Patent Application: US-30158902-A

Abstract:
the present invention provides two unique monoclonal antibodies directed against a portion of the human glycine n - methyltransferase , and methods of use for the monoclonal antibodies in detecting , monitoring and diagnosing malignancies characterized by down - regulation of expression or inappropriate expression of the glycine n - methyltransferase .

Description:
glycine n - methyltransferase ( gnmt , ec 2 . 1 . 1 . 20 ) was originally revealed to be an enzyme regulating the ratio of s - adenosylmethionine ( sam ) to s - adenosylhomocysteine by catalyzing the synthesis of sarcosine from glycine and sam [ kerr s j , et . al . 1972 , ogawa h , et . al . 1982 ]. gnmt is conservative among different animal species [ bork p . et . al . 1992 , chen y m . et . al . 1998 , lai m c . et . al . 1999b , ogawa h . et . al . 1993 ]. in halophilic methanoarchaea , gnmt plays a major role in osmoregulation [ lai m c . et . al . 1999a ]. in rabbit and rat livers , gnmt comprises 1 - 3 % of the cytosolic proteins , and it has been suggested to play an important role in the metabolism of methionine [ cook r j , et al . 1984 , heady j e , et al . 1975 , ogawa h , et . al . 1982 ]. previously , through mrna differential display and northern blot analysis of liver tumors and non - tumorous liver tissue from human hepatocellular carcinoma ( hcc ) patients , we reported that the expression level of gnmt was diminished in tumorous tissue and hcc cell lines [ chen y m , et al . 1998 ]. subsequently , the human gnmt gene was isolated , sequenced and mapped to chromosome 6p12 [ chen y m , et al 2000 ]. additionally , genotypic analyses of different polymorphisms of the human gnmt gene demonstrated that 36 - 47 % of the genetic markers showed loss of heterozygocity in tumorous tissues of hcc patients [ tseng t l , et al .]. functional characterization of gnmt showed that gnmt is able to bind benzo ( a ) pyrene ( bap ) and decrease bap - dna adduct formation [ chen s y , et al ., raha a , et al . 1994 ]. therefore , gnmt can be classified as a tumor susceptibility gene . furthermore , gnmt has been shown to have diverse functions and the ability to compete with the ah ( dioxin ) receptor for binding benzo ( a ) pyrene ( bap ), down - regulating bap - dependent cytochrome p450 1a1 expression and decreasing bap - dna adduct formation [ chen s y , et al ., krupenko n i , et al .]. that the activity of gnmt is significantly decreased in rat hepatoma has also been shown [ houser w h , et al . 1985 ]. in addition , a rat hepatoma model induced through n - 2 - fluorenylacetamide , the gnmt enzyme activity gradually decreased and became undetectable in the liver tumor after 8 months of treatment [ zhang y j , et al . 1991 ]. from these studies , we can conclude that the down - regulation of the gnmt gene expression is present in both naturally - occurring and carcinogen - induced liver cancers . recently , mudd et al . reported that two italian siblings who had mild hepatomegaly and chronic elevation of serum transaminases were diagnosed to have gnmt deficiency [ mudd s h , et al 2001 ]. both children had compound heterozygotes of the gnmt gene with missense mutations [ luka z , et al 2002 ]. therefore , the present invention fuirther provides monoclonal antibodies for detecting , monitoring and diagnosing the decreased level of gnmt by immunohistochemistry and eia methods . all of the documents or publications recited in the text are incorporated herein by reference . that the gnmt gene is differentially expressed between normal and tumorous cells with a significant distinction is surprisingly found in the present invention . an objective of the present invention is to provide a method of detecting abnormalities of cells by determining the relative levels of gene expression of gnmt . furthermore , another objective of the present invention is to provide a method of correcting the abnormalities of cells by delivering gnmt into the abnormal cells . in this invention , we used monoclonal antibodies against gnmt to establish a sensitive method to monitor the correlation between the gnmt in serum or plasma and the pathogenesis of hcc formation . the polyclonal antibodies against human gnmt as capture antibody were used to capture the human gnmt in serum or plasma , and further analyze by monoclonal antibody grl1 as indicating antibody . we setup a quantitative enzyme immunoassay to measure the quantity of human gnmt in blood . additionally , the monoclonal antibodies posses immunogenic epitopes that can be used as a reference to make synthetic peptides for the generation of antisera or for the production of other diagnostic assay . further details of this invention are illustrated in the following examples . for the construction of the pgex - gnmt , a full - length gnmt cdna fragment was cleaved from pbluescript - gnmt - 9 - 1 - 2 phagemid dna [ chen y m , et al . 1998 ] by using smai and saii restriction enzymes ( stratagene , la jolla , calif ., usa ). this 1 . 2 - kb dna fragment was ligated to a vector , pgex - kg [ guan k l , et al . 1991 ] that had previously been digested with smai and xhoi . for the construction of pgnmt - his , as shown in fig1 b , plasmid pcmv - gnmt [ chen s y , et al .] was used as a template in the polymerase chain reaction ( pcr ). a 1 . 2 - kb dna fragment containing gnmt cdna sequence and restriction enzyme sites on both ends was amplified . twenty pcr cycles were performed in a dna thermal cycler ( perkin elmer cetus , foster city , calif ., usa ) using their amplitaq gold taq dna polymerase . the upstream primer ( 5 ′- gaggaattcatggtggacagcgtgt ac ) consisted of a 3 - bp “ clamp ” ( gcg ) at the 5 ′ end , followed by one restriction enzyme site ( ecori ) and a gnmt cdna sequence . the downstream primer ( 5 ′- gcgctcgaggtctgtcctcttgagcac ) contained a similar structural sequence motif as the upstream primer , except that it consisted of negative strand sequences from the terminal region of gnmt cdna and a different restriction enzyme site ( xhoi ). after the pcr , the 1 . 2 - kb dna fragment was gel - purified , digested with ecori and xhoi and ligated to a vector pet29a ( novagen , inc ., madison , wis ., usa ) that had been digested with the same pair of restriction enzymes . the dna sequences of both plasmids were confirmed by automated dna sequencing using an abi prism dye terminator cycle sequencing core kit ( perkin - elmer cetus ). the escherichia coli strain jm109 or bl21 was used as the recipient in the transformation and expression experiments for pgst - gnmt . in addition , plasmid pgex - kg [ guan k l , et al . 1991 ] was used for the induction and purification of glutathione s - transferase ( gst ) protein that served as a parallel control in the enzyme immunoassay . all the gst , gst - gnmt and gnmt - his recombinant proteins ( rps ) were induced in jm109 or bl21 cells through isopropyl - beta - d - thiogalactopyranoside ( iptg ). the transformants of gst - gnmt and his - gnmt had induction optical densities of 0 . 6 - 0 . 7 , 0 . 7 - 0 . 8 , respectively . their respective induction times were 3 . 5 hours and 1 hour . both the gst and gst - gnmt rps were purified using glutathione - sepharose 4b beads ( pharmacia , uppsala , sweden ), as described by guan et al . [ guan k l , et al . 1991 ]. the gnmt - his rp was purified using ni 2 + - charged histidine ( his )- bind resin column according to the procedures provided by the manufacturer ( novagen ). the bound gst - gnmt - rp was eluted from the glutathione - sepharose 4b beads using a 5 mm reduced glutathione buffer . the thrombin digestion method was used to purify gnmt rp from the bead - bound gst - gnmt fusion protein . concentrations of the rps were measured using the pierce bca protein assay reagent ( pierce , rockford , ill ., usa ) and purity was analyzed by running the samples on a 12 . 5 % sds - polyacrylamide mini - gel ( bio - rad laboratories , richmond , calif ., usa ). 1 . 4 . production of a rabbit anti - gnmt antiserum to raise rabbit antibodies against gnmt , purified gst - gnmt rp was mixed with freund complete ( for the initial immunization ) or incomplete ( for the booster injections ) adjuvant ( sigma co ., st . louis , mo ., usa ) and the resultant mixture was used as an immunogen to inoculate 8 - week - old nzw rabbits ( 150 - 200 μg rp per rabbit ) subcutaneously . rabbits received booster injections every 3 weeks after the initial injection with additional doses of the same rp . rabbit sera were collected before the immunization and 1 week after each injection . all sera were heat inactivated at 56 ° c . for 30 min and stored at − 20 ° c . murine mabs were produced by a hybridoma technique commonly used in our laboratory . briefly , balb / c mice were immunized with purified gst - gnmt and gnmt - his rps mixed with complete ( for primary immunization ) or incomplete ( for booster injections ) freund adjuvant ( sigma ), at 10 days intervals by intra - peritoneal ( i . p .) injection with a dosage of about 25 μg of rp per inoculum . serum samples were collected from the tail vein before the immunization and 1 week after each injection . three days after the last intravenous injection ( i . v .) of rp , a fusion of the splenocytes of immunized mice with mouse myeloma cells ns1 ( american type culture collection , rockville , md .) was performed with peg1500 ( roche diagnostics gmbh , mannheim , germany ), and cultured on ninety - six - well plates [ chu t m , et al 1993 ]. the culture supernatants were screened using the gst / gnmt eia and also with wb strips blotted with gst , gst - gnmt , gnmt - his rp . selected hybridoma cells were expanded and cloned at least twice by limiting dilution , and grown as ascitic tumors in 0 . 5 ml pristine ( sigma )- primed balb / c mice . mabs were purified and concentrated in protein - a antibody purification kits ( pro - chem inc . acton , mass ., usa .) and centricon plus - 80 columns ( millipore , bedford , mass ., usa ). the hybridoma cell line was cultured in rpmi 1640 medium ( gibco - brl , gathersburg , md ., usa ) supplemented with 10 % heated - inactivated fetal calf serum , 2 mm l - glutamine , penicillin ( 100 iu / ml ) and streptomycin ( 100 iu / ml ) as described in [ chu t m , et al . 1993 ]. two human hepatoblastoma cell lines - hepg2 [ aden d p , et al 1979 , javitt n b . 1990 ], huh 6 [ nakabayashi h , et al . 1982 ], and five hcc lines huh 7 [ nakabayashi h , et al . 1982 ], ha22t [ chang c , et al 1983 ], plc / prf / 5 [ luka z , et al 2002 ], hep3b and sk - hep1 [ aden d p , et al . 1979 , fogh j , et al . 1976 , fogh j , et al . 1977 ] were used in this study . these cells were cultured in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( gibco brl , grand island , n . y .) with 10 % heat - inactivated fetal bovine serum ( hyclone , logan , utah ), penicillin ( 100 u per ml ), streptomycin ( 100 μg per ml ), nonessential amino acids ( 0 . 1 mm ), fungizone ( 2 . 5 mg per ml ), and l - glutamine ( 2 mm ) in a humidified incubator with 5 % co 2 . eia was used to monitor the antibody titers of the immunized animals and to screen for mabs in the supernatant of different hybridomas . ninety - six - well plates , coated with either gst - gnmt or gnmt - his rp at a concentration of 1 μg per ml ( 100 μl per well ) were used . antibody titers of either rabbit or mouse serum were determined at a serial 10 - fold dilution . to screen the hybridomas from animals immunized with gst - gnmt rp , eia plates coated with gst were also used to rule out those mabs reactive with the gst . additionally , to screen the hybridomas from animals immunized with gnmt - his rp , gst - gnmt - coated plates were used to confirm those positive clones screened by the gnmt - his - coated plates . a rabbit anti - gnmt antiserum ( r4 ) and multiple mouse anti - gnmt anti - sera were used as the positive controls in the eia . details of the procedures have been described previously [ chen y m , et al . 1991 ]. the concentrations of mabs were determined by mouse igg eia quantitation kits ( bethyl laboratory , montgomery , tex ., usa ) and the immunoglobulin concentrations were analyzed using eia eix808 readers with 4 - parameter logistic regression ( bio - tek instruments , winooski vt ., usa ). all the isotyping and light chain determination was done using mouse immunoglobulin isotyping elisa kits ( bd biosciences pharmingen , san diego , calif ., usa ). the antibodies were diluted with 0 . 1m nahco 3 ( ph 8 . 6 ) to a concentration of 100 μg / ml , and added to 5 ml sterile polystyrene petri dishes . after coating overnight at 4 ° c . in a humidified container , the plates were blocked with blocking buffer ( 0 . 1 m nahco 3 ph 8 . 6 , 5 mg / ml bsa , 0 . 02 % nan 3 , with a sterilized filter , stored at 4 ° c .) and incubated for at least 1 hour at 4 ° c . m13 phages displaying random heptapeptides at the n - terminus of its minor coat protein ( piii ) were used ( ph . d .- 7tm phage display peptide library , new england biolabs inc . beverly , mass ., usa ). the selection of specifically bound phages was done according to the manufacturer &# 39 ; s instructions . the 5 ′- end nucleotides of gene iii from the chosen phages were sequenced and then encoded peptides were deduced as show in table 1 [ cortese r , et al 1996 ]. [ 0049 ] b . grl7 gnmt sequence r s 10 l g v a a 15 m13 phage display # 1 a — — t — — f # 2 r w — t — — f # 3 t w — t — — w # 4 q a — — i — — # 5 k t — — y — — # 6 a m — — — f r consensus sequence : a l g t a a the carboxymethyl dextran ( cmd ) sample cuvettes were purchased from thermo labsystems . in initial experiments , coating conditions were optimized with regards to the ph . the surface was equilibrated in 10 mm sodium acetate buffers , with ph ranging from 4 to 6 in 0 . 5 unit steps for 6 min . samples of 9 μg human gnmt rp ( thrombin - cleaved ) in the same buffer were then added to the cuvette . to test , optimized ph conditions were created . the binding of the rp to the cuvette , which is due to electrostatic attraction between the negative charged carboxyl groups on the dextran and the positively charged protein , was performed for 6 min . optimized response occurred under a ph 5 . 0 in a 10 mm acetate buffer as shown on fig3 a . nine μg gnmt rp cleaved by thrombin were used to couple the cmd surface cuvette in 10 mm sodium acetate buffer under optimized ph by a 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide ( edc )/ n - hydroxysuccinimide ( nhs ) method shown in fig3 b . following the immobilization of gnmt , a pbst / tween 20 ( pbst ) baseline was established , and the stirring rate in the cuvette was kept constant , 100 rpm for all reactions . a series of 2 fold dilutions ( in 200 μl pbst ) of mab from culture supernatant were added to the cuvette , and responses were measured by iasys control software 3 . 01 as shown in fig4 a , b . kinetic analysis of the binding data was undertaken using the curve - fitting kinetic analysis software fast fit ( thermo labsystems , affinity sensors division , cambridge , uk ) designed for the iasys , and kd , determined as previously described [ morgan c l , et al . 1998 ]. wb was used for the confirmation of the mabs against gnmt . three rps - gst - gnmt ( 58 kd in size ), his - gnmt ( 37 . 4 kd in size ), gst ( 26 kd in size ), as well as human and mouse liver proteins were used as the antigens in the wb . in the strip experiment and after incubation of mabs grl1 , grl7 and r4 , normal mouse and pre - immune rabbit serum washed the strip and reacted with horseradish peroxidase - conjugated goat - anti - murine immunoglobulin ( sigma ). finally , the resultant serum was developed with 3 , 3 ′- diaminobenzidine tetrahydrochloride solution ( zymed laboratories inc . calif ., usa . ( gst , gst - gnmt , gnmt - his rp )). in the experiment of nitrocellulose membrane and after incubation of mabs grl1 and grl7 , anti - β actin ( sigma ) washed the membrane and reacted with horseradish peroxidase - conjugated goat - anti - murine immunoglobulin ( sigma ) and was finally developed with ecl reagent ( amershan ) as described previously [ chen y m , et al . 1988 ]. the results were shown in fig5 a , b . two sets of tumorous and non - tumorous liver tissue from hcc patients were used for the immunohistochemical procedures with mabs and r4 . the first set included 13 non - tumorous and 9 tumorous tissues ( 7 pairs ) and the second set included 13 non - tumorous and 16 tumorous tissues ( 9 pairs ). all the cancerous and non - cancerous tissue specimens were confirmed by pathologic examination and shown in table 2 a , b . the tissue blocks fixed in paraffin were sliced into 6 - μm - thick sections , de - paraffinized and immersed in a 3 % solution of hydrogen peroxide in distilled water for 5 min to abolish the endogenous peroxidase reaction . primary antibodies , mab grl1 ( 1 : 100 dilution of the ascites ) or mab grl7 ( 1 : 25 dilution of the ascites ) or r4 ( 1 : 200 dilution ), were applied to the tissues . a ready - to - use biotinylated secondary antibody was applied to bind the primary antibody . a streptavidin - peroxidase conjugate was applied to the same tissue slide as shown in fig6 ( histost5050 detection kit , zymed laboratories inc .). the presence of peroxidase was revealed by addition of 3 , 3 ′- diaminobenzidine tetrahydrochloride solution for color reaction as described in chen et al [ chen y m , et al . 1998 ]. table 2a rates of positive staining antibody a tumorous tissue non - tumorous tissues grl1 0 % ( 0 / 9 ) 38 . 5 % ( 5 / 13 ) grl7 6 . 3 % ( 1 / 16 ) 61 . 5 % ( 8 / 13 ) total 4 . 0 %( 1 / 25 ) 50 % ( 13 / 26 ) [ 0059 ] table 2b no . of cases in the tissue pairs with different gnmt staining a antibody a n +/ t + n +/ t − n −/ t + n −/ t − total pairs grl1 0 3 0 4 7 grl7 1 3 0 5 9 total 1 6 0 9 16 * the dilutions for mabs grl1 and grl7 ( ascites ) were 1 : 100 and 1 : 25 , respectively . a total of 545 sera or plasma samples from taipei municipal jen - ai hospital were collected in our laboratory between august 2000 and february 2002 , 413 from healthy people , 90 from chronic hepatitis patients who had viral hepatitis , 20 from patients who were diagnosed as liver cirrhosis by pathological method , and 22 from hcc patients who were confirmed by physical , ultrasonic and pathological methods . gnmt concentrations in human serum or plasma were measured by an indirect eia method . briefly , ninety - six well plates ( costar 3590 96 well assay plate , corning , n . y ., usa ) were coated with ammonium sulfate precipitated rabbit serum anti - gnmt ( r4 ) 0 . 1 μl / well ( concentration 7 . 13 mg / ml ) in 0 . 02 m sodium carbonate buffer ( ph 9 . 6 ) for 1 hour at 37 ° c . the wells were post - coated with 5 % skim milk in tbs ( 50 mm tris , 0 . 14 m nacl ) at 37 ° c . for 2 hours . the standards and samples were in a sample diluent ( tbs with 0 . 05 % tween - 20 and 1 % bsa ). the plates were put into a two - fold dilution of standard from 500 ng / ml to 7 . 8 ng / ml and two - fold dilution of serum samples at 37 ° c . for 1 hour . the goat anti - mouse peroxidase 1 : 3000 dilution was added to the well plate at 37 ° c . for 1 hour . after washing , opd was added and a wait of 30 min at rt was made . the reaction was stopped by adding 100 μl 3m h 2 so 4 . the absorption was read at 490 nm by eia reader , biotek elx808 . the results of standards were calculated by the 2 - parameter logistic curve fit method , and extrapolated to the concentration of serum samples . statistical analyses were performed using the spss statistical software ( spss inc . chicago , ill .). one - way anova was performed to determine statistical significance among the differences of gnmt protein concentrations in sera among the 4 groups : normal , patients with chronic hepatitis , cirrhosis , hcc . a post hoc test was used , when the p value was near 0 . 5 , and lsd was used to demonstrate significance between the groups shown in fig7 . 1 . aden d p , fogel h , plotkin s , damjanov i , knowles b b . nature 282 : 615 - 616 ; 1979 2 . bork p , ouzounis c , sander c , scharf m , schneider r , sonnhammer e . protein sci 1 : 1677 - 1690 ; 1992 . 3 . chang c , lin y , o - lee t , chou c , lee t , liu t , p &# 39 ; eng f , chen t , hu c . mol cell biol 3 : 1133 - 1137 ; 1983 . 4 . chen s y , wong f h , lin j r , liu t y , lin c h , lin p p , hsieh j t , chen y m . functional characterization of a putative tumor susceptibility gene - gnmt in the benzo [ a ] pyrene - detoxification pathway . 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