pipette and dissolved in 2 ml of 1% deoxycholate (DOC) in 0.01 M phosphate buffered saline (PBS) containing 0.1 mM PMSF. After two hours of gentle mixing at 4 ° C, 2 ml of 0.01 M PBS are added to bring the DOC concentration to 0.5%. This mixture is centrifuged at 108,000 xg for 30 minutes and the supernatant is collected.

The supernatant is dialyzed in Visking tubing 8-1 / 32 at 4 ° C for 48 hours with 3 changes in the dialysate (0.01 M PBS and 0.1 mM PMSF). This is effective for removing most of the deoxycholate. Aliquots of dialyzed cancer cell membrane extract can be stored at -20 ° C for at least one month.

The dialyzed supernatant, which contains TSAs, can be further purified by passing it through columns of Sepharose beads coupled with an immobilized lectin (wheat germ agglutinin). Wheat germ agglutinin in covalent coupling to columns of Sepharose pearls is effective for glycoprotein separation and can be used in the presence of low concentrations of detergents.

Aqueous, TSAs and HLAs contain extracts that are run through such columns, bind TSAs to the lectin on the beads, but not the HLAs. The TSAs are cleaned using a relatively simple one-step process.

B. Production of Wheat Germ Agglutinin Columns

Wheat germ agglutinin (available from Pharmacia Fine

Chemicals, Piscataway, NJ / USA) is coupled to cyanbromide-activated Sepharose 4B (Pharmacia Fine Chemicals). 2 g of cyan-activated Sepharose 4B gel is swollen and washed for 15 minutes in 500 ml of 1 mM HCl on a glass frit filter. 10-50 mg of wheat germ agglutinin are dissolved in 10 ml of the coupling buffer (0.1 M NaHC03, 0.1 M NaCl, pH 8.3), which contains 2% N-acetylglucosamine.

The washed swollen gel is added to the coupling buffer and mixed for 2 hours at room temperature (19 ° C). Unbound material is washed off with the coupling buffer and all remaining active groups are reacted with 1 M ethanolamine (pH 8.0) for 1 hour. Three wash cycles are used to remove non-covalently absorbed protein, each cycle consisting of a wash at pH 4.0 (0.1 M sodium acetate, 1.0 M NaCl) and a subsequent wash in the coupling buffer. This method causes 70-80% of the lectin to bind to the Sepharose as estimated by the optical density at 280 nm (OD 280) of added and unbound lectin.

C. Elution of purified TSA extract

5 ml of washed lecting-coupled gel are placed in a plastic syringe on a small piece of glass wool. The column is loaded with loading buffer (0.01 M NaHP04,

10-5 CaCl2, 10-5M MnCl2, 0.1 mM PMSF, 0.85% NaCl, 0.2% DOC, pH 7.3) brought into equilibrium and operated at 25 ° C. Solubilized cancer cell membrane extract freed from deoxycholate by dialysis is loaded into a 2 ml volume and equilibrated for 30 minutes. The column is then washed with loading buffer until no further protein (determined by OD 280 of the effluent) is detected. Three washes, which contain unbound components, are combined. The elution buffer, which contains 2% N-acetylglucosamine, is then run into the column.

After 30 minutes equilibrium, the column is washed with elution buffer, and the washing solutions are collected again, pooled, and then dialyzed against 0.01 M PBS for 48 hours and concentrated by vacuum dialysis. The dialyzed purified solubilized TSA extract can be in an aqueous medium, e.g. in RMPI-1640 tissue culture medium aliquots, stored and stored at -20 ° C. TSA can be divided into single Ali-20 quotas by O.H. Loyry's protein determination method (J. of Biol. Chem. 193: 265-709, 1971).

II. Production of cancer cell membranes with a strong specific antigen effect.

Usable cancer cells obtained from finely-sliced and finely screened, fresh, surgically removed, usable human cancer or usable in vitro cultured human cancer cell masses of known pathohistological definition are washed three times with sterile saline solution and then in saline solution, the 0 Contains 1 mM phenyl-methylsulfonyl fluoride, suspended. After standing at room temperature for 15 minutes and a final wash with saline, the sedimented cancer cells are placed in an aqueous medium which consists of 150 mM NaCl, 1.0 mM CaCl2.1.0 mM MgCl2 and 50 mM 35 sodium borate at 7.2 pH consists and adjusted to give a pellet of about 1.2 ml at the end. Each pellet is then suspended in 5 ml of 2.5 mM sodium borate and 0.2 mM EDTA at pH 9.6 and quickly mixed with 200 ml of the same solution. After a period of 10 minutes, during which the cells burst and surface membrane separation takes place, the extraction is stopped by adding sodium borate at pH 9.6 to a final concentration of 20 mM. The white gel, which mainly consists of precipitated core material, is removed with a sieve, 45 and centrifugation at 5000 xg for 10 minutes concentrates the surface membrane phantoms. These are suspended in saline and then centrifuged at 5000 rpm for 10 minutes. The supernatant is dumped, and the pellet is suspended in 20 ml of saline and then centrifuged at 5000 rpm for 10 minutes. After decanting, the pellets are washed three times with phosphate-buffered (pH 7.4) culture medium RPMI-1640 and evenly suspended in 5 ml of this medium and irradiated by the energy of HF radiation, equivalent microwaves or ultrasound and stored in the refrigerator before they are used as antigens or liposome-encapsulated antigens in the cancer test.

III. Extraction of TSAs from cancerous tissue by radio frequency energy, equivalent microwave energy or

60 Ultrasound irradiation can also be carried out according to a modification of the method according to Reisfeld and Kahan, Fed. Proc., Vol. 29, page 2034 (1979), after which 3 M KCl hypertonic salt is slowly added to a cell suspension over 30 minutes to reach a final concentration of 3 M. The extraction mixture is incubated at 4 ° for 16 hours with constant stirring. Insoluble cell components are sedimented by ultracentrifugation at 164,000 xg for 50 minutes. The above

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Standing liquid containing solubilized TSAs is concentrated by dialysis against 50% sucrose solution and then dialyzed against 200 volumes of 0.15 M saline for 16 hours. Precipitates formed during concentration and dialysis are removed by centrifugation at 48,000 xg. The supernatant liquid containing TSAs and HLAs is diluted with 15 volumes of phosphate buffered saline and further purified by absorption on and eluted from wheat germ agglutinin columns as described above.

IV. Human fetal antigens, e.g. Human lung tumor-fetal associated antigen (LTFA) can be extracted from human tumor tissue and purified by a combination of salt precipitation and ion exchange chromatography according to the article by Ercole Sega et al “Specific Blastogenic Response of Peripheral Blood Lymphocytes from Lung Cancer Patients to a Fetal Lung Antigen ", Journal of the National Cancer Institute, 64: 1001-1006 (1980) (" Specific blastogenic response of peripheral blood lymphocytes from lung cancer patients to a fetal lunar gene "),

Fetal antigens are remarkably heat resistant and the preferred upper limits for heating other tumor antigens listed above do not necessarily apply to the best TSA preparations derived from tumors rich in fetal antigens. After heating by high frequency radiation, human lung cancer tissue or lung tissue from 3 to 5 month fetuses is homogenized in distilled water. After centrifuging the homogenate at 25,000 xg for 30 minutes, ammonium sulfate was added to the supernatant until 40% saturation was reached, after which the precipitate was discarded. Then the salt concentration of the supernatant was increased to 60%; the second precipitate was dissolved in 0.01 M K2HP04, dialyzed extensively and finally stored at -20 ° C. The protein concentration of the stored solution (30 mg / ml) was determined by the Lowry et al method using bovine serum albumin as a standard.

1 ml of a solution of protein, which had been precipitated from both tumor and fetal extracts at 60% ammonium sulfate saturation, was extracted by extractive DEAE cellulose chromatography (DE52 Whatman) on a 1 x 30 cm column using 180 ml of a 0.01-0.2 M K2HPO4 linear gradient (pH 8.2) fractionated as an eluent. The flow rate was 7 ml / hour and 2 ml fractions were collected.

The optical density of the eluate was monitored at 280 and 260 nm using an LKB Uvicord III device (LKB Products AB, Bromma, Sweden).

Elution of LTFA was followed by subjecting single fractions to a double immunodiffusion test using an anti-LTFA specific rabbit anti-serum. Fractions containing the antigenic material were pooled, dialyzed against phosphate (0.01 M) buffered saline (0.15 M, pH 7.2) and concentrated to 2 mg / ml under negative pressure. Before use in peripheral blood lymphocyte cancer tests, the preparations were sterilized on 0.22 micron Millipore filters (Millipore Corp., Bedford, Mass.). Higher concentrations of TSA extracts can be obtained using the hollow fiber concentration method (Bio-Rad Lab .; Richmond, California / USA).

Malignant human tumors, obtained surgically or by autopsy, are a limited source of cancer cell extracts. In order to obtain larger amounts of the TSAs derived from human cancer cells of sufficient uniformity and in larger amounts, are in vitro

Mass cultures of known tumor cell lines are necessary. Fermentation facilities and / or the cytogenerator described by S. Graff and H. Moser in "New Interprétations in Cancer Biology", Journal of the Hospital for Joint Diseases 23: 59-79 (1972) ("New 5 Interpretations in Cancer Biology") can be used to cultivate cancer cells in large numbers. The TSA extraction methods described above are applicable to cancer cells grown in mass cultures.

10 The expression "extracted from the named cancer material" denotes TSAs with increased antigen activity, which were obtained from irradiated cancer material ", which contains cancer cell membranes. This can be expressly extracted using the methods described here. The TSAs can also be extracted simply by elution from the cancer cell membrane into the aqueous culture medium during the irradiation indicated above.

Methods and materials for the production of TSAs containing liposomes.

Lipid beads (droplets), referred to as liposomes, have been known as carriers for water materials in an aqueous intermediate phase since their original description by A.D. Bangham et al "Diffusion of Univalent Ions 25 Across the Lamellae of Swollen Phospholipids", Journal of Molecular Biology, 13: 238-252 (1965) ("Diffusion of monovalent ions through the lamellae of swollen phospholipids"). Recent information on encapsulation in liposomes is contained in publications by W.E. Magee 30 et al, "The Interaction of Cationic Liposomes Containing Entrapped Horseradish Peroxidase with Cells in Culture", Journal of Cell Biology, 63: 492-504 (1974) ("The interaction of cationic liposomes, which include horseradish peroxidase, with Cells in culture »); 35 S. Sone et al, "Rat Alveolar Macrophages are Susceptible to Activation by Free and Liposome-Encapsulated Lymphognes", Journal of Immunology, 124: 2197-2002 (1980) ("Al veolen macrophages of the rat can be activated by free and Lymphokines encapsulated in liposomes «); and F. Sakai 40 et al, "Association of Gross Virus-Associated Cell-Surface Antigen with Liposomes", British Journal of Cancer, 41: 227-235 (1980) ("Association of Gross Virus-Associated Cell Surface Antigen with Liposomes »).

Liposome particles with TSAs are made by dispersing dried films of phospholipids in an aqueous phase containing TSAs that firmly adhere to the lipid layers of the liposomes.

Sphingomyelin liposomes can be made by dissolving 50 mg sphingomyelin (highly purified, 50 from bovine brain), 10 mg cholesterol and 6 mg stearylamine in 18 ml chloroform, along with a small amount of methanol. The solution is divided into six 50 ml round bottom flasks or three 100 ml round bottom flasks and the solvent is removed by flash evaporation on a 55 rotary evaporator at room temperature. The flasks are flushed with N2 and 0.3-0.5 ml of the aqueous suspension containing the extracted TSAs are added to each bottle.

Another method of producing liposomes 60 is to use egg phosphatidylcholine, bovine brain phosphatidylserine and lysolecithin in a molar ratio of 4.95: 4.95: 0.01 in a total weight of 66 mg. This amount is dissolved in 18 ml of chloroform together with a small amount of methanol. The solution is distributed into six 65 50 ml round-bottom flasks, and the solvent is removed by flash evaporation, the flasks are washed with N; rinsed and 0.3-0.5 ml of the aqueous phase containing TSAs are added to each bottle.

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The films are stripped from the glass using a vortex genie mixer. Two or three glass beads can be added to promote loosening of the lipid film. The liposomes are removed from the flasks and the flasks are rinsed with a small amount of the aqueous phase. The liposomes are treated with ultrasound at intermittent 20 second intervals at 4-10 C for a total of 1-2 minutes with a Branson Biosonic III vibrator equipped with a micro-attachment. The preparation is diluted with a salt phosphate solution (0.15 M NaCl and 0.01 M phosphate, pH 6.9) and the liposomes are collected by centrifugation at 60,000 xg for 30 minutes in a 50 Ti rotor in a Beckman ultracentrifuge. The pelletized liposomes are resuspended and washed twice more by centrifugation and stored at 0-5 ° C in saline phosphate solution for use in various aliquots in the cancer tests.

Macro plant fusion test

A. The macrophages used are pulmonary alveolar macrophages obtained from experimental animals, preferably rabbits. The manufacturing method follows the procedure described in the Q.N. Myrvik et al “Studies on Pulmonary Alveolar Makrophages from the Normal Rabbit”, J. of Immunology 86: 123-132 (1961) (“Examination of pulmonary alveolar macrophages from normal rabbits”). Normal adult rabbits are sacrificed by injecting 4 ml of IV veterinary nembutal. The chest is opened and 40 ml of isotonic tissue culture fluid are introduced into the lung bronchial branch through a cannula into the trachea. The entry of blood into the surgical field is carefully avoided. The washing liquid is withdrawn from the lungs and centrifuged at 1500 rpm for 20 minutes. Aliquots of the sedimented alveolar macrophages are tested for usability (viability) by staining with 0.02% trypan blue solution. Useful macrophages are suspended in RPMI-1640 without serum, washed twice and obtained after centrifugation at 2000 xg for 10 minutes. Macrophage suspensions in culture medium are counted in hemocytometers and their concentration adjusted to 9 x 106 cells / ml.

B. Lymphocytes from cancer patients and others who are being tested for cancer are obtained by venipuncture. Mononuclear cells are separated from 15 ml of heparinized or defibrinated blood from cancer patients or other persons by A. Boyum's method using a Ficoll-Hypaque solution with a density of 1.076. After pipetting off the interface cells that are retained, the underlying Ficoll-Hypaque solution is discarded. The erythrocyte pellet fraction is resuspended in medium in a volume equal to the volume of the blood originally stratified on the gradient. In order to obtain lymphocytes, this pellet fraction is pipetted onto a fresh Ficoll-Hypaque gradient (density 1.083) and centrifuged at 750 xg for 25 minutes at room temperature.

The interface fraction cells of the original 1.076 flotation layer and the interface fraction obtained from the second 1.083 Ficoll-Hy-paque flotation layer are combined and washed twice with RPMI-1640 culture medium. The sedimented lymphocytes are suspended in 30 ml of RPMI medium containing 10% heat-inactivated human serum.

The monocytes present are removed by incubating the cell suspension in 75 cm 2 culture flasks (Falcon Plastics No. 3024) at 37 ° C. for 30 minutes. Monocytes bind to the tissue culture plastic surface and the lymphocytes are washed off by rinsing with 30 ml of culture medium.

After checking the usability of the lymphocytes with the trypan blue exclusion test, aliquots of 106 lymphocytes / ml are suspended in RPMI-1640 culture medium containing 60% normal human serum, 0.1 mM L-glutamine, 40 micrograms gentamicin and 2-mercaptoethanol in 60 Micromolar concentration is supplemented.

io Single lymphocyte samples in test tubes,

16 x 100 mm, with round base and screw cap (Kimax 45066, Kimble Prod., Toledo, Ohio, USA) in aliquots of 1 ml are in a 5% C02 humidified atmosphere at 37 ° C for 24 hours with the addition of 3 to 15 to 25 Micro-15 grams of TSA / ml or with 0.5 ml aliquots of uniformly suspended cancer cell membranes / phantoms / ml, which are prepared as described under II, on page 15/16. After 24 hours, the test tubes are centrifuged (500 xg) and the cell-free supernatant is removed and replaced with 1 ml of fresh culture medium of the same composition, after which the incubation is continued for a further 48 hours. The supernatant is then aspirated, filtered through 0.22 micron Millipore filters, and if not used immediately, stored at 4 ° C.

25th

C. Obtaining normal alveolar macrophages

Healthy, non-sensitized rabbits are killed and their alveolar macrophages are obtained by washing. The alveolar macrophages are washed twice in 30 RPMI-1640 medium without serum and obtained by centrifugation at 200 xg for 10 minutes. The macro-phage cell suspensions are counted with a hemocytometer chamber and adjusted to a concentration of 9 x 106 macrophage cells / ml.

35 0.5 aliquots of the above-mentioned cell culture supernatant filtered through Millipore filters are placed separately in Waserman tubes. A 0.1 ml sample of rabbit alveolar macrophage suspension (1.5 x 105 cells) is added to each Wasserman tube. The 40 cell suspensions obtained are distributed into 5 wells of No. 3034 Mi-krotest tissue culture plates (Falcon Plastics, Oxnard, California / USA). The plates are then incubated under aseptic conditions in a CO 2 incubator (95% air, 5% CO 2) at 37 C in a humidified atmosphere and examined for macrophage fusion 45 after 24 hours and then at intervals of up to 72 hours. Instead, the lymphocyte response to antigens can be increased by culturing at 40 C as described in the publication by J.B. Smith et al “Human Lymphocyte Re-50 sponses are Enhanced by Culture at 40 C”, The Journal of Immunology, Vol. 121, No. 2, pages 691-694 (1978) ("Hu-man lymphocyte reactions are increased by culture at 40 C"). As a routine, incubation should be carried out for at least 24 hours (however, occasional incubations should be carried out up to 72 hours). In positive cultures, after 24 hours of incubation, fused macrophages or multinucleated giant cells are present at the bottom of the culture wells. The number of individual macrophages after 1 hour of culture and 60 after 24 hours, as well as the number of fused (fused) macrophages or giant cells (with more than 5 nuclei per cell) are counted and photographed at 100 times magnification. The ratios and respective numbers of single and fused macrophage cells as well as the ratios of single and giant cells are recorded.

More than 30% merged macrophages, including multinucleated giant cells, which after 24 hours of Incuba

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tion are considered to be very positive (3+) results. Tests with less than 10% fused macrophages are considered (1 +). Cultures containing 10% to 30% fused macrophages, including multinucleated giant cells, are rated as (2+). However, the presence of any number of multinucleated giant cells is a positive result for cancer of a tumor type of histology similar to the TSAs used in the test, to which the patient's lymphocytes responded by forming lymphokines that melted added macrophages.

Instead, macrophage cells can be placed in an aqueous suspension and aqueous solutions of assumed lymphokines can be added to the cell suspensions, and the speed and extent of the possible association of macrophages in such media can be monitored and quantitatively evaluated using the aggregometer test method developed by B. Rouveix et al., "A sensitive technique for measuring specific macrophage aggregation", Immunology, 36; 589-594, 1979) ("A sensitive method for measuring specific macrophage gene aggregation"). Another quantitative aggregometric method for testing for the presence or absence of lymphokines that are generated when sensitized platelets are exposed to the sensitizing antigen in aqueous medium has been described by K.J. Lavelle et al "Identification of a New Platelet Aggregating Factor Released by Sensitized Leucocytes", Clinical Immunology and Immunopathology, 3; 492-502, 1975) ("Identification of a new platelet aggregating factor that is released by sensitized leukocytes").

The aggregometric methods of Rouveix et al or Lavelle et al are preferred over the original visual method used in the study of macro-phage fusion reactions by B. Galin-do et al "Infection and Immunity" 9; 212-216, 1974).

Cy to fluorometric test

The presence of cancer-sensitive lymphocytes in peripheral blood drawn from cancer patients can be determined using the cyto-

fluorometric analysis and more specifically by flow cytofluorometry. The degree of lymphocyte stimulation by tumor TSAs is determined by the result of the method "Quantitation of Transformed 5 Lymphocytes by Flow Cytofluorimetry", Fédération Pro-ceedings, given by Braunstein et al. Vol. 34, No. 3 (1975) ("Quantitative determination of transformed lymphocytes by flow cytofluorometry") as follows:

Mononuclear cells separated from peripheral human blood by combined fractions obtained by Ficoll-Hypaque gradients as mentioned above are at 37 C or at 40 C in 5% CO 2 atmosphere in RPMI-1640, the 20 mM HEPES -Buffer and 10% normal human serum and 20 mM L-glutamine at a cell concentration of 100,000 cells / ml in the presence of TSAs in various (0.1 to 100 micrograms) amounts. The samples are harvested at intervals of up to 72 hours, fixed in 1: 1 ethanol / acetone and stained with 10 ~ 5 g ml of acridine orange in a pH 6 buffer. Cytofluorimetric 20 measurements (cytofluorograph 4801 connected to Nova 1220 minicomputer) of blank samples from stimulated cells show increased red fluorescence per cell. The percentage of transformed cells is determined by determining the number of cells with fluorescence intensity that falls outside 25 of the locus of the unstimulated lymphocytes. The percentages of stimulated cells increase with time and change with the individual optimal sensitization dose of the TSAs. A fluorescence intensity that is higher than that of unresponsive cells means a positive test for cancer, which is related to the cancer type and or histological character of the cancer with respect to the TSAs used as sensitizers.

The cancer tests described here indicate. whether the lymphocytes used in the tests originated from a person who has a malignant tumor or has had cancer of the type or histological character in the past such as the TSA used in the cancer test.

Compositions that contain highly antigenic tumor-specific substances can be applied by extracorporeal or parenteral activation of small and / or large mononuclear cells from immunocompetent human cancer patients.

C.

Claims (10)

662 573 2nd PATENT CLAIMS 1. A process for the preparation of substances with strong tumor-specific antigen activity, characterized in that living and reactive human cancer tissue, cancer cells or cancer cell membranes with a certain pathohistological character are irradiated in vitro with radio frequency or microwave radiation so that when they are in the presence of a suspension of living and reactive macrophage cells obtained from rabbit lungs are mixed with an aqueous suspension of living and reactive lymphocytes, which are obtained from a host that has cancer of the same or related pathohistological character as the irradiated cancer tissue, cancer cells or cancer cell membranes, and the mixture incubated in aqueous cell culture medium in a humidified atmosphere containing 5% carbon dioxide for 24 to 72 hours at 37 ° C, fusions between macrophages occur. 2. The method according to claim 1, characterized in that the temperature of the cancer material is increased to above 22 ° C by the irradiation with radio frequency radiation or microwave radiation. 3. The method according to any one of claims 1 to 2, characterized in that the radiation consists in the inductive or dielectric application of radio frequency or microwave radiation. 4. The method according to claim 2, characterized in that substances with strong tumor-specific antigen activity are generated in the irradiated cancer cells or cancer cell membranes and emerge or are extracted from the cancer cell material in aqueous culture medium, whereby an aqueous substance composition is formed. 5. The method according to claim 4, characterized in that the aqueous composition is dispersed with substantially dried lipoid films which lipoid films form liposome particles when an aqueous phase is dispersed therein to form liposomes which are lipoid spheres which are the Encapsulate aqueous substance composition that contains strongly antigenic tumor-specific substances. 6. The lipoid-encapsulated substance compositions, produced by the method according to claim 5. 7. A method for the detection of cancer using the substance produced by the method according to claim 3 or 5, characterized in that a lymphocyte sample of blood or tissue of a host to be examined, which is essentially free of suppressor cells, with a according to The method according to claim 3 or 5, produced, highly antigenic tumor-specific material is brought into contact and incubated in vitro in the aqueous cell culture material and that it is determined whether lymphokines have been secreted by the lymphocytes, whereby if lymphokines are secreted, the test is positive and indicates, that the host has cancer that is histopathologically related to the cancer from which the highly antigenic tumor-specific material is derived. 8. The method according to claim 7, characterized in that aqueous eluates or extracts of the irradiated human cancer tissue, cancer cells or cancer cell membranes are incubated with the lymphocytes. 9. The method according to claim 7, characterized in that the lymphocyte sample taken from the host to be examined is mixed in the presence of an aqueous suspension of macrophages with the material with strongly tumor-specific antigen activity in the form of eluates or extracts and that it is determined whether lymphokines in the Suspension have been generated so that cell fusions occur between added macrophages, indicating that the lymphocytes present in the mixture originate from a host who has or had a cancer of the pathohistologically same or similar character as the substances with highly tumor-specific antigen activity. 10. The method according to claim 9, characterized in that the lymphocyte sample taken from the host to be examined is mixed with the material with highly tumor-specific antigen activity or extracts thereof and cultivated in vitro with the addition of living and reactive macrophages obtained from rabbit lungs and that it is determined whether macrophage fusion occurs in the mixture.