THE USE OF A SPECIFIC CARCINOM ASSOCIATED ANTIGEN (HAPTEN), FUCOSYLSIALOSYLGANGLIOTETRAOSE (FUC-GM1) IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES RELATED TO HUMAN LUNG CANCER (SMALL CELL CARCINOMAS)

Abstract: This invention relates to the use of a specific carcinoma associated antigen/hapten of carbohydrate nature, fucosyl­ sialosylgangliotetraose-IV²αFucll³-αNeuAcGgOse₄-(IUPAC­ IUB Lipid Document, 1977) for diagnostic and therapeutic procedures related to human lung cancer (small cell carci­ nomas).

Description: Field of the Invention This invention relates to the use of a specific carcinoma associated antigen/hapten of carbohydrate nature, fucosylsialosylgangliotetraose-IV²αFucII³αNeuAcGgOse₄-(IUPAC-IUB Lipid Document, 1977) for diagnostic and therapeutic procedures related to human lung cancer (small cell carcinomas). Background It is well established that the transformation of normal tissue cells to tumor cells is associated with a change of carbohydrate structures (glycoconjugates) on the cell surface. These new or modified glycoconjugates may serve as antigens and may then represent a type of so-called tumor-associated antigens. The cell surface carbohydrates are linked either to a lipid moiety, in which case they are called glycolipids, or to peptides (proteins), in which case they are called glycopeptides (glycoproteins). Tumor-associated glycoconjugate antigens are previously known in relation to human cancer disease. In melanomas the carbohydrate structures disialosylgangliotetraose and disialosyllactose have been identified linked to a lipid portion. Two carcinoma associated antigens CEA ( c arcinoembryonal a ntigen) and GlCA ( g astro i ntestinal c ancer a ntigen) have been demonstrated particularly in gastrointestinal carcinomas while a third antigen, CA-50, seems to have a more general distribution. All the antiqens are shedded from the cell surface or secreted from the tumor cell and can be demonstrated in blood serums by means of immunological methods. The present invention is based on the discovery that patients with certain forms of lung cancer (small cell carcinomas or oat-cell carcinoma) express in the primary lung tumor and its metastases an acidic glycolipid, which is not expressed in normal lung tissue. This glycolipid antigen is a ganglioside with the complete chemical structure Fucα1-2Galβ1-3GaINAcβ1-4(NeuAcα2-3)Galβ1-4Glc-Cer or IV²αFucII³αNeuAcGgOse₄Cer with the short designation Fuc-GM1 (IUPAC-IUB Lipid Document, 1977). This ganglioside was first isolated from bovine brain (Ghidoni et al., J. Neurochem. 27, 511-515, 1976) but indications for its occurrence in other mammalian organs as adipose tissue, spleen and testis have also been presented. Hakomori and co-workers (Baumann et al, Cancer Res. 39, 2637-2643, 1979) isolated from rat hepatomas a ganglioside which was tentatively identified as Fuc-GM1. A definitive establishment of the chemical structure of this ganglioside was performed by Svennerholm and collaborators (Fredman et al., Eur. J. Biochem. 116, 553-564) on gangliosides isolated from porcine nervous tissue. They also showed that chronic chloroquine treatment of the pig caused an increased lysosomal storage of Fuc-GM1 in nervous tissue (Klinghardt et al., J. Neurochem. 37, 897-908, 1981) but also in several other organs (Fredman et al., Biochem. J. 201, 581-588, 1982). Although this ganglioside has a wide distribution in pig, we have not been able to show its occurrence in normal human lung tissue or in carcinomas from a large number of human organs. In patients, died in lung small cell carcinoma, all lung tumors contained Fuc-GM1 ganglioside, and in other patients Fuc-GM1 was shed to blood serum. With the assay method for Fuc-GM1 developed by us the tumor antigen was detected on the blood serum in patients with lung small cell carcinoma but not in the blood serum of a control population or of patients with other forms of cancer. Within the scope of the present invention procedures have been elaborated for the isolation of the tumor associated glycolipid, fucosylsialosylgangliotetraosylceramide with the short designation, ganglioside Fuc-GM1, in pure form from human cancer tissue and from animal organ. The purified ganglioside has by a special device obtained an increased immunogenic activity and then been used for the production of monoclonal antibodies. Procedures have, within the scope of the invention, been developed for the identification and quantitative determination in tissues, tissue sections, cytological preparations and various body fluids. All of these methods for diagnostic purposes are based on the fact that the antigen can be detected and quantitatively determined by the reaction with specific antibodies produced against the pure ganglioside antigen, Fuc-GM1. The following non-limiting examples illustrate the various sources where the ganglioside antigen can be measured: surgical or autopsy material of lung, liver, brain or similar organs, cytological specimens obtained by biopsy of lung or other organs, where signs of cancer metastases can be suspected, cells, obtained by aspirations of the respiratory tract, expectorations, or by puncture of pleural or abdominal cavity, biological fluids like whole blood, blood plasma or serum, lymph, cerebrospinal fluid, urine, saliva or similar. The antigen which is used in the present invention is termed, fucosylsialosylgangliotetraose and has the following chemical structure: Fucα1-2Galβ1-3GaINAcβ1-4(NeuAcα2-3)Galβ1-4Glc or IV²αFucII³αHeuAcGgOse₄ [IUPAC-IUB Lipid Document, 1977). Within the scope of the present invention all antigens, which have been modified by the removal of the terminal glucose moiety or by the substitution of the sialic acid (NeuAc) moiety to the N-acetylgalactosamine (GaINAc) moiety instead of to the galactose (Gal) moiety are covered by this patent application. The monoclonal antibodies referred to in the present invention have all been produced from antigens bound to a lipid group (acylsphingosine) but this claim will also include the binding of the antigenic group to every other compound. The antigen which has been used in the present invention can be isolated from tumor tissue, obtained by surgery or autopsy of patients with lung small cell carcinoma. The concentration of the antigen in the tumor tissue is in general relatively low, particularly when the patient has been treated with cytostatic drugs, and the isolation of the antigen will then be laborious. We have also found another biological material, which can be used for the isolation of the cancer antigen. When a certain strain of miniature pig is foddered with chloroquine in the diet during 3-6 months, high concentrations of the ganglioside antigen is formed in the pig's central nervous system, particularly In the dorsal root ganglia. The cancer associated ganglioside antigen can then be relatively simply isolated from brain and dorsal root ganglia. By this device one is not limited for the isolation of the antigen to surgery or autopsy material and a large number of practical and ethical difficulties are avoided. It has previously been found that gangliosides - it means glycolipids containing sialic acid - are poor immunogens and it has been difficult to produce antibodies with high affinity and specificity against them. This difficulty has been circumvented in the present invention by the adsorption of the ganglioside antigen to a bacterial membrane with known high immunogenic activity. By this device a large number of monoclonal antibodies with high specificity and affinity against the cancer antigen have been produced. The present invention concerns in accordance herewith a procedure for the induction of the cancer associated antigen fucosylsialosylgangliotetraose in the nervous system of a particular type of miniature pig, isolation of the antigen with chromatographical methods and production of monoclonal antibodies against the purified antigen adsorbed to a certain bacterial membrane. The invention includes also the use of the compound fucosylsialosylgangliotetraose and its above mentioned derivates and all types of antibodies specifically directed against them for the diagnosis of small lung cell cancer, and for the treatment of the cancer disease in patients. Example 1. ISOLATION OF FUCOSYLSIALOSYLGANGLIOTETRAOSE ANTIGEN IN THE FORM OF FUC-GM1 GANGLIOSIDE FROM MINIATURE PIG, TYPE GÖTTINGEN Chloroquine treatment of Pig Miniature pigs, type Göttingen, with a weight of 20-30 kg, were given a diet, in which the regular fodder was supplemented with 30 g chloroquine diphosphate per kg fodder. The different ingredients were mixed under the addition of water and after careful mixing the fodder was pelletted and dried. The pigs were given this fodder for 2 months and were then slaughtered (bleeding under narcosis). The whole nervous system was collected, including retina and dorsal root ganglia. Brain and dorsal ganglioa + retina were processed separately, since dorsal ganglia and retina had approx. 20 times higher concentration of Fuc-GM1 than brain. Extraction of gangliosides from tissue The tissue was homogenized after the addition of 3 volumes of water to one volume of tissue material. After homogenization with a knife homogenizer at 1500 rpm during 3 min., 10 volumes of methanol was added under continuous stirring. Then 5 volumes of chloroform were added. After extraction for 1 h the clear supernatant was isolated by centrifugation at 2000 rpm. To the supernatant was added water to give a final chloroform-methanol-water ratio 4:8:5.6 (by volume). After mixing in a separatory funnel two distinct phases appeared. The upper phase was collected and evaporated to dryness after the addition of 0.1 volume butanol to prevent foaming. The evaporation was performed with a rotaratory evaporator under gentle warming on a water bath, the temperature not exceeding 50°C. The residue was added 0.5 volume methanol per volume original tissue and 0.5 volume of 1.0 M potassium hydroxide and was left overnight at room temperature. The alkaline solution was neutralized to pH 8 with 1.0 M hydrochloric acid and was then allowed to dialyse against running tap water for 48 hours. Isolation of a monosialoganglioside fraction by anion exchange chromatography The dialysed crude ganglioside fraction was evaporated (lyophilized) and dissolved 1 volume of chloroform-methanol-water with a volume ratio of 60:30:4.5 per volume original tissue. A column (inner diameter 20 mm) was packed with Spherosil-DEAE-dextran, 0.2 volume of resin per volume of original tissue. The dialysed ganglioside extract was added slowly to the column and the column was then eluted with 10 bed volumes of chloroform-methanol-water (60:30:4.5, by volume). The monosialoganglioside fraction was eluted with 10 bed volumes of 0.02 M potassium acetate in methanol. The monosialoganglioside fraction was evaporated and desalted by dialysis against distilled water. Purification of Fuc-GM1 by silica gel chromatography For the chromatography 1 g of silica gel (latrobeads ®) was used for each g of original nervous tissue. The gel was suspended in chloroform-methanol (volume ratio 4:1) and poured into a glass column with a sintered glass filter and a diameter of 15-20 mm depending on the amount of gel. The ganglioside fraction was dissolved in 10 ml of chloroform-methanol-water (65:25:4, by volume) and applied to the column. It was eluted first with 15 ml of chloroform-methanol-water (65:25:4, by volume) for each g (weight/volume) of gel, which was collected in one fraction. The elution continued then with chloroform-methanol 2.5 M ammonia (60:40:9, by volume) and 5 ml fractions were sampled on a fraction collector. The elution was monitored by testing 10 µl of each fraction by high performance thin-layer chromatography with the same solvents as used for the column chromatography, chloroform-methanol-2 M ammonia (60:40:9) and the gangliosides were detected with resorcinol reagent. The fractions which contained Fuc-GM1 were pooled and evaporated to dryness. The final purification of Fuc-GM1 was achieved by preparative thin-layer chromatography. Ganglioside (5 µmoles) was applied on a thin-layer plate (20x20 cm, 0.25 mm thick, Merck, AG., Darmstadt), which was developed for 60 min with chloroform-methanol-2.5 M ammonia at 21°C. After completed chromatography the plate was sprayed with 0.01% bromthymol blue in water and the Fuc-GM1 band was scraped off. The ganglioside was eluted from the gel with chloroform-methanol-water (30:60:20, by volume) and was added one drop of 0.1 M potassium hydroxide dissolved in methanol and evaporated to dryness. The pure ganglioside Fuc-GM1 was kept dry in a vacuum desiccator or was dissolved in a small volume of chloroform-methanol (2:1, by volume) and was kept in a teflon capped tube in the dark at +4°C. Example 2. PRODUCTION OF MONOCLONAL ANTIBODIES DIRECTED AGAINST FUCOSYLSIALOSYLGANGLIOTETRAOSE BY MEANS OF GANGLIOSIDE FUC-GM1 The purified ganglioside Fuc-GM1 from Example 1 was adsorbed to acid-washed Salmonella Minnesota bacteria before immunization according to the principle, described by Young, W.W. and collaborators (J. Exp. Med. 150, 1008-1019, 1979). Balb/c mice, 6-8 weeks old, were immunized intravenously with 5 µg of ganglioside Fuc-GM1, adsorbed to 50 µg acid-washed Salmonella Minnesota bacterial membranes, suspended in 100 µl of phosphate-buffered physiological saline, pH 7.4. A new immunization was performed after two weeks with the same dose of immunogen under identical conditions (booster dose). Three days after the booster dose, the spleen cells taken from the immunized mice were fused with mouse Sp 2/0 myeloma cells. The resulting hybridomas were expanded, tested and cloned according to the protocol, devised by de St. Groth and Scheidegger (J. Immunol. Methods, 1-21, 1980). The culture medium was collected from each microtiter well with growing hybridomas to determine those secreting antibody specific for Fuc-GM1. The determination was performed in an ELISA system according to the following procedure: Fuc-GM1, 50 pmol dissolved in 50 µl of methanol, was added to the wells of a polyvinylmicrotiter plate and evaporated to dryness. The wells were incubated overnight with 100 µl of culture medium diluted 1:2 with 0.01 M phosphate buffered saline (0.14 M) (PBS), pH 7.5. After thorough washing with PBS the wells were incubated for 4 h with 100 µl of peroxydase (HRP)-conjugated rabbit anti-mouse immunoglobulin diluted 1/200 on PBS-1% bovine serum albumin (BSA). The absorbance at 450 nm was determined in a Titertek® microtiter scanner (Flow Laboratories, USA) after incubation for 5 min with 50 µl of substrate (0.1% ortophenyldiamine in 0.1 M citrate buffer, pH 4.5, mixed with 0.3% H₂O₂). Hybrids were regarded as positive if the absorbance exceeded that of the mean medium blank by a 2-fold or more. Medium from positive hybrids was collected for further screening and the hybrids were frozen in liquid nitrogen. Culture medium from hybrids positive in the initial ELISA screening against Fuc-GMl was tested against normal human lymphocytes for cross-reactivity. Human lymphocytes (HuLy-c) were isolated by centrifugation on Lymphopre® of blood from five donors of all ABH-blood groups, pooled and used as antigen in an ELISA-test as described by Lindholm et al. (infect. Immun. 40, 570-576, 1983). Hybrids that produced antibodies cross-reacting with HuLy-c were not further expanded. The Fuc-GM1 positive/HuLy-c negative hybrids were cloned by limited dilution and expanded. The culture medium was collected and used for additional tests without further processing. The isotype of the antibodies produced by each clone was determined by single radial immunodiffusion in agarose containing isotype specific antimouse immunoglobulins. The Fuc-GM1 positive/HuLy-c negative hybridomas were cloned and screened to determine those secreting antibody specific for Fuc-GM1. The determination was performed in a double antibody solid phase radioimmuno assay. Fuc-GM1 and a large number of different neutral glycolipids and gangliosides were dissolved in methanol and pipetted into the wells of microtiter plates. After evaporation of the solvents the antigen coated wells were incubated with the monoclonal antibodies. Bound antibody was detected with 125 I-labelled antimouse immunoglobulin; 125 I-antimouse Igm or 125 I-antimouse IgaF(ab)₂ fragment. Each monoclonal antibody was tested against Fuc-GM1; a standard mixture of human brain gangliosides, normal lung tissue, neutral glycolipids and gangliosides. The specificity of the monoclonal antibodies were also determined by thin-layer chromatographic immunostaining of the glycolipid antigens. The total neutral and acidic glycolipid fractions from a number of organs were separated on aluminia-backed high performance thin-layer plates (HPTLC) and incubated with the monoclonal antibodies of the Fuc-GM1 positive hybridomas, diluted with Tris buffer-1% BSA (0.05 M Tris, 0.14 M NaCl, pH 7.8, containing 10 g bovine serum albumin/l and 100 mg merthiolate/l). Bound monoclonal antibody was detected with ¹²⁵I-labelled antimouse immunoglobulin and then exposing it to X-ray film for 12-24 h. When different monoclonal antibodies were tested on the same HPTLC-plate the antibodies were applied to the plate by soaking cellulose acetate strips in the antibody solution and covering one lane of the HPTLC-plate with the strip. Only the antibodies which showed an exclusive specificity for Fuc-GM1 have been used in the subsequent studies. Example 3. DETERMINATION OF FUCOSYLSIALOSYLGANGLIOSYLTETRAOSE ANTIGEN IN LUNG SMALL CELL CARCINOMA The same principle method was adapted as described in Example 1, but the method has been modified to be optimal for small tissue samples. Tissue, approx. 0.1 g, was homogenized together with 0.5 ml of water in a small conical tube tissue grinder with teflon pestle in an ice bath. After finished homogenization 1.6 ml of methanol and 0.8 ml of chloroform were added. After thorough mixing the tube was centrifuged at 800xg for 10 min. The clear supernatant was transferred to a Kimax-tube with teflon cap. The sediment was suspended in 0.5 ml of water and then 1.6 ml of methanol and 0.8 ml of chloroform were added. Mixing and centrifugation were performed as before. The two supernatants were combined and water was added to give a final chloroform-methanol-water ratio in:8:5.6 (by volume). After mixing the tube was centfigued at 100xg for 5 min. The upper phase was transferred to a small round flask with ground neck and after addition of 0.1 volume of isobutanol, the supernatant was evaporated. The residue was the heated with 0.1 M sodium hydroxide in methanol for 1 h at room temperature. After finished transesterification 4 ml of chloroform and 1 ml of methanol were added and the content was poured into a small column with 1 g of Sephadex G 25, packed in chloroform-methanol-water 60:30:4.5 (by volume). The eluate was slowly added to a column with 1 .0 g of DEAE-Sepharose® (Pharmacia Fine Chemicals, Uppsala, Sweden) in acetate form. After rinsing the column with 10 ml of methanol, the tumor antigen was eluted with 0.02 M potassium acetate in methanol. After evaporation and resolution in chloroform-methanol-water 65:25:4 (by volume) the simple monosialogangliosides were separated from the antigen fraction by elution with 15 column volumes of 65:25:4 (by volume), whereafter the antigen fraction was eluted with 10 volumes of chloroform-methanol-water 50:40:10 (by volume). A portion of the antigen fraction was dissolved in methanol and 50 µl of extract was pipetted into the wells of a polyvinyl microtiter plate. Standard solutions of 0.5 to 5 pmol of pure Fuc-GM1 in methanol was added to other wells. The solvent was evaporated under a stream of nitrogen. To the unknown samples and standards 10-100 µg of monoclonal antibody, specific for Fuc-GM1 and produced as described in Example 2, dissolved in PBS/BSA was added. The samples were incubated for 1 h at room temperature. The wells were washed 3 times with PBS solution and then ¹²⁵I-labelled antimouse-immunoglobulin was added and allowed to react for 3 hours. After repeated washing with PBS the wells with unknowns and standards were cut out from the microtiter plate and counted in the gamma counter. The concentration of fucosylsialosylgangliotetraosyl antigen of the unknowns were calculated from the standard curve of pure Fuc-GM1. Under the described conditions Fuc-GM1 has been detected in 19/20 lung small cell carcinoma. The antigen has not been identified in other forms of lung carcinomas (squamous cell, large cell or adenocarcinoma) neither in carcinomas of mammary gland, stomach, colorectal mucosa, urinary bladder or uterus. Traces of the antigen has been shown in normal and carcinomatous pancreatic tissue. Example 4. DETECTION OF FUCOSYLSIALOSYLGANGLIOTETRAOSE ANTIGEN IN BLOOD SERUM OF PATIENTS WITH LUNG SMALL CELL CARCINOMA The following procedure was used for the demonstration of the fucosylsialosylgangliotetraose antigen in blood serum or plasma. 1.0 ml of serum was slowly dropped into a small test tube with 4 ml of methanol under continuous agitation. Then 2 ml of chloroform was added and the content thoroughly mixed and centrifuged at 800xg for 10 min. The clear supernatant was transferred to a new tube and the solvent evaporated in a stream of nitrogen under gentle warning on a water bath of 40°C. The residue was dissolved in 5 ml of chloroform-methanol-water 60:25:4 (by volume) and put into a small column (inner diameter 8-10 mm) with 1 g of silica gel (Silica Gel 60, 230-400 mesh Merck AG, Darmstadt, FRG). The column was eluted with 20 ml of chloroform-methanol-water 65:25:4, (by volume), and then the tumor antigen was eluted with 10 ml of chloroform-methanol-water 50:40:10 (by volume). After evaporation, dialysis for two days against water, and lyophilization, the residue was dissolved into 5 ml of chloroform-methanol-water 60:30:4.5 (by volume) and added slowly to a small column with 1 g of DEAE-Sepharose in acetate form. The column was eluted with 10 ml of methanol and then the tumor antigen was eluted with 10 ml of 0.02 M potassium acetate in methanol. The salt was removed by dialysis and after evaporation the residue was dissolved in a small volume of methanol. The concentration of tumor antigen was determined with a double solid phase antibody method, at which the antigen was adsorbed to the solid phase and determined with the specific monoclonal antibody (Example 2) and antimouse-immunoglobulin. Fuc-GM1 (20 pmol) and the purified antigen fraction of unknown serum samples were dissolved in methanol and serial dilutions of them (112-112048) were pipetted in the wells of polyvinyl microtiter plates (Flow Laboratories art.nr. 77-173-05) and were evaporated. Unspecific binding of the wells was blocked with 20 µl of 1% BSA in PBS and then 50 µl of specific monoclonal antibody, diluted in 1% BSA in PBS, was added. After incubation for 4 hours, the wells were rinsed 3 times with 200 µl of PBS. After the rinsing rabbit antimouse immunoglobulin labeled wiih 125 I, peroxydase or β-galactosidase was added. Measurement of the antigen amount was performed in a gamma counter or after addition of enzyme substrate (ortophenyl diamine, see Example 2), or methylumbelliferyl-β-galactoside in citrate buffer, pH 4.2) reading of the absorbance in a spectrometer or spectrofluorimeter, respectively. The concentration of cancer antigen in blood serum was calculated from the standard curve of Fuc-GM1. Under the conditions specified above, the antigen was only detected in blood serum of patients with lung small cell carcinoma.

Claims: Diagnostic method in vitro to detect in a patient a inng small lung cell carcinoma (= oat cell carcinoma) or its metastases, characterized by the occurrence of (i) fucosylsialosylgangliotetraose (= Fucul-2Galβ1-3GalNAcβl-4(NeuAcα2-3)Gaβ1-4Glc), (ii) fucosylsialosylgangliotetraose in which the terminal glucose is removed or the sialic acid (NeuAc) moiety being substituted on the N-acetylgalactosamine (GalNAc) moiety instead of on the galactose (Gal) moiety, and (iii)the compounds of (i) or (ii) bound in form of a glycolipid, glycoprotein or glycopeptide. in a specimen from the patient being taken as an indication of the patient having small lung cell carcinoma or metastases thereof. Diagnostic method according to claim 1, characterized by the occurrence of said compound being detected immunochemically by the means of an antibody directed thereagainst, preferably a monoclonal antibody. Diagnostic method according to claim 2, characterized by said compound being detected immunochemically on cellular level in a histological preparation. Use of an antibody being specifically directed against (i) fucosylsialosylgangliotetraose (= Fucal-2Galβ1-3GalNAcβ1-4(NeuAcα2-3)Gaβ1-4Glc), or (ii) fucosylsialosylgangliotetraose in which the terminal glucose is removed or the sialic acid (NeuAc) moiety being substituted on the N-acetylgalactosamine (GalNAc) moiety instead of on the galactose (Gal) moiety, or (iii)the compounds of (i) or (ii) bound in form of a glycolipid, glycoprotein or glycopeptide. for the manufacture of a composition for the treatment and diagnosis of small lung cell carcinoma.

Inventors: HOLMGREN, JAN ROLAND, LINDHOLM, LEIF GUSTAV, SVENNERHOLM, LARS TORVALD
Applicants: PHARMACIA AB
Application year: 1985
Main classification: A61K39
IPC codes: A61K39, A61K51, G01N33, C07H15, C07K16, C07H13, C07K14