Patent Application: US-44623182-A

Abstract:
gm2 is a ganglioside present on the surface of tumors and stimulates an appreciable immune response in mammals . it is useful when coupled with a non - toxic protein carrier or mixed with an adjuvent and injected parenterally of raising the anti - gm2 titer in serum . gm2 is also valuable as a diagnostic agent .

Description:
the general method for the production of the antigenic ganglioside gm2 , which is free of other antigenic determinants ( hereinafter pure gm2 ) has been set forth above . such method &# 39 ; s starting point is gm1 which is relatively easy and inexpensive to obtain . the gm1 is first dissolved in an aqueous solution . the terminal galactose residue of gm1 is removed , to form gm2 , by using an enzyme effective amount of beta - galactosidase ( preferably lysomal beta - galactosidase ). the thus formed gm2 may be separated from the solution by any of a number of methods including chromatography on silica gel . we have found that the reaction is much more efficient if it is conducted in the presence of a surfactant . we have also found that a very effective surfactant is a metal ( preferably an alkali metal ) salt of taurocholic acid . the surfactant is preferably present in an amount of between about 3 to 10 parts by weight , based on one part by weight of the starting ganglioside . in the preferred method the beta - galactosidase used is bovine testes beta - galactosidase prepared as described in reference 16 , table i . a reaction solution ( 1 . 0 ml ) is prepared containing 1 mg . of gm1 and 1 mg . of delipidized bovine serum albumin in 0 . 01 solar potassium acetate ( ph 5 . 0 ), 1 wt . % sodium taurocholate and 0 . 4 units of bovine testes beta - galactosidase . the solution is incubated at 37 ° c . the reaction is greater than 95 % in 29 hours . the reaction mixture , after 29 hours , is lyophilized and the gangliosides , gm1 and gm2 , are desalted on a column containing 1 . 0 gram sephadex g - 25 and eluted . the eluate is purified by preparative thin layer chromatography in chlorofrom : methanol : water ( 1 . 0 : 2 . 0 : 1 . 4 , vol .). pure gm2 is eluted from the silica by extraction into chloroform : methanol ( 1 : 2 , vol .) overnight at room temperature ( 25 ° c .). as noted hereinbefore the gm2 vaccine may be of two types : ( 1 ) gm2 conjugated to a non - toxic protein carrier and ( 2 ) an antigenic composition consisting essentially of pure gm2 and a stimulative immune response adjuvent such as liposome or methylated human serum albumin . the pure gm2 is conjugated to an appropriate non - toxic carrier such as human serum albumin using the oligosaccharide of gm2 which is released by ozonolysis . one mg . of gm2 is dissolved in 1 . 5 ml . of absolute methanol . ozone is bubbled through the solution at room temperature . the methanol is then evaporated off and after the methanol is removed , one ml . of 0 . 1 molar sodium hydroxide is added . after 16 hours at room temperature , the sample is neutralized by passage through a column of dowex 50w - x8 resin ( h - form ). the eluate is lyophilyzed , the residue is dissolved in 0 . 002 molar pyridine acetate buffer , ph 5 . 4 , and applied to a column of deae - cellulose equilibrated with the same buffer . the oligosaccharide is eluted with 1 . 0 molar pyridine acetate , ph 5 . 4 , and the eluate lyophilyzed . this yields about 0 . 5 mg . of the oligosaccharide portion of gm2 . the oligosaccharide is reacted with beta ( p - aminophenethyl ) ethyl amine by mixing the two together at room temperature to form a schiff &# 39 ; s base conjugate . sodium borohydride in ethanol is then added to the conjugate to reduce the conjugate , yielding an irreversible bond . after destruction of excess sodium borohydride by lowering the ph to 5 . 0 with glacial acetic acid , the ethanol is removed under vacuum and the resulting conjugate is separated from the free amine by chromatography on a column of sephadex g - 10 . the now - purified conjugate is linked with thiophosgene and after removal of the excess thiophosgene by extraction with chloroform , the isothiocyanate derivative is added to an equal volume of 0 . 3 molar sodium chloride in 0 . 1 molar sodium carbonate ( ph 9 . 5 ) containing human serum albumin . the mixture is incubated about 18 hours at room temperature and the gm2 oligosaccharide - albumin conjugate is purified by chromatography on a column of sephadex g - 75 . in order to use the conjugate as a vaccine for humans it should be dissolved in an aqueous solution which is non - toxic ( e . g . about one to 10 mg . per ml . of saline ). the solution is parenterally injected ( about 5 cc .) every two weeks until high antibody titer appears in the blood . in order to use the conjugate as a diagnostic agent for skin tests , e . g . to determine the effectiveness of the vaccine in cell mediated delayed hypersensitive , the vaccine solution is injected intradermally ( about 0 . 5 ml . per site ). after about two days the patient is examined to see if red spots appear at the injected sites , which indicate the presence of cell mediated immune response . the gm2 antigenic composition may be made by mixing pure gm2 with liposome consisting of dimyristoylphosphatidylcholine ( dmpc ), cholesterol and dicetyl phosphate in molar ratios of 2 : 1 . 5 : 0 . 22 in a ratio of 0 . 15 mg . gm2 to one mg . of dmpc . the resulting mixture is a vaccine which is used in the same way as the protein conjugate . the liposome - gm2 antigenic composition may , in general , consist of one part by weight of gm2 and from about one to 20 parts by weight of liposome or any other adjuvent known to stimulate immune response . another adjuvant very useful in the present invention is non - toxic lipid a which may be used per se with gm2 or , preferably , with the liposome - gm2 antigenic composition . for example , 5 weight parts of lipid a may be mixed with the liposome composition to produce an excellent vaccine for therapy of cancer patients or in prevention of cancer . pure gm2 is adsorbed on the walls of polystyrene microtiter plates by contacting with an aqueous phosphate buffered saline solution of gm2 ( about 1 . 5 mg . per ml .). the thus coated microtiter plates are then contacted with 0 . 05 ml . of serum from a human patient and allowed to incubate at room temperature for about twelve hours . the serum is washed off the plates with phosphate buffered saline solution and the washed microplates are then overlayed with a solution containing a marker for anti - gm2 . the plates are then incubated for an hour at 25 ° c . the overlay is removed by washing any marker remaining on the plate measured by an approximate instrument which will show the presence or absence of anti - gm2 in the serum . 1 . irie , r . f . and morton , d . l . : a new membrane antigen on human cultured cells . proc . amer . assoc . cancer res . 16 : 171 , 1975 . 2 . irie , r . f . irie , k . and morton , d . l . : a membrane antigen common to human cancer and fetal brain tissues . cancer res . 36 : 3510 - 3517 , 1976 . 3 . irie , k ., irie , r . f . and morton , d . l . : humoral immune response of patients with malignant melanomas : melanoma associated membrane antigens demonstrable by indirect membrane immunofluorsecence . cancer immunol . immunother . 6 : 33 - 39 , 1979 . 4 . irie , r . f . : oncofetal antigen ( ofa - i ): a human tumor - associated fetal antigen immunogenic in man . in : serclogic analysis of human cancer antigens ( s . rosenberg , ed .) academic press , new york , 1980 , pp . 493 - 513 . 5 . rees , w . v ., irie , r . f . and morton , d . l . : oncofetal antigen ( ofa - i ): distribution in human tumors . j . natl . cancer inst . 67 : 557 - 562 , 1981 . 6 . irie , r . f ., giuliano , a . e . and morton , d . l . : oncofetal antigen ( ofa ): a tumor associated fetal antigen immunogenic in man . j . natl . cancer inst . 63 : 367 - 373 , 1979 . 7 . sidell , n ., irie , r . f . and morton , d . l . : immune cytolysis of human malignant melanoma by antibody to oncofetal antigen - i ( ofa - i ). ii . antibody - dependent cell - mediated cytotoxicity . cancer immunol . immunother . 9 : 49 - 54 , 1980 . 8 . sidell , n ., irie , r . f . and morton , d . l . : oncofetal antigen : a target for immune cytolysis of human cancer . brit . j . cancer 40 : 950 - 953 , 1979 . 9 . sidell , n ., irie , r . f . and morton , d . l . : immune cytolysis of human malignant melanoma by antibody to oncofetal antigen - i ( ofa - i ). i . complement dependent cytotoxicity . cancer immunol . immunother . 8 : 211 - 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