Patent Application: US-15154702-A

Abstract:
disclosed is a receptor - mediated protein delivery system using a ligand derived from the region ii of malarial circumsporozoite protein which recognizes receptors specifically localized on the surface of liver cells in vivo and many types of cultured cells grown in vitro . using the present invention , a “ suicidal gene product ”, cytosine deaminase , has been successfully fused to cs protein . the recombinant fusion protein possesses both cell type targeting specificity of cs as well as cytosine deaminase enzymatic activity which catalyzes the conversion of prodrug 5 - fluorocytosine into antitumor drug 5 - fluorouracil and elicit cell killing capacity . moreover , the fusion protein exhibits prolonged stability and sustained cell killing activity , due to the entrapment of the recombinant protein in a particular cellular compartment . thus , the present invention provides technology for improved cell - type specificity and enhanced favorable pharmacokinetics of drug delivery .

Description:
the present invention includes a fusion protein comprising a ligand and an enzyme . as used herein , a fusion protein can be defined as one or more molecules fused by physical or chemical means . the fusion protein can be synthesized in living organisms using expression recombinant dna technologies . the living organisms can be bacteria , yeasts , plants , animals and mammalian cells . as used herein , the term “ ligand ” is intended to refer to any molecule that binds to another molecule or macromolecule . the term enzyme is intended to refer to any molecule or gene product that possesses biological function . the biological function is referred to any disease - related or health - related therapeutic or cytoprotective means in human health . in the present invention , the ligand comprises a malarial protein known as circumspoprozoite ( cs ) protein . cs covers the surface membranes of malarial sporozoites . the amino acid sequence of cs protein can be divided into three domains : ( from the n - terminal ) region i ( ri ), repeat and region ii + ( rii +). the function of ri is not clear . the repeat domain which consists of three copies of nanpnvdp and 21 copies of nanp , is immunodominant and confers the major antigenicity of cs protein ( nussenzweig , 1997 ; miller et al ., 1994 ). rii + contains an evolutionarily conserved stretch of 18 amino acid residues among different species of plasmodium , i . e ., human malarial parasite plasmodium falciparum [ ewspcsvtcngiqvrik ], murine parasite p . berghei [ ewsqcnvtcgsgirvrkr ] and p . yoelii [ ewsqcsvtcgsgvrvrlr ]. these amino acid sequences also share similarities with human cellular proteins , i . e . thrombospondin i [ ewtrcstrcgngiqqrgr ] and thrombospondin ii [ ewsscsvtcgdgvotror ]. ( robson , et al ., 1988 ; prater et al ., 1991 ; goundis et al ., 1988 ). because of these similarities , it has been suggested that the parasites can the present invention includes a fusion protein comprising a ligand and an enzyme . as used herein , a fusion protein can be defined as one or more molecules fused by physical or chemical means . the fusion protein can be synthesized in living organisms using expression recombinant dna technologies . the living organisms can be bacteria , yeasts , plants , animals and mammalian cells . as used herein , the term “ ligand ” is intended to refer to any molecule that binds to another molecule or macromolecule . the term enzyme is intended to refer to any molecule or gene product that possesses biological function . the biological function is referred to any disease - related or health - related therapeutic or cytoprotective means in human health . in the present invention , the ligand comprises a malarial protein known as circumspoprozoite ( cs ) protein . cs covers the surface membranes of malarial sporozoites . the amino acid sequence of cs protein can be divided into three domains : ( from the n - terminal ) region i ( ri ), repeat and region ii + ( rii +). the function of ri is not clear . the repeat domain which consists of three copies of nanpnvdp and 21 copies of nanp , is immunodominant and confers the major antigenicity of cs protein ( nussenzweig , 1997 ; miller et al ., 1994 ). rii + contains an evolutionarily conserved stretch of 18 amino acid residues among different species of plasmodium , i . e ., human malarial parasite plasmodium falciparum [ ewspcsvtcngiqvrik ], murine parasite p . berghei [ ewsqcnvtcgsgirvrkr ] and p . yoelii [ ewsqcsvtcgsgvrvrlr ]. these amino acid sequences also share similarities with human cellular proteins , i . e . thrombospondin i [ ewtrcstrcgngiqqrgr ] and thrombospondin ii [ ewsscsvtcgdgvotror ]. ( robson , et al ., 1988 ; prater et al ., 1991 ; goundis et al ., 1988 ). because of these similarities , it has been suggested that the parasites can bypass the host defense mechanisms during their invasion . moreover , because of its free of the immunodominant repeat , rii + is nontoxic to the hosts it is this region of the cs protein that specifically recognizes and binds to the receptors on the cell surface and confers targeting specificity ( cerami et al ., 1992 ). the cognate receptor protein for cs is predominantly expressed in on the surface of liver , with low levels expression in intestines and kidney , but not in brain , bladder , thyroid , spleen , stomach , lung , and heart . however , a number of non - hepatocytic cell lines also express receptor recognizable by the circumsporozoite region ii - containing polypeptide ( rich et al ., 1990 ; ding et al ., 1995 ). these observations suggest that cs protein can be up - regulated during in vitro cell culture conditions . these observations also suggest that cs protein can be used as a ligand carrier for the delivery of recombinant proteins into these cells . in preferred embodiments , the ligands would be proteins that recognize cell surface receptors . these include epidermal growth factor ( fan and mendelsohn , 1998 ), tumor necrosis growth factors , fibroblastic growth factor , cd - 28 ( schwarzenberger et al ., 1996 ), interleukin - 2 ( junbo , et al ., 1999 ), and folate ( lee and huang , 1996 ), etc . the ligands could be used to targeting malignant cells overexpressing their cognate receptors . in preferred embodiments , the therapeutic gene products could be enzymes that convert prodrugs into drugs . these include cytosine deaminase which catalyzes the conversion of 5fluorocytosine ( 5 - fc ) into 5 - fluorouracil ( 5 - fu ) ( danielsen et al ., 1992 ), thymidine kinase which phosphorylates the prodrug ganciclovia to its cytotoxic derivatives , human β - glucuronidase which converts prodrug epirubicin - glucuronide ( houba et al ., 1996 ; murdter et al ., 1997 ). additional embodiments would be gene products of tumor suppressor genes , e . g ., p53 , p21 , brca1 , brca2 , rb , nf - 2 , wt - 1 , wt - 2 , men - 1 , men - 22 , vhgl , mcc , and fcc , nf - 1 , etc . in still further embodiments , the gene products would be those involve in cell death / cell survival mechanisms , including bax , bcl - 2 , bcl - xl , etc . the fusion protein comprising cell ligand for receptor - mediated protein delivery also creates a situation that recombinant fusion protein is internalized by a cell type - specific manner . once internalized , the fusion protein is entrapped in a particularly cellular compartment , most likely remains in the endosommal and lysosomal compartments . the exact mechanism of such entrapment remains to be investigated , but most likely due to the possibility that the recombinant fusion has changed the protein conformation of the ligand , thereby altered the normal endocytotic process . this results in enhanced protein stability because the entrapped protein is sequestered from cellular protein degradation machinery . this has been demonstrated in the prodrug strategy using cs protein fusion with cytosine deaminase ( lin - lee , et al . 2002 ). therefore , the invention is generally applicable to prolong protein stability for sustaining long - term enzymatic activities . it is also proposed that the invention is generally applicable to any situation where one desires to introduce fusion configuration to interfere with cell growth by interfere normal receptor - mediated endocytotic physiology . many malignant cells overexpress receptors for various growth factors ( fan and mendelsohn , 1998 ). one may use fusion protein to intervene the normal receptor physiology of these overexpressed receptors , thereby controlling the growth of cancer cells . on the other hand , the therapeutic gene may be directed to a non - cancerous disease state . for example , cystic fibrosis ( cf ) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator ( cftr ). furthermore , therapy of genetic diseases such as hyperchlesterolemia , phenylketonuria and hemophila could be achieved by the introduction of a corresponding gene products . the underlying deficiency of these diseases may also be alleviated by the introduction of these relevant proteins using the present invention . moreover , the present invention may be applicable to the treatment of infectious diseases , i . e ., various forms of hepatitis . interferon has been used in the treatments of hepatitis viral infections . however , interferon causes sever adverse side effects if administered systemically . the strategy of hepatic targeting using cs protein as a ligand could drastically reduce the toxicity of interferon otherwise delivered systematically and thus enhance the therapeutic efficacy of the treatment . pcs27i c6 × his encodes a cs protein with deletion of internal repeats but retaining the receptor binding domain ( region ii +) and six histidine residues . this plasmid dna also contains an isopropylthio - β - d - thiogalactoside ( iptg ) regulatable promoter element followed by a ribosomal binding side in front of the cs gene and a transcriptional terminal signal from chloramphenicol acetyltransferase gene behind the translational termination codon . cytosine deamninase gene was synthesized by polymerase chain reaction ( pcr ) using oligo ( 5 ′ agt ggatcc acgtttgtaatcgastggc , underscored nucleotides contain bamhi site ) and oligo ( 5 ′ aca ggatcc aataacgctttacaaaca ) and plasmid template of psd112 ( a gift of dr . jan neuhard , university of copenhagen , denmark ) which contains the escherichia coli cd gene ( danielsen et al ., 1992 ). the pcr product was purified and digested by restriction enzyme bamhi , and ligated into plasmid pcs27i c - 6 × his at the bamhi site which is located at the 5 ′ end of the cs gene . the resulting recombinant , designated pcd5 - 73 . 15 . 5 , encoded cd - cs fusion protein with the full length cd inserted in frame after the third amino acid residue of the cs protein . similarly , bamhi - digested pcr product was cloned into the bamhi site of a pqe - 60 vector ( qiagen ) and the resulting recombinant was designated as pcdp6 which also contains 6 histidine residues at the c - terminus . all the recombinant plasmids were verified by dna sequencing . the plasmid dna was transformed into e . coli sg13009 ( prep4 ) hosts . bacterial cells carrying various recombinant plasmids were grown in one liter of 2 × yt both containing 16 g bactotryptone , 10 g yeast extract , 5 gm nacl , 100 mg ampicilin and 25 mg kanamycin for 2 to 3 hr . when the cultures reached an a600 of 0 . 6 , 2 mm of iptg was added to the medium . three hours after induction , the cells were harvested by centrifugation and resuspended in 10 ml of buffer mcac - 0 ( 20 mm tris - hcl , ph 8 . 0 , 0 . 5 m nacl , 10 % glycerol and freshly prepared 1 mm phenylmethylsulfonyl fluoride , 1 μg / ml of aprotinin and 1 μg / ml of leupeptin ). cells were sonicated and the resulting cell lysates were centrifuged at 12 , 000 rpm in the hb4 rotor of a sorval high - speed rc5b centrifuge for 35 min . clear lysate was mixed gently with 4 ml of ni - nta agarose in ice for 1 hour . after washing the agarose with buffer mcac - 20 ( mcac - 0 containing 20 mm imidazole ), the recombinant protein was eluted with buffer mcac - 200 ( mcac - 0 plus 200 mm imidazole ). fractions containing cdcs , cd or cs were analyzed by 10 % polyacrylamide gel electrophoresis , pooled , and dialyzed extensively in phosphate - buffered saline ( pbs ). proteins were concentrated and frozen in aliquots at − 70 ° c . until used . typical yields ( per liter ) were 1 - 3 mg , and 4 - 5 mg of cd - cs and cd proteins , respectively . the expression of these proteins was under the control of the lac repressor . upon induction by iptg , these bacterial cultures produced recombinant proteins consisting 10 - 30 % of total cellular proteins . these recombinant proteins contained six histidine tags at their respective c - termini . therefore , purification of these proteins was achieved by affinity column chromatography through a ni - nta column . a one - step fractionation by passing the crude extracts from the induced cultures through the column resulted in 120 - to 130 - fold purification ( data not shown ). the purified proteins were then analyzed by sds - page . single bands corresponding to the molecular mass of 45 kda and 34 kda were observed for the cd and cs preparations , respectively ( fig1 a ). a major band corresponding to molecular mass of 87 kda was found in the purified cd - cs sample . this molecular mass is consistent with that for the fusion between cd and cs . cd - cs preparations also contained a minor component with molecular mass of 65 kda . the identity of this minor band remains unknown ; it may represent a degradation product of cd - cs . the specific activity of the cd - cs fusion protein , as measured by the conversion of [ 3 h ] cytosine to [ 3 h ] uracil , was 2 , 493 nmol / min / mg protein , about 75 % that of the cd protein ( 3 , 308 nmol / min / mg ). this reduction in specific activity was presumably due to the fusion . these results indicated that the recombinant fusion protein retained the enzymatic activity of cd . the repeat - deleted cs protein and cd were similarly prepared and used in parallel in the cd - cs experiments ( fig1 b ). [ 35 s ] cd - cs - labeled protein was prepared by the method described previously ( giovane et al . 1997 ). in brief , bacterial cultures harboring recombinant plasmids encoding cd - cs were growth in 2 × yt media until a600 of 0 . 6 . cells were pelleted and re - cultured in 100 ml of mem ( life technologies , bethesda , md .) supplemented with 1 mm glutamine , 25 mm hepes , ph 7 . 5 , 1 . 5 mm iptg , and 0 . 1 ml of l -[ 35 s ] methionine ( 1000 ci / mmol . 10 mci / ml ). after culturing cells at 37 ° c . for an additional 2 . 5 hr , cells were harvested and the recombinant proteins were purified according to the procedure as described above . crude cell extracts were prepared by sonicating cells in a buffer ( 50 mm na phosphate , ph 7 . 8 and 0 . 3 m nacl ) followed by centrifugation . cytosine deaminase activity was determined by measuring the production of uracil in a reaction mixture ( 50 μl ) containing 5 mm , 6 -[ 3 h ] cytosine ( 0 . 45 ci / mol ), 50 mm tris - hcl ph 7 . 8 , 1 mm dtt , 1 mm edta and cellular crude extract ( 10 - 20 μg / ml ) or eluted fractions . after 1 - 2 hour of incubation at 37 ° c ., 10 μl aliquots were withdrawn and analyzed by thin layer chromatography ( silica gel 60 , selecto scientific , norcross , ga .). chromatograms were developed in 1 - butanol / water ( 86 / 14 , v / v ). positions of cytosine and uracil were identified by tv light . the corresponding spots for cytosine and uracil were cut out and radioactivities were determined in a scintillation counter . in some cases , 6 -[ 3h ] 5 - fc ( 4 . 0 ci / mmol ) was used as a substrate in the assay . human hepatoma ( hepg2 ), rat hepatoma ( h4iie ), and mouse colorectal carcinoma mca - 26 cells were grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) supplemented with 10 % fetal calf serum ( fcs ) ( life technologies , inc ) containing penicillin ( 100 units / ml ) and streptomycin ( 100 μg / ml ). el - 60 cells were maintained in rpmi 1640 medium containing 10 % fcs , penicillin ( 100 units / ml ) and streptomycin ( 100 μg / ml ). all cultures were maintained in a 37 ° c . incubator maintained with 5 % co 2 in air . cells were plated in costar 24 - well culture plate at a density of 1 - 2 × 10 5 cells / well . cells were treated with different amounts of purified recombinant proteins for 4 hrs . the medium was then removed , cells were washed and replaced with fresh dmem containing 1 mm 5 - fc . after 3 - 4 days incubation , cells were gently detached by trypsin . viable cells were determined by trypan blue exclusion staining and counted using hemacytometer . cultured cells were incubated with 30 μl of [ 35 s ] cd - cs ( 5 × 10 4 cpm / μg ). at different time intervals , cells and culture media were collected . aliquots of collected cells and medium were resuspended in 0 . 1 ml of ncs ii solubilizer ( amersham corporation ) and incubated at 50 ° c . in a water bath overnight . radioactivities were determined by a scintillation counter . protein contents were measured by the bio - rad protein assay kit . to determine whether cd - cs could be internalized by hepatoma cells , we treated hepg2 cells with [ 35 s ]- labeled cd - cs protein . at different time intervals , cells were extensively washed with regular medium . uptake of the label was determined by scintillation counting . the kinetics of protein uptake reached a plateau approximately 4 hr after the treatment . in a parallel experiment , the treated cells were further washed with a solution containing 0 . 25 m acetic acid . the kinetics of cd - cs uptake in the mild acid - washed cells followed the kinetics similar to that of regular washed conditions , suggesting that the cd - cs proteins were internalized , because these mild acid - washing conditions should have removed cell surface - associated ligand ( runegar et al ., 1997 ). the uptake of recombinant protein can be competitively inhibited by a peptide ( e35 ) containing the 23 amino acid sequence in the rii + region that recognizes cs receptor ( fig2 triangles ) but not the unrelated sequence ( a 128 , not shown ). these results demonstrated that cs - cs could be internalized by hepatoma cells by a receptor - mediated mechanism . similar results were obtained with mouse colorectal metastatic cells ( mca26 ). to determine whether the cd - cs protein could exert cell type - specific killing activities in the presence of exogenously added 5 - fc , we performed the following experiments using a pair of cell lines , i . e ., hepg2 which contains receptors for the cs protein and hl60 which does not ( ding , et al ., 1995 ; rich et al ., 1990 ). first , hepg2 and hl60 cells were treated with recombinant proteins cd - cs , cs , and cd for 4 hrs . each set of treated cells was divided into two parts : one part was extensively washed with the regular medium to remove the recombinant proteins ; the other part was not . cells in both parts were then treated with 5 - fc . as expected , unwashed hepg2 cells treated with cd - cs or cd alone were sensitive to the subsequent 5 - fc treatments , owing to the manufacture of cytotoxic 5 - fu from 5 - fc by the cd activities in the medium ; the cs - treated hepg2 cells were not sensitive to 5 - fc ( fig3 a . hatched bars ). in the washed cultures , only cd - cs - pretreated cells were sensitive to the treatment of 5 - fc ( solid bars ). these results suggested that cd - cs could be taken up by hepg2 cells and exerted cell killing activity whereas cd could not . however , in similar experiments with hl - 60 cells ( fig3 b ), removal of cd - cs or cd from the treated cells failed to induce cell death upon subsequent addition of 5 - fc . the failure of cell killing was not due to intrinsic resistance of hl - 60 cells to 5 - fu , because cell death was observed in the unwashed control . these results indicate that both cd - cs and cd could not be taken up by hl - 60 cells , consistent with the finding that these cells lacks cs receptor . cd - cs - mediated cell killing was concentration - dependent with an ld 50 for hepg2 , rat hepatoma h - 4 - ii - e , and mca26 cells of approx . 0 . 22 μm ( data not shown ). to demonstrate that the observed cell killing was related to the production of 5 - fu from 5 - fc , hepg2 and hl60 cells were treated with cd - cs protein for 4 hrs . cells were then washed and incubated in regular medium supplemented with [ 3 h ] 5 - fc . two days later , cell extracts were prepared . the amounts of [ 3 h ] 5 - fc and [ 3 h ] 5 - fu in the cell extracts were measured using thin - layer chromatography . likewise , the amounts of these radioactively labels in the medium was measured . as shown in fig4 & gt ; 30 % [ 3 h ] 5 - fu conversion were found in the cultured medium and in cell extracts derived from hepg2 cells , whereas only & lt ; 5 % conversion was seen in bl60 cells . the percentages of conversion indicated here were normalized by cell numbers . it is not likely that the substantial amounts of [ 3 h ] 5 - fu found in the medium was contributed significantly by cell lyses , because no apparent cell death was observed under these conditions . these results indicate that the internalized cd - cs in hepg2 cells was functional , i . e ., capable of synthesis 5 - fu from 5 - fc . more important , the synthesized 5 - fu could leak into the medium , which could exert bystander effect of cell killing ( huber et al ., 1993 ; huber , et al ., 1994 ). to demonstrate the bystander effect , we treated hepg2 cell with cd - cs for 4 hrs . cells were harvested and extensively washed with the regular medium . the pretreated cells were mixed in different ratios with freshly prepared hepg2 or hl60 cells . the populations were cultured in fresh dmem containing 5 - fc for 3 days . in parallel , cd - cs - untreated cells were similarly mixed with fresh cells as controls . as shown in fig5 mixed populations containing 0 %, 10 %, 50 %, and 100 % cd - cs - treated hepg2 cells resulted in 8 %, 50 %, 85 %, and 100 %, respectively , of total hepg2 cell death ( fig5 solid bars ) whereas the controls showed less than 10 % total cell death in all cases ( fig5 hatched bars ). similar results were obtained using hl60 cells for mixing ( data not shown ). together , these results demonstrated the bystander effect of the cd - cs / 5 - fc strategy . prolonged stability and sustained prodrug cell killing activity using cd - cs fusion protein two experiments were carried out to demonstrate the stability of the internalized cd - cs : first , confluent hepg2 cells were treated with [ 35 s ] cd - cs or with [ 35 s ] cs for 4 hrs . cells were washed to remove the radioactive labels and replenished with fresh medium . at different time intervals from 2 hrs to 7 days , cell extracts were prepared and total proteins were separated by sds - page . gels were stained with coomassie blue to view the protein loading in different samples ( fig6 panels c and d ) and then autoradiographed to view the stability of the labeled protein ( fig6 panels a and b ). densitometric analyses were used to quantify the remaining intracellularly labeled proteins using the 2 - hr time point as reference of 100 % ( fig6 panel e ). these analyses revealed that the internalized cd - cs proteins were reduced approximately 50 % 2 days after the delivery and remained stable thereafter for at least 7 days , whereas the internalized cs protein continued to decline to about 5 % at 7 days post - delivery . these results demonstrated that the recombinant cd - cs protein exhibited enhanced intracellular stability . second , to demonstrate that the internalized cd - cs remains functional several days after delivery , we carried out cell killing experiments . hepg2 cells were treated with 0 . 5 μm of cd - cs or cd for 4 hrs and the recombinant proteins were removed from the medium and the cells were maintained in fresh medium thereafter . at different time intervals , cells were treated with 5 - fc . sustained cell killing was observed even 28 days after the removal of the recombinant protein ( fig7 ). since cd was not internalized , only basal level of cell killing (& lt ; 10 %) was observed in the cd - treated cells . these results , collectively , indicated the sustained stability and prolonged cell killing properties of the recombinant fusion protein . demonstration of hepatic targeting specificity of cd - cs in animals after intravenous injection previous study has demonstrated that intravenously injected recombinant cs protein containing rii + sequence rapidly reached hepatocytes ( cerami et al ., 1994 ). moreover , the injected cs protein was not detected in organs including bladder , heart , kidney , lung , and spleen ( cerami et al ., 1994 ). to demonstrate that cs can target the cd - cs fusion to the liver , we carried our investigation o the targeting profile of cd - cs fusion protein in balb / c mice . all the recombinant proteins including radioactively labeled protein prepared from bacteria were free of endotoxin contamination . this was accomplished by passing the purified recombinant protein through an affi - prep polymyxin support ( bio - rad ). the labeled protein ( 20 , 000 cpm ) was injected into the tail veins of mice . at different time intervals , animals was exsanguinated , different organs were removed , rinsed with tris - buffered saline , blotted on filter paper , and counted for radioactivity . the amount of labeled cd - cs protein in the blood was also determined . the contribution of the labeled protein in the blood to various tissues were subtracted according to a previously described method ( cerami et al ., 1994 ). as shown in fig8 it is demonstrated that 20 minutes after the injections , approximately 80 % of the labeled protein were distributed in the liver . twenty four hr later , a great majority of the counts (˜ 75 %) remains in the livers , another 5 ˜ 10 % each were in intestines and kidney , whereas the remaining were in various organs . the distribution of the labeled counts was in a general agreement with that of the labeled cs protein published previously ( cerami et al ., 1994 ). these results indicated that cd - cs exhibits the similar targeting specificity as cs protein . demonstration of cs - protein binding to the human liver metastasis of colorectal carcinoma biopsy specimens to demonstrate whether cs protein can bind to liver metastases of colorectal cancer , we prepared frozen sections of liver mcc from six patients . the tissue sections were incubated with full - length recombinant cs protein . the tissue - bound cs was probed with a monoclonal antibody ( mab2a10 ) reacting with the repeat domain of the ligand detected . fitc conjugated rabbit anti - mouse igg antibody was used as secondary antibody to detect the binding . the tissue sections were counterstained by propidium iodine . by this fluorescence immunomicroscopy , strong staining was observed in the section incubated with cs protein with concentration ranging from 0 . 64 to 0 . 06 μm ( fig9 d ). the staining is mainly distributed at the surface of the cell . all 6 patients showed greater than 90 % positive staining cells . moreover , the staining could be blocked by e35 peptide containing the 18 amino acids recognized by the receptor ( fig9 c ). as a control , frozen sections of rat livers were similarly stained . in an agreement with the previous results , the staining closely followed the contours of the sinusoidal spaces of the hepatic lobules ( fig9 b ). as other controls , staining was not observed in spleen , lung , heart , where no receptors for the cs protein have been noted ( not shown ). these results demonstrate that mcc containing binding activities to cs protein . these results are consistent with the previous reports showing the presence of heparan sulfate proteoglycan ( hspg ) in human liver metastases of colorectal cancers ( tovari et al ., 1997 , lozzo , et al ., 1989 ) and crc ( jozzo et al ., 1990 ). binding of cs protein to primary hepatocellular carcinomas has been noted previously ( ding et al ., 1995 ). as the molecular identity of cs receptor has not been fully revealed and because dna probe or antibody for the receptor is not available at the present . it is difficult to quantify the exact amounts of cs receptor in these tissues . nonetheless , our indirect immunoprobing shows that normal liver sections and mcc from patient biopsies display similar levels of staining intensity ( fig9 ). these results provide a justification for the development of targeted prodrug therapy of mcc . cerami , c ., fravert , u ., sinnis , p ., takacs , b ., and nussenzweig , v . rapid clearance of malaria circumsporozoite protein ( cs ) by hepatocytes . j . exp . med . 179 : 695 - 701 , 1994 . cerami , c ., frevert , u ., sinnis , p ., takacs , b ., clavijo , p ., santos , m . j ., and nussenzweig , v . the basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of plasmodium falciparum sporozoites . cell 70 : 1021 - 1033 , 1992 . cohen , a . m ., winawer , s . j ., friedman , m . a ., and gunderson , l . l . 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