Patent Application: US-94626007-A

Abstract:
this invention provides methods for detecting serum gep level . this invention further provides methods for determining whether a subject is afflicted with hepatocellular carcinoma by measuring serum gep level . in another embodiment , this invention provides methods for the suppression of hcc growth and progression both in vitro and in vivo by treating a patient with anti - gep monoclonal antibody a23 .

Description:
reference will now be made in detail to the presently preferred embodiments of the invention , which , together with the examples and figures following the detailed description , serve to explain the principles of the invention . from earlier cdna microarray analysis ( 11 ), the inventors identified gep as a potential tumor marker of hcc . the inventors have further validated the observation in a separate set of patient samples and confirmed that gep protein is upregulated in hcc tissue ( 12 ). in addition , the inventors have also shown that gep level positively regulates cancer cell proliferation and tumor invasiveness ( 12 ). as gep is a secretory autocrine growth factor , the inventors hypothesized that the upregulation of gep in hcc tumor tissues would also lead to an elevation of serum gep protein level in patients and hence act as a useful diagnostic marker for the disease . in the present study , the inventors report the generation of gep specific monoclonal and polyclonal antibodies . using the newly isolated monoclonal antibody , gep protein level was shown to be upregulated in hcc tumor tissues , which is in agreement with previous observation ( 11 , 12 ). from immunohistochemical study , gep protein was expressed in the neoplastic hepatocytes but not the other tumor components . the inventors then evaluated if gep protein would be secreted from hcc cells , by performing immunoblotting from hcc cell line conditioned medium . the inventors have shown that gep was detectable from the culture supernatant , suggesting that gep could be a secretory protein detectable in hcc patient sera . to detect the gep serum protein , a specific gep elisa has been established using the newly isolated antibodies . monoclonal antibody targeting the c - terminus of gep was used as the capture antibody and a polyclonal antibody targeting the center part of gep as the detection antibody . the use of these antibodies combination which target two different epitopes of the gep full - length protein enhanced the specificity of the assay as confirmed by the immunoprecipitation experiment ( fig1 b ). nonetheless , due to the heterogeneity of hcc ( 20 ), it is questionable if there would be a tumor marker that expressed in all hcc tissues . however , the combination use of two to three markers will enhance the sensitivity of detection . in the current study , the inventors demonstrated that serum gep level has no correlation with serum afp level in hcc patients . sensitivity of hcc diagnosis by either one marker was only 58 . 0 % ( afp alone ) to 60 . 7 % ( gep alone ), but the sensitivity increased to 87 . 9 % by combination use of these two markers . the high fatality - to - case ratio associated with hcc is partially caused by the lack of symptoms in its early stages . curative resection can only be the treatment of choice for 20 % of hcc patients . early detection of hcc is therefore essential to improve survival . in the current study , serum gep was also detectable in early - stage hcc patients ( 56 . 6 %), suggesting this maker would be useful for early diagnosis which is important to improve patient survival . thus , serum gep determination would enhance early detection of hcc , allowing for better treatment option and survival outcome . the inventors previously have shown that the down - regulation of gep using the antisense approach can significantly reduce the tumorigenicity of hcc in athymic nude mice model ( 12 ). this observation suggested that gep is an attractive target for cancer therapy . however , the mode of gene delivery and infection / transfection efficiency remains as the main obstacle in successful cancer gene therapy . the use of gep antibody compare to gene therapy is a more practical and feasible approach for targeted cancer therapy . as gep is a secretory autocrine growth factor , therefore the inventors hypothesized that neutralizing the extracellular gep by gep - specific antibody a23 may hinder the proliferation function of gep . unlike targeting by antisense approach , antibody targeted therapy , like herceptin and anti - vegf , has higher efficacy and lower toxicity and make targeted therapy feasible in cancer patients . in order to investigate the inhibitory effect of anti - gep antibodies , e . g . a23 , they were added to culture supernatant of hepg2 and hep3b cells in the presence of 1 % fbs . proliferation of the cancer cells were significantly inhibited by the mabs a23 when compare to no treatment control in a dose dependent manner ( fig5 a and 5b ). concentration of gep in the culture supernatant was measured by sandwiched elisa . hep3b has a higher concentration of gep in the culture supernatant than hepg2 ( fig5 c ). after 72 hours of a23 treatment , the concentrations of gep in the culture supernatant were reduced in both cell lines ( fig5 c ). this result indicated that addition of a23 could effectively neutralize the gep secreted into the culture supernatant . gep has been shown to stimulate the phosphorylation of p44 / 42 mitogen activate protein kinase ( mapk ) in the extracellular regulated kinase signaling pathway . to investigate whether the inhibition of proliferation by anti - gep treatment is related to the phosphorylation of p44 / 42 mapk , western blot analysis was performed on cultured cell lysate after treatment with a23 . as shown in fig5 d , the addition of anti - gep a23 in the culture supernatant for 72 hours significantly reduced the phosphorylation of mapk in both hepg2 and hep3b cell lines suggesting that the reduction of cell proliferation is dependent on the reduced phosphorylation of p44 / 42 mapk . in animal study , the antitumor effect of anti - gep mabs a23 was confirmed with hep3b tumor implanted on nude mice . antibody treatment of 50 and 100 μg / injection was started once the tumor size reached ˜ 0 . 3 cm 3 . nine doses of treatments were given twice a week and the tumor sizes were monitored . after 5 weeks of treatment , the median tumor volume of mice treated with anti - gep a23 were 1 . 57 cm 3 ( range 1 . 44 - 2 . 53 cm 3 ) and 1 . 21 cm 3 ( range 0 . 79 - 1 . 97 cm 3 ) for 50 μg and 100 μg treatments , respectively , whereas that of the median tumor volume of the control mice was 2 . 20 cm 3 ( range 1 . 65 - 3 . 04 cm 3 ). analysis of variance by t - test demonstrated that difference between treated and untreated animal were statistically significant ( p & lt ; 0 . 05 ) ( fig6 ). this experiment indicated that in objects treated with a23 resulted in a dose - dependent suppression of hep3b tumor growth . moreover , this model mimics the situation in the clinic when most hcc patients were diagnosed at late stage and become in - operable . the marked decrease in tumor volume from the antibody treatment , suggested that neutralizing gep using anti - gep antibody can significantly delay tumor proliferation even in an established tumor . the current study demonstrated that anti - gep therapy is feasible for stabilizing the disease and / or delay tumor progression . as the anti - gep mabs a23 was injected intraperitoneally , the antibody titer was measured in order to evaluate the actual amount of antibody found in the mice blood circulation . the antibody titer of anti - gep mabs a23 in the mice serum were measured by direct elisa . as expected , the level of a23 in the control group was undetectable , but remained high in treatment group . for the 100 μg treatment group , the median level of a23 was 74 . 61 μg / ml ( range from 4 . 50 μg / ml to 145 . 48 μg / ml ). for the 50 μg treatment group , the median level of a23 was 8 . 87 μg / ml ( range from 1 . 35 to 16 . 24 μg / ml ) ( fig7 a ). in order to examine the effectiveness of a23 in the clearance of serum gep , the concentration of gep in mice serum was measured by sandwiched elisa . for the pbs control group , the serum gep level was highest with the median level of gep of 21 . 46 ng / ml ( ranged from 8 . 33 to 137 . 50 ng / ml ). however , after a23 treatment , the serum gep level was significantly lowered ( p & lt ; 0 . 05 ). after 100 μg treatment , the serum gep level was barely detectable ( median = 0 ng / ml , range from 0 to 2 . 5 ng / ml ). after 50 μg treatment , the median level of gep was reduced to 7 . 08 ng / ml ( range from 0 to 10 . 83 ng / ml ) ( fig7 b ). histologic examination of xenografts at the end of the treatment showed marked difference in the tumor from animals receiving a23 compared with tumor from animals receiving control therapy . in the 100 μg a23 - treated group , massive necrotic areas were found and there were substantially more cell - sparse regions compared with the control group ( fig8 a ). there was no gross histological difference in the non - tumor liver from the treatment and control group ( fig8 b ). immunohistological examination of xenografts was performed using ki - 67 antibody , there was a marked decrease in ki - 67 positive cells in 100 μg a23 - treated mice compared to the control group ( fig9 a ). however , there was no difference in the number of positive cell from the tunel assay in the treatment and control group ( fig9 b ). these results indicated that the decrease in tumor volume by the a23 treatment was caused mainly by a decrease in proliferation but not an increase in apoptosis . to investigate the mechanism of anti - gep antibody actions on tumor cell proliferation in mouse xenograft , the phosphorylation level of the key proliferative gene , mark and akt were examined . the phosphorylation of both mapk and akt at ser473 were significantly reduced upon anti - gep treatment suggesting that anti - gep antibody treatment delay tumor cell proliferation via the mapk and akt pathway ( fig1 ). these observations showed that anti - gep delay tumor cell proliferation both in vitro and in vivo . it inhibited p44 / 42 mapk phosphorylation and akt phosphorylation in a dose dependent manner . in summary , the inventors have shown that gep is a novel serum marker of hbv - related hcc . the combination of afp and gep improves the diagnostic sensitivity of hcc in both early - stage and late - stage tumors . the availability of this simple and reliable immunoassay for measuring serum gep concentration may provide a valuable tool to further evaluate the clinical usefulness of serum gep for the management of hcc . furthermore , the inventors have shown that anti - gep antibodies are able to inhibit the growth of established hcc tumors . these results indicated that gep is a target for hcc therapy and the potential application of anti - gep antibodies for treatment of hcc . the study protocol was approved by the institutional review board of the university of hong kong and signed consents were obtained from the patients and controls . between march 1999 and october 2004 , blood samples were obtained from 107 patients diagnosed with primary hcc , 38 chronic hepatitis b patients ( only those with no indication of malignancy for more than 2 years of follow - up were included in the current study ) and 72 healthy donors who were hepatitis b surface antigen ( hbsag ) negative . serum hbsag was positive in 96 ( 89 . 7 %) hcc patients , and therefore control groups included chronic hepatitis b patients and healthy volunteers . serum samples were frozen at − 70 ° c . until use . tumor and adjacent non - tumor liver tissues from hcc patients were collected and snap frozen in liquid nitrogen and stored at − 70 ° c . until use . parallel sections were formalin - fixed and paraffin embedded for histological examination and immunohistochemical study . clinical and pathological data including the serum afp level of all patients and control subjects were prospectively collected . the human hcc cell lines hep3b , hepg2 and huh7 ( american tissue culture collection , manassas , va .) and japan health science research resources bank , osaka , japan ) were maintained in dulbecco &# 39 ; s modified eagle medium ( dmem ) supplemented with 10 % fetal bovine serum ( gibco brl , carlsbad , calif .). gep - specific antibody was generated by immunizing balb / c mice with 33 μg of keyhole limpet hemocyanin ( klh )- conjugated custom - made gep specific peptide seq id no : 3 subcutaneously with complete freund &# 39 ; s adjuvant ( sigma - aldrich , dorset , uk ). for subsequent booster , the same amount of antigen was injected intraperitoneally in incomplete freund &# 39 ; s adjuvant biweekly . serum antibody activity to the immunizing antigen was monitored after each boost using elisa against peptide antigen . mice showing high serum antibody titer to the antigen were given a final boost of intravenously injected antigen 3 days prior to harvesting the spleens . spleen was harvested from mice shown high titre of antibody in their serum . fusion of the spleen cells with a nonproducer myeloma line , ns0 , was carried out according to the standard protocols originally derived from kohler and milstein ( 21 ). ns0 was maintained in dmem supplemented with 10 % fetal bovine serum ( gibco brl , carlsbad , calif .). briefly , lymphocytes were harvested from the mouse spleen and fused with ns0 using polyethylene glycol 1500 ( roche diagnostics gmbh , mannheim , germany ). hybridoma was selected by plating into dmem medium contained hat and 20 % fbs . antibody secreting hybridoma were selected by elisa and subsequently subcloned by limited dilution . isotypes of the monoclonal antibody were determined using the mouse monoab id kit ( hrp ) ( zymed laboratories , inc ., san francisco , calif .). new zealand white rabbits were immunized with 100 μg of keyhole limpet hemocyanin ( klh )- conjugated gep specific peptide seq id no : 4 ( zymed laboratories , inc ., san francisco , calif .) using standard procedures ( 22 ) the rabbit antisera were affinity purified using the immobilized antigen column , dialysed against 1 × pbs and concentrated to 1 mg / ml . to generate the gep monoclonal antibodies , a synthetic peptide of 16 - amino acid , seq id no : 3 , designated at the gep carboxyl - terminal was used as an immunogen to generate the antibodies . the clones were then subjected to another round of elisa screening against full - length recombinant gep and hep3b cell lysate . the supernatants of these clones were then subjected to western blot analysis and subcloned by limited dilution . clone a23 was identified , as the only antibody that recognized the gep glycosylated form at 88 - kda from the gep recombinant protein ( fl ), hcc cultured cell lysate ( hep3b and hepg2 ) and patients &# 39 ; tissue lysate ( fig1 a ). to increase the specificity of the sandwiched elisa against full - length gep , the inventors custom - made another gep specific polyclonal antibody specifically recognizing the center parts of gep , seq id no : 4 . to determine the specificity of the polyclonal and monoclonal gep antibodies , immunoprecipitation was performed . the monoclonal and polyclonal gep antibodies recognized the 88 - kda glycosylated gep from the culture lysate ( fig1 b ). to determine whether gep was a secretory protein , gep was examined in the conditioned medium from the hcc cell lines using the gep monoclonal antibody . as shown in fig1 c , the 88 - kda glycosylated gep was detectable in the supernatant of hcc cells . gep localization was revealed by immunohistochemistry on tumor tissue paraffin sections . the protein signals were found to be uniformly associated with neoplastic hepatocytes but not in the endothelial cells or fibroblasts in the tumor tissues , while hepatocytes in the non - tumor tissues revealed no signals ( fig2 ). hcc cell lines , hcc and adjacent non - tumor liver tissues were subjected to western blot analysis . total proteins were extracted by homogenizing snap frozen patients &# 39 ; samples in buffer a ( 8 m urea , 50 mm tris - hcl ph 8 . 0 ) containing 1 mm pmsf . protein extracts , totally 10 μg , were separated by 10 % sds - page gel followed by western blotting . the blot was blocked with 5 % skim milk in pbs / 0 . 1 % tween 20 and probed with the appropriate monoclonal antibodies . polyclonal goat anti - β - actin antibody was used as 1 : 1000 dilution ( dako , glostrup , denmark ). secondary anti - mouse and anti - goat horseradish peroxidase ( hrp ) conjugated antibodies respectively were used in 1 : 3000 dilution ( ap biotech , chalfont st . giles , uk ). ecl was performed according to the manufacturer &# 39 ; s instructions ( ap biotech , chalfont st . giles , uk ). immunoprecipitation was performed with 500 μg of cell lysate and incubated with 1 μg of the purified monoclonal antibodies . the immunocomplexes were separated on an sds - page and immunoblotted with the polyclonal anti - gep antibody . immunohistochemistry study was performed on paraffin - embedded hcc and adjacent non - tumor liver tissues . protocol was described previously with modification ( 12 ). antigen retrieval was performed by microwave with sections immersed in citrate buffer , followed by endogenous peroxidase blocking and biotin blocking reagents ( dako , glostrup , denmark ). appropriate monoclonal antibodies were used as 2 μg / ml . signal was detected by anti - mouse hrp conjugated secondary antibody and color development with diaminobenzidine ( dab ) as the chromogen . tissue sections were counterstained with hematoxylin . ninety - six - well elisa plates ( nalge nunc international , rochester , n . y .) were coated with 0 . 5 μg of anti - gep mab a23 in 50 μl of pbs per well . the plates were blocked for 1 hour with 300 μl of blocking buffer ( 1 × pbs , 1 % bsa , 5 % surcose , 0 . 05 % nan 3 ), then 50 μl of 1 : 5 diluted serum samples was added and incubated at room temperature for 2 hours . after washing the unbound material with 0 . 05 % tween 20 in 1 × pbs , bound gep was detected using an affinity purified anti - gep rabbit polyclonal antibody ( 1 : 2000 , 1 mg / ml ) followed by incubation with horseradish peroxidase - conjugated goat anti - rabbit igg ( zymed laboratories , inc , san francisco , calif .) using tmb ( pierce biotechnology inc ., rockford , ill .) as substrates . to quantify the gep present in the serum , a calibration curve of purified gep diluted in pbs with 10 % fetal bovine serum was performed in parallel . each sample was measured 3 times by quadruplicates . the dynamic range of the gep sandwich elisa was 469 pg / ml to 30 ng / ml . a pooled serum sample of patients was included in each assay and used for adjustment of plate - to - plate variation . the variations within and between assays were 2 . 9 % ( range 1 . 1 - 5 . 5 %) and 5 . 0 % ( range 1 . 3 - 10 . 8 %), respectively . the serum gep protein levels were measured by a specific elisa in 107 hcc patients , 72 healthy individuals and 38 patients with chronic hepatitis b ( fig3 ). the median and mean levels of serum gep in healthy subjects were 4 . 59 ng / ml and 5 . 63 ng / ml , respectively ( range , 0 to 20 . 46 ng / ml ). the median and mean concentrations of serum gep in patients with chronic hepatitis b were 6 . 03 ng / ml and 6 . 85 ng / ml , respectively ( range , 0 . 17 to 28 . 36 ng / ml ). the median and mean serum gep levels in hcc patients were 10 . 53 ng / ml and 16 . 09 ng / ml , respectively ( range , 0 to 113 . 59 ng / ml ). the serum gep levels measured in hcc patients were significantly higher than those in healthy controls ( p & lt ; 0 . 001 ) and patients with chronic hepatitis b ( p & lt ; 0 . 001 ). an roc curve for gep was also constructed ( fig4 ), showing an auc of 0 . 74 ( 95 % cl 0 . 67 - 0 . 81 , p & lt ; 0 . 001 ). to discriminate hcc from controls including chronic hepatitis b carriers and healthy individuals , the youden index was employed to determine the optimal cutoff for class prediction . the optimal cutoff value was 9 . 07 ng / ml , which achieved a sensitivity and specificity of 60 . 7 % and 82 . 5 %, respectively . serum afp levels were also measured in the same set of samples and compared with the serum gep data . when using serum afp levels for hcc diagnosis , the cutoff value of 100 ng / ml was used which was considered as relatively high and specific ( tables 1 and 2 ). a lower cutoff value of serum afp at 20 ng / ml was also examined and data in comparison with serum gep was presented in the supplementary tables 1 and 2 . the sensitivity of hcc diagnosis by serum afp ( 58 . 0 %, 62 / 107 , cutoff at 100 ng / ml ) and serum gep ( 60 . 7 %, 65 / 107 , cutoff at 9 . 07 ng / ml ) was comparable ( table 1 ). there was no correlation between gep and afp serum levels ( r =− 0 . 113 ; p = 0 . 243 ) in hcc patients . the majority of hcc patients ( 87 . 9 %, 94 / 107 ) demonstrated elevation of either serum gep (& gt ; 9 . 07 ng / ml ) or afp (& gt ; 100 ng / ml ). importantly , the simultaneous use of these two markers increased the sensitivity of hcc diagnosis from 58 . 0 % ( elevation of afp alone ) to 87 . 9 % ( elevation of either afp or gep , or both ). early diagnosis of hcc with combined screenings of serum afp and gep early diagnosis is the key to enable hcc patients to receive curative treatment and to improve survival . the performance of the serum markers were examined according to tumor stages . in early - stage hcc patients , the sensitivity of detection by serum gep ( 56 . 6 %, 43 / 76 ) and serum afp ( 55 . 3 %, 42 / 76 ) was similar ( table 2 ). in late - stage patients , the sensitivity of hcc detection by serum gep ( 71 . 0 %, 22 / 31 ) was slightly better than serum afp ( 64 . 5 %, 20 / 31 ). elevation of either serum gep or afp was observed in 84 . 2 % ( 64 / 76 ) of early - stage patients and 96 . 8 % ( 30 / 31 ) of late - stage hcc patients . thus , the use of two markers would increase the sensitivity of diagnosis in both the early - stage and late - stage hcc patients . cellular proliferation was measured via 3 -( 4 , 5 - dimethylthiazol - 2 . yl )- 2 , 5 - diphenylthtrazolium bromide ( mtt ) assay . briefly , 5 × 10 3 cells were seeded to a 96 - well plate in 100 μl dmem medium containing 1 % fbs either with or without mabs a23 as indicated . for every 24 hours , the medium was replaced with 100 μl dmem containing 0 . 5 mg / ml mtt and incubated for 3 hours at 37 ° c . crystal was dissolved by 100 μl mtt solvent ( 0 . 1n hcl in isopropanol ) and absorbance was plot as the measurement at 540 nm subtracted the background absorbance at 650 nm . each data point represented results from 3 independent experiments , each performed in triplicates . anti - gep mabs a23 was added to culture supernatant of hepg2 and hep3b cells in the presence of 1 % fbs , proliferation of the cancer cells were significantly inhibited by the mabs when compare to no treatment control ( fig5 a and b ). this inhibition is in a dose dependent manner ( fig5 b ). concentration of gep in the culture supernatant was measured by sandwiched elisa . hep3b has a higher concentration of gep in the culture supernatant than hepg2 ( fig5 c ). after 72 hours of a23 treatment , the concentrations of gep in the culture supernatant were reduced in both cell lines ( fig5 c ). this result indicated that addition of a23 could effectively neutralize the gep secreted into the culture supernatant . total proteins were extracted by homogenizing mouse xenografts and hep3b cells in cell lysis buffer ( cell signaling technology inc ., beverly , mass .) containing 1 mm pmsf . protein extracts , totally 10 g , were separated by 10 % sds - page gel followed by western blotting . the blot was blocked with 5 % skim milk in pbs / 0 . 1 % tween 20 and probed with the appropriate antibodies . polyclonal goat anti - β - actin antibody was used as 1 : 1000 dilution ( dako , glostrup , denmark ). polyclonal rabbit anti - gep antibody was used as 1 : 500 dilution ( 12 ). antibody against p44 / p42 mapk and phospho - p44 / 42 mapk ( thr202 / tyr204 ) were used according to manufacturers &# 39 ; instruction ( cell signaling technology , inc ., beverly , mass .). secondary anti - mouse , anti - rabbit and anti - goat hrp conjugated antibodies were used in 1 : 3000 dilution respectively ( ap biotech , chalfont st , giles , uk ). ecl was preformed according to manufacturer &# 39 ; s instructions ( ap biotech , chalfont st . giles , uk ). gep has been shown to stimulate the phosphorylation of p44 / 42 mitogen activate protein kinase ( mapk ) in the extracellular regulated kinase signaling pathway ( 23 ). to investigate whether the inhibition of proliferation by anti - gep treatment is related to the phosphorylation of p44 / 42 mapk , western blot analysis was performed on cultured cell lysate after treatment with a23 . as shown in fig5 d , the addition of anti - gep a23 in the culture supernatant for 72 h ours significantly reduced the phosphorylation of mapk in both hepg2 and hep3b cell lines suggesting that the reduction of cell proliferation is dependent on the reduced phosphorylation of p44 / 42 mapk . this study protocol was approved by the committee on the use of live animals in teaching and research of the university of hong kong . mice ( n = 15 ) were housed in barrier facilities that provided 12 - hour light - dark cycles and received food and water . all manipulations were performed while mice were under isoflurance gas anesthesia . no mouse showed signs of wasting or other signs of toxicity . hep3b cells ( 2 × 10 6 cells / mouse ) were injected subcutaneous to 5 - to 6 - week - old male athymic nude mice . tumor sizes were determined by vernier caliper measurements and the tumor volume was calculated according to the formula ( a × b 2 )/ 2 , where a and b are the largest and smallest diameters respectively ( 24 ). treatments were started as the tumor size reaches a mean tumor volume of ˜ 0 . 3 cm 3 and mice were randomized into 3 groups ( n = 5 ). antibodies were injected intraperitoneally twice weekly for the duration of the study . from our preliminary study , the half - life time ( t 1 / 2 ) of serum a23 antibody in the mice was longer than 72 hours after intraperitoneal injection ( data not shown ), therefore a treatment regime of 100 μg and 50 μg intraperitoneally twice weekly was chosen . group 1 mice were treated with either 100 μg purified mouse igg ( zigma - aldrich , saint louis , mo .) or pbs . in preliminary studies , the inventors found no difference between mouse igg or pbs on tumor growth . group 2 and 3 mice were treated with 50 μg and 100 μg a23 mabs , respectively . the antitumor effect of anti - gep mabs a23 was examined on hep3b tumor implanted on nude mice . antibody treatment of 50 and 100 μg injection was started once the tumor size reached ˜ 300 mm 3 . nine doses of treatments were given twice a week and the tumor sizes were monitored . after 5 weeks of treatment , the median tumor volume of mice treated with anti - gep a23 was 1 . 57 cm 3 ( range 1 . 44 - 2 . 53 cm 3 ) and 1 . 21 cm 3 ( range 0 . 79 - 1 . 97 cm 3 ) for 50 μg and 100 μg treatments , respectively , whereas the median tumor volume of the control mice was 2 . 20 cm 3 ( range 1 . 65 - 3 . 04 cm 3 ). analysis of variance by t - test demonstrated that difference between treated and untreated animal were statistically significant ( p & lt ; 0 . 05 ) ( fig6 ). treatment with a23 resulted in a dose - dependent suppression of hep3b tumor growth . mice serum was collected for measurement of antibody concentration and serum gep concentration using elisa . since the anti - gep mabs a23 was injected intraperitoneally , the antibody titer was measured in order to evaluate the actual amount of antibody found in the mice blood circulation . the antibody titer of anti - gep mabs a23 in the mice serum were measured by direct elisa . as expected , the level of a23 in the control group was undetectable , but remained high in treatment group . for the 100 μg treatment group , the median level of a23 was 74 . 61 μg / ml ( range from 4 . 50 μg / ml to 145 . 48 μg / ml ). for the 50 μg treatment group , the median level of a23 was 8 . 87 μg / ml ( range from 1 . 35 to 16 . 24 μg / ml ) ( fig7 a ). in order to examine the effectiveness of a23 in the clearance of serum gep , the concentration of gep in mice serum was measured by sandwiched elisa . for the pbs control group , the serum gep level was highest with the median level of gep of 21 . 46 ng / ml ( ranged from 8 . 33 to 137 . 50 ng / ml ). however , after a23 treatment , the serum gep level was significantly lowered ( p & lt ; 0 . 05 ). after 100 mg treatment , the serum gep level was barely detectable ( median = 0 ng / ml , ( range from 0 to 2 . 5 ng / ml ). after 50 mg treatment , the median level of gep was reduced to 7 . 08 ng / ml ( range from 0 to 10 . 83 ng / ml ) ( fig7 b ). mice were euthanized by the end of 5 weeks . xenografts and liver tissues were collected and snap frozen in liquid nitrogen and stored at − 70 ° c . until use . parallel sections were formalin - fixed and paraffin embedded for histological examination and immunohistochemical study . histologic examination of xenografts at the end of the treatment showed marked difference in the tumor from animals given a23 compared with tumor from animals receiving control therapy . in the 100 mg a23 - treated group , massive necrotic areas were found and there were substantially more cell - sparse regions compared with the control group ( fig8 a ). there was no gross histological difference in the non - tumor liver from the treatment and control group ( fig8 b ). immunohistological examination of xenografts was performed using ki - 67 antibody , there was a marked decrease in ki - 67 positive cells in 100 mg a23 - treated mice compared to the control group ( fig9 a ). however , there was no difference in the number of positive cell from the tunel assay in the treatment and control group ( fig9 ). these results indicated that the decrease in tumor volume by the a23 treatment was caused mainly by a decrease in proliferation but not an increase in apoptosis . to investigate into the mechanism of a23 on cell proliferation , the phosphorylation level of the key proliferative protein , mapk and akt were examined using the total protein lysate from mouse tumor xenograft after treatment . antibody against p44 / p42 mapk , phospho - p44 / 42 mapk ( thr202 / tyr204 ), akt and phospho - akt ( ser473 ) were used according to manufacturers &# 39 ; instruction ( cell signaling technology , inc ., beverly , mass .). the phosphorylation of both mapk and akt at ser473 were reduced upon anti - gep treatment ( fig1 ), suggesting that anti - gep antibody treatment delayed tumor cell proliferation via the mapk and akt pathway in mouse tumor xenografts . gep - specific antibodies were generated by immunizing balb / c mice or new zealand white rabbits with gep specific peptide sequence located at and around seq id no . 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , or 13 ( fig1 ). the anti - gep monoclonal antibodies or anti - gep polyclonal antibodies were used to detect serum gep levels or suppression of tumor growth . 1 . el - serag h b : hepatocellular carcinoma : an epidemiologic view . j clin gastroenterol 2002 ; 35 : s72 - 8 . 2 . montalto g , cervello m , giannitrapani l , et al : epidemiology , risk factors , and natural history of hepatocellular carcinoma . ann n y acad sci 2002 ; 963 : 13 - 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