Patent Application: US-94978001-A

Abstract:
the invention relates to the field of apoptosis . the invention provides novel therapeutic substances , for example novel therapeutic proteinaceous compounds that can contain apoptin alone or jointly with other proteinaceous protein or protein fragments , especially in those cases when cells are derailed such as cancer -, auto - immune - derived cells .

Description:
the invention provides a system to produce and deliver intracellularly ( recombinant ) proteinaceous substances comprising apoptin or functional equivalent or functional fragments there of , or recombinant proteinaceous substances with apoptin - like activity . the invention further provides for the addition of further optional modular peptides . this can include an epitope tag , allowing easy detection and immuno - precipitation without direct steric hindrance of associations of apoptin with cellular proteins . the invention provides or describes all steps needed for the production of recombinant apoptosis inducing agent apoptin , or derivatives of apoptin that have a similar tumor specificity . the recombinant protein can be produced in e . coli , insect cells by means of a baculovirus - based vector , or in yeast strains ( such as pichia pastoris ). the invention provides evidence that the apoptin or apoptin - like proteinaceous substance does not need to be folded properly in the producer cell , enabling the production of recombinant apoptin or apoptin - like proteinaceous substances in transgenic plant cells , for mass production . the invention proposes a modified metal affinity tag for optimal purification of the recombinant protein . by using a double his - tag next to the transduction domain , a significantly better binding to nickel beads is achieved , resulting in the possibility to wash the recombinant apoptin or apoptin - like proteinaceous substances under very stringent conditions resulting in an optimal purification of apoptin or apoptin - like proteinaceous substances . the invention describes the use of a transduction domain fused to recombinant apoptin protein . this domain allows the recombinant protein to pass through the cellular membrane . this domain can consist of a transduction domain derived from hiv tat , or of any other known transduction domain . the invention describes the use of cav - derived vp3 ( apoptin ) proteinaceous substance as part of a fusion protein , or derivatives of apoptin proteinaceous substances that are selected on their ability to specifically induce apoptosis in tumor cells . the invention also provides a therapy for cancer , autoimmune diseases or related diseases which is based on apoptin or apoptin - like proteinaceous substances . the invention also provides a method to remove aberrant cells in their first stages of transformation and pre - malignant lesions . the invention describes a research tool based on apoptin or apoptin - like proteinaceous substances to unravel apoptosis and transformation pathways or as diagnostics for determining the tumorigenic status of patient material . 1 . production and purification of mbp apoptin ( mbp - vp3 ) protein 1 . 1 mbp - vp3 expression construct the vp3 gene was fused in frame in a bacterial expression vector encoding the maltose - binding protein ( mbp ), a 10 asn linker and a thrombin - cleavage site . the expression system is based on a modified pmal - c2 plasmid vector ( new england biolabs , usa ), in which the factor xa site has been replaced by a thrombin - cleavage site . this modified vector was named pmaltb . a pcr fragment consisting of the complete apoptin or vp3 sequences and at the 5 ′- end a bamhl and at the 3 ′- end a sali site , was cloned in pmaltb . the resulting fusion product consists of a n - terminal mbp moiety that is separated from the vp3 part by a 10 - asn linker and a thrombin - cleavage site as shown in fig1 . the dna and protein sequence of the fusion product is given in fig2 and fig3 respectively . the resulting plasmid is called pmbp - vp3 and the proteinaceous substance encoded by this plasmid is designated mbp - vp3 . the correct sequence of the essential parts of the mbp - vp3 construct was confirmed by means of the sanger method ( sanger et al ., 1973 ) and carried out by base - clear , leiden , nl . the plasmid pmbp - vp3 was transformed in bacteria derived from strain bl21 ( de3 ), and initial expression studies showed that mbp - vp3 protein constitutes roughly 10 % of the soluble cytoplasmatic protein , after 3 hours induction with 1 mm iptg . purification was carried out on amylose beads at ph 7 . 4 and 1m nacl . subsequently , elution in buffer containing 20 mm hepes 8 . 0 , 50 mm nacl , 1 mm edta , 1 mm dtt , 1 mm maltose , yielded about 100 mg protein per liter of bacterial culture . this purified protein was loaded on a uno - s1 chromatography column ( biorad ), and the fractions that elute at 400 - 500 mm nacl , 20 mm hepes ph7 . 4 , 1 mm edta were pooled , dialyzed against pbs and concentrated with millipore ultrafree spin filters . the negative control preparation being the maltose binding protein ( mbp ) was produced and purified in the way as been described for mbp - vp3 . vp3 lacking a stop codon was cloned in the ndei site and noti site of the iptg - inducible bacterial expression plasmid pet22b , which provides in frame a 6 - histidine tag and a stop codon . the essential regions of the final pvp3h6 dna construct were sequenced according to the sanger method and carried out by base clear , leiden , nl . the map of the essential regions of pvp3h6 is shown in fig4 a and the dna and protein sequence of the his - tagged vp3 ( apoptin ) region is given in fig5 and 6 , respectively . the vp3h6 construct was transformed in bl21 ( de3 ) bacteria ( novagen ) and a colony was grown at 37 ° c . to an od600 of ca . 0 . 6 . expression was then induced by adding 1 mm iptg and the cells were grown for an additional 3 hrs . after harvesting by centrifugation , the cells were lysed in a bead - beater ( biospec inc .) in lysis buffer ( containing 50 mm nahepes ph 7 . 4 , 100 mm nacl , 1 mm edta , 1 mm dtt and protein inhibitors ( complete , boehringer )). the inclusion bodies were harvested by centrifugation and made soluble by suspending in solubilisation buffer ( containing 50 mm hepes ph 7 . 4 , 20 mm glycine , 1 mm edta , 10 mm dtt , 8 m urea ). the cleared supernatant was loaded directly on uno - s12 ( biorad ), pre - equilibrated with : 20 mm kpo 4 , 5 mm imidazole , 6 m urea , 1 mm gsh . the vp3h6 protein was eluted with a nacl gradient ( 0 - 1 m nacl at 3 ml / min with a total volume of 200 ml ). vp3h6 eluted between 400 and 650 mm nacl . it was loaded directly on ni - nta ( qiagen ) ( pre - equilibrated in 20 mm kpo 4 ph 7 . 4 , 5 mm imidazole , 500 mm nacl , 6 m urea at 4c ). next , the column was washed with 20 mm kpo 4 ph 7 . 4 , 20 mm imidazole , 500 mm nacl , 6 m guhcl . the guhcl was removed by washing with 20 mm kpo 4 ph 7 . 0 , 400 mm nacl , 2 mm mgcl2 , 1 mm gsh , and vp3h6 protein eluted with 20 mm kpo 4 ph 7 . 4 , 400 mm nacl , 500 mm imidazole , 2 mm mgcl2 . the vp3h6 protein containing peak fractions were pooled and 5 mm edta was added to remove nickel traces . the sample was dialysed ( 1 volume to 200 ) to 20 mm kpo 4 ph 6 . 5 , 400 mm nacl , 2 mm mgcl2 , 1 mm dtt . finally , the vp3h6 protein was concentrated on centricon ym3 filters ( millipore ) to at least 7 mg / ml . all dynamic light scattering ( dls ) measurements were recorded on a dynapro - ms / x ( protein solutions inc . ), at room temperature . bsa was used as a control ( 0 . 5 mg ml − 1 in pbs , 0 . 5 mm edta ). mbp - vp3 was measured at 10 mm ( pbs , 0 . 5 mm edta ), refolded vp3h6 at 35 mm ( in 20 mm kpo 4 ph 6 . 5 , 400 mm nacl , 2 mm mgcl 2 ). per experiment , between 20 and 40 measurements were collected . protein molecular weight ( kda ) was estimated from hydrodynamic radius ( rh ) by : mw ( kda )=[ 1 . 68 × rh ] 2 . 34 . biotin labeling . fresh mbp - vp3 ( 20 mg ml − 1 , 0 . 1 m nahco 3 ph 8 . 3 ) was incubated with 5 mm sulfo - nhs - lc - biotin ( molecular probes , inc .) at room temperature for 3 hours . the reaction was terminated by adding 10 mm ethanolamine . the labeled protein was passed over a pd - 10 desalting column ( pharmacia ), and equilibrated in 20 mm hepes ph 7 . 4 , 0 . 1 mm edta , to remove unincorporated label . electron microscopy . both labeled and unlabeled mbp - vp3 ( 30 mg ml − 1 , in 20 mm hepes ph 7 . 4 , 0 . 1 mm edta ) were filtered over 0 . 22 mm and adsorbed to a carbon - coated polioform layer grid . both samples were incubated with concentrated streptavidin - gold conjugate ( 5 nm ) ( kpl inc .) and stained with 3 % uranyl acetate . electron microscopy was performed on philips tem 410 transmission electron microscope . in e . coli , mbp - vp3 ( or mbp - apoptin , the terms are used interchangeably herein ) is abundantly expressed in a soluble form . after affinity chromatography on amylose resin and cation exchange chromatography , mbp - vp3 migrates as a single species on a size exclusion chromatography column ( superose 6 hr 10 / 30 ). its molecular weight was calculated to be 2 . 5 ± 0 . 3 mda . this feature was found to be independent of protein concentration ( 0 . 5 to 25 mg ml − 1 ). increased ionic strength ( 2 × pbs ) and the presence of detergent ( 0 . 5 % chapso or 1 % triton x - 100 ) do not disrupt the mbp - vp3 complex . its size was corroborated by dynamic light scattering ( dls ), which showed only one solute species with an average hydrodynamic radius ( rh ) of 17 . 9 ± 2 . 0 nm ( corresponding to an estimated molecular weight of 2 . 9 ± 0 . 6 mda ). the variation in the average diameter of the mbp - vp3 particle between different protein batches remained within experimental error ( 16 to 20 nm ). as demonstrated by reducing and non - reducing sds - page , mbp - vp3 is a non - covalently linked complex . in addition to the full - length 58 . 5 kda species , three less abundant expression products of 56 . 7 , 53 . 3 and 50 . 1 kda consistently co - purify with mbp - vp3 . all three are detectable by anti - apoptin monoclonal antibody ( mab ) 111 . 3 ( epitope : residues 18 to 23 , danen - van oorschot et al ., 1997 ). since mbp was found to be highly protease - resistant , these products arise from partial c - terminal degradation of the apoptin moiety . the smallest degradation product is an mbp fusion to an n - terminal fragment of apoptin of approximately 70 residues . under native conditions , these products cannot be removed by conventional methods , which demonstrates that the degradation products are still incorporated into the mbp - vp3 complex . in the linker peptide that connects the mbp and apoptin moieties , there is a thrombin cleavage site . when the fusion protein is digested with thrombin , apoptin remains part of the complex whereas mbp is released . hence , all biophysical and functional studies are conducted with intact mbp - vp3 . trace amounts of unfused mbp do not co - elute with mbp - vp3 , showing that the apoptin moiety has a negligible affinity for mbp . we conclude that the multimerization behaviour of mbp - vp3 depends , probably , entirely on the apoptin moiety . electron microscopic analysis of purified mbp - vp3 shows a uniform population of globular particles with a diameter of around 20 nm . for identification , mbp - vp3 is labeled with sulfo - nhs - lc - biotin and incubated with a streptavidin - gold conjugate ( 5 nm gold ). between 5 and 10 % of globules show gold binding , while a selective binding is less than 0 . 1 %. vp3h6 ( or apoptin - h 6 , the terms are used interchangeably herein ) was expressed at 20 to 40 mg l − 1 as inclusion bodies . reducing growth temperature or induction time , lowering of the iptg concentration or varying the lysis conditions did not increase its solubility . after solubilization in 8 m urea , vp3h6 was purified to near homogeneity using cation exchange chromatography . immediately afterwards , it can be refolded in one step while bound to ni 2 + - nta with a net efficiency of around 50 %. supplementing the expression medium with zn 2 + did not promote the expression of soluble vp3h6 , nor did it increase its refolding yields . nevertheless , the presence of certain divalent cations did improve the solubility of purified , refolded vp3h6 . in this respect , mg 2 + was the most effective of all cations tested . however , refolded vp3h6 has only a mild preference for mg 2 + , when compared to ca 2 + , zn 2 + or mn 2 + . hence , we assume that the stabilizing effect of mg 2 + relies on a specific shielding of charged residues . on superose 6 hr 10 / 30 , refolded vp3h6 migrates as a single species with a molecular weight of 400 ± 50 kda . the particle size is independent of protein concentration ( 1 to 7 mg ml − 1 ). there are two additional peaks at 30 and 10 kda , but these do not contain any mab 111 . 3 - reactive species . the particle size of vp3h6 is confirmed by dls , indicating a single solute species with an rh of 8 . 7 ± 1 . 0 nm . the variation in complex size between protein batches remained within experimental error ( 8 to 10 nm ). the apparent molecular weight of vp3h6 on sds - page is 18 kda . there are a number of additional species migrating at around 8 , 34 and 48 kda , which can be detected by mab 111 . 3 . all of these additional species co - elute with the complex during size exclusion chromatography . the vp3h6 complex is non - covalent , as shown by non - reducing sds - page . the smallest mab 111 . 3 - reactive species corresponds to an n - terminal fragment , which lacks the c - terminal hexahistidine tag . like in the mbp - vp3 complexes , fragments truncated at the cterminus remain associated and co - purify . we demonstrate , with a range of techniques , that recombinant constructs of apoptin form multimeric globules of a distinct size . the average sizes of mbp - vp3 and vp3h6 homoglobules suggest a monomer content of 45 and 30 subunits , respectively . however , the presence of an 18 - residue linker between mbp and apoptin in the mbp - vp3 complex will lead to an overestimation of its hydrodynamic radius . probably both types of apoptin homoglobules consist of 30 to 40 subunits , which suggests that they are structurally equivalent . our em studies indicate that apoptin multimers have a roughly spherical shape . our findings could indicate that apoptin is active as a multimeric species . we suggest that apoptin &# 39 ; s propensity to form globular multimeric aggregates of a well - defined size is crucial for its biological function . also other apoptosis associated proteins form multimers ( bax , apaf - 1 ), and these multimeric complexes probably have a well - defined internal structure . bax oligomers form pores in the mitochondrial outer membrane , releasing cytochrome c into the cytoplasm ( antonsson etal , 2000 and 2001 ). apaf - 1 activates caspase - 9 upon oligomerisation ( cain et al , 1999 ). if apoptin homoglobules have a well - defined internal structure , they may likewise exhibit structural or enzymatical characteristics . fluorescent labeling : fluorescent labels used : 1 , 5 - iadeans ( ia , dissolved in pbs ), fluorescein - maleimide ( fm , dissolved in 20 mm na 3 po 4 ), pyrene - n - maleimide ( pm , dissolved in dmso ) ( all : molecular probes ). a fresh stock solution of label ( around 10 mm ) was diluted to a final concentration of 1 to 2 mm in 2 ml of dialyzed mbp - vp3 ( 5 to 10 mg / ml ) in pbs , 0 . 1 mm edta . the mixture was incubated overnight in the dark at 4 ° c . for ia and fm co - labeling , an equimolar amount of label ( 1 mm ) was used . the reaction was stopped by adding 10 mm dtt . the non - conjugated label was removed by passing the sample twice over a 5 ml p6dg cartridge ( biorad ), equilibrated in pbs . the level of label incorporation was determined from : [ a c / e label ]×[ mw ( kda )/ c protein ( mg / mil )]. all samples were stored in the dark at 4 ° c . fluorescence emission and excitation spectra were recorded on a perkin elmer ls - 50b . settings : 2 . 5 to 6 nm slit width , 120 nm / min scan speed , final average of 3 separate spectra . background emission and excitation spectra were recorded of the respective filtered dialysis buffers . intrinsic tyr fluorescence measurements . free l - tyr ( sigma ) was used as a control , diluted in dialysis buffer from a stock solution of 100 mm in 1 m hcl . the refolded vp3h6 sample was prepared as described before , spectra were first recorded in the presence of 2 mm mgcl 2 , 0 . 1 mm zncl 2 and then after a 2 h incubation at rt - in the presence of 5 mm edta . the protein concentration of the native sample was determined at 55 mm . in all cases , l exc was 280 nm . pm fluorescence . pm - b - me was used as a control : a stock solution of pm ( 5 mm ) was reacted with a 10 × molar excess of b - mercaptoethanol ( b - me ). this was first diluted 1 : 100 in meoh and then to 1 mm in pbs + 0 . 1 edta . mbp - vp3 - pm was also diluted to 1 mm in the same buffer . for time course measurements , mbp - vp3 - pm was diluted to 10 mm ( 0 . 6 mg / ml ) and incubated in the dark at 4 ° c . to compensate for concentration changes due to protein precipitation , all spectra were normalized to the 377 nm fluorescence emission peak , which was not affected by monomer / excimer formation . for pm fluorescence , the l exc was 341 nm . for trp - pm fret , l exc was 280 nm . ia / fm fret . for fret measurements , mbp - vp3 - fm was mixed with mbp - vp3 - ia at a 1 : 10 label ratio , after which the total protein concentration was adjusted to 10 mm ( 0 . 6 mg / ml ). the mixture was then incubated in the dark at 30 ° c . after 1 , 3 , 6 and 24 h , samples were filtered ( 0 . 22 mm ) and diluted to 1 mm , after which fluorescence spectra were recorded ( l exc = 338 nm ). to compensate for protein precipitation , the fret was expressed as the ratio between the ia emission ( 485 nm ) and the fm emission ( 518 nm ), denoted as f 518 / f 485 . in order to determine whether the architecture of mbp - vp3 homoglobules is either static or dynamic , a method based on fluorescence resonance energy transfer ( fret ), for studying subunit exchange between homoglobules was developed . fret originates from the electronic interaction between a fluorescent donor probe and a fluorescent or non - fluorescent acceptor . the excited state of the donor can decay by transferring its excitation energy to the acceptor probe . usually , the effective range of fret is between 10 and 100 å ( wu and brand , 1994 ). the distance - dependency of fret is summarized in the forster radius ( r 0 ), which is defined as the distance at which energy transfer between a specific donor / acceptor pair is 50 % effective . any useful fret - based approach to monomer exchange in mbp - vp3 requires site - specific labeling of either the mbp or apoptin moiety . since apoptin has four cys residues and mbp has none , the apoptin moiety can - in principal - be labeled selectively . the solvent exposure of protein sulphydryl groups can be quantified by reaction with dtnb ( riddles et al ., 1983 ). under native conditions , the dtnb - reactivity of freshly prepared mbp - vp3 is around 65 % of one molar equivalent of cys - hcl . in comparison , purified mbp alone showed no reactivity at all with dtnb . this demonstrates that not more than one cys residue in apoptin is solvent exposed . since c30 , c47 and c49 are likely to be buried due their localization in the n - terminus of apoptin ( see further ) and when the presence of the apoptin degradation products is taken into account , it can be argued that the single exposed cys residue in apoptin is c90 . therefore , cys - labeling of mbp - apoptin is thought to occur at a specific location on the protein surface . mbp - vp3 was labeled with 1 , 5 - iadeans ( ia ) and fluorescein - 5 - maleimide ( fm ). the ia label can act as a fret donor to fm with an r 0 of 46 å , which compares well with the probable dimensions of the apoptin portion of the mbp - vp3 particle ( garzon - rodriques et al ., 1997 ). when mbp - vp3 - ia is mixed with mbp - vp3 - fm , any exchange of subunits between homoglobules is expected to result in the formation of a new effective fret contact between an ia and fm label . therefore , an increase in the ratio between fm and ia fluorescence ( f 518 / f 485 ) can be interpreted as being the result of subunit exchange . however , prolonged incubation of mbp - vp3 at 30 ° c . can lead to the formation of a specific apoptin aggregates which are prevented from precipitating by the solubilizing propensity of mbp . this can introduce an error in ia / fm fret measurements . in order to evaluate the relevance of this effect , mbp - vp3 was first labeled with n -( 1 - pyrene ) maleimide ( pm ). a well - documented feature of pyrene fluorescence is the formation of so - called ‘ excimer ’ pairs ( lehrer , 1997 ; sahoo et al ., 2000 ). pyrene excimer fluorescence is characterised by a broad emission peak around 465 nm and occurs when an excited pyrene comes into close proximity with a ground - state pyrene (& lt ; 10 å ). when the pyrene - labeled apoptin domain in mbp - vp3 - pm undergoes progressive a specific aggregation , this is likely to give rise to an increase in excimer fluorescence ( panse et al ., 2000 ). therefore , an increase in the monomer / excimer ratio can be regarded as a measure of apoptin intradomain aggregation . an additional informative property of mbp - vp3 - pm stems from the fact that trp can act as a fret donor for pm ( johnson et al ., 2001 ). since mbp contains eight trp residues and apoptin contains none , any a specific aggregation of apoptin with mbp is expected to result in a rise in trp / pm fret . in proteins , the r 0 of the trp / pm donor / acceptor pair is between 20 and 30 å ( wu and brand , 1994 ). since the trp residues in mbp are evenly distributed throughout the molecule , any increase in trp / pm fret is a clear indication of interdomain aggregation ( quiocho et al ., 1997 ). in the pyrene emission spectrum of fresh mbp - vp3 - pm , there is a significant amount of excimer fluorescence , when compared to pm - me ( pm - b - mercaptoethanol ). the average pm incorporation level in mbp - vp3 is 30 %, which shows that - on average - a fraction of the exposed cys sites are less than 10 a apart . the ratio of monomer / excimer fluorescence is essentially unchanged after 6 h at 30 ° c . after 24 h at 30 ° c ., the excimer fluorescence is increased by about 25 % ( fig1 b ). the initial trp - pm fret in mbp - vp3 - pm is very small and after 24 h at 30 ° c ., the changes in trp / pm fret are negligible . both lines of evidence indicate that in mbp - vp3 , a specific aggregation only begins to be a detectable event after more than 6 h of incubation at 30 ° c . moreover , aggregation takes place only within the apoptin domain . also , any increase in f 518 / f 485 can only be ascribed to subunit exchange exclusively up to 6 h of incubation . the incorporation levels for mbp - vp3 - ia and - fm were 150 and 75 %, respectively . since the fusion protein contains an amount of degradation products that lack c90 , 75 % incorporation is likely to represent near complete cys - labeling by fm . a fraction of the ia label reacts with a lys residue on mbp , which was deduced from a strong trp / ia fret in mbp - vp3 - ia ( data not shown ). the r 0 of the trp / ia couple is 22 å , which is comparable to that of trp / pm ( wu and brand , 1994 ). so , the trp / ia fret can only be caused by the ia label located on mbp and not on apoptin . since the distance dependence of fret is in the order of r − 6 , the contribution of mbp - ia to apoptin - ia / fm fret is thought to be minimal . indeed , co - labeled mbp - vp3 - ia / fm has no significant trp / ia ® fm fret , although that was expected in view of the intensity of ia fluorescence . the fact that the ia label is not entirely specific for the apoptin moiety means that a reliable estimate of the ia / fm distance is not possible , since a fraction of donor label does not effectively participate in fret . besides , the observation that mbp - vp3 - pm displays clear excimer fluorescence shows that the cys - labeling sites are well within the r 0 of ia / fm . in all , the heterogeneity of ia - labeling does not affect the relationship between f 518 / f 485 and subunit exchange in mbp - vp3 . furthermore , after 24 h incubation at 30 ° c ., the rh of labeled mbp - apoptin complex was indistinguishable from unlabeled and unincubated mbp - vp3 , as was shown by dls . it was also established that mbp - vp3 - ia and mbp - vp3 - fm are equally sensitive to denaturation at 30 ° c . mbp - vp3 - ia was combined with mbp - vp3 - fm ( 10 : 1 label content ) and incubated at 30 ° c . in pbs , in the presence of either 0 . 5 mm edta , 0 . 1 mm zncl 2 , 2 . 5 mm mgcl 2 . there is a clear increase in f 518 / f 485 over the course of 6 h at 30 ° c ., in pbs / edta . this demonstrates that mbp - vp3 homoglobules are capable of exchanging either monomers or oligomeric subunits under physiological conditions . the presence of either mg 2 + or zn 2 + does little to change the exchange rate . however , the presence of edta greatly improves thermal stability of the fusion protein . to test for the influence of detergents on the exchange rate , labeled mbp - vp3 was assayed in pbs , 0 . 5 mm edta with either 1 % chapso or 1 % triton x - 100 . the increase in f 518 / f 485 in pbs / edta + chapso is only around 30 % faster than it is in pbs / edta . clearly , the presence of detergents has relatively little effect on the dynamics of the mbp - apoptin homoglobule . nevertheless , chapso is clearly favored over triton x - 100 . at the concentration used here , the tendency of triton x - 100 to form micelles probably outweighs interaction with mbp - vp3 , more so than in chapso . overall , these results indicate that the multimeric mbp - vp3 or vp3h6 complexes are dynamic complexes . dna - affinity chromatography . mbp - apoptin was applied to a ss or dsdna - cellulose column ( 1 . 5 × 2 cm ; pharmacia ), equilibrated in 20 mm hepes ph 7 . 4 , 50 mm nacl , 1 mm edta ( or 0 . 1 mm znso 4 or 2 . 5 mm mgcl 2 ). the column was then eluted with a nacl step gradient ( 5 cv &# 39 ; s per step ), 50 to 2000 mm nacl . idna fragment elution . idna ( ci857 , strain 7 ) ( roche ) was digested with rsai ( 0 . 3 mg unite − 1 , 2 h 30 minutes ) ( neb ), after which rsai was deactivated by heating at 65 ° c . for 20 minutes . the digest ( 10 mm bis - trishcl ph 7 . 0 , 10 mm mgcl 2 ) was mixed directly with mbp - vp3 ( 50 mg dna / mg protein ) and incubated on ice for 15 minutes . the mbp - vp3 - dna complex was loaded on an amylose column ( 0 . 5 × 1 . 0 cm ), which was eluted with a nacl step gradient ( 5 cv &# 39 ; s per step ), 50 to 2000 mm nacl . fractions were desalted using qiaquick spin filters ( qiagen ) and separated on 1 . 2 % agarose . when apoptin is expressed in situ in transformed cells or introduced via microinjection , apoptin forms intranuclear aggregates . noteborn ( et al ., 1994 ) has suggested that apoptin associates with heterochromatin in apoptotic cells . we evaluated the dna - binding of mbp - apoptin in vitro by means of ss and dsdna - affinity chromatography . at ph 7 . 4 , 50 mm nacl , all mbp - apoptin binds to both ss and dsdna adsorbed to cellulose with a binding capacity of 1 . 5 and 2 . 2 ( mg protein / mg of dna ) respectively . the elution profiles of mbp - vp3 on ss and ds dna are essentially the same . in both cases , mbp - vp3 elutes as a heterogeneous species with an optimum around 200 mm nacl . supplementing the buffer with either mg 2 + or zn 2 + does not have any effect on the binding capacity and elution profile of mbp - vp3 , nor does it induce specificity for either ss or dsdna . next , we devised a method for detecting any sequence specificity in the dna - binding properties of mbp - vp3 . ldna was digested with rsai , to yield a set of blunt - ended dna fragments ranging in size between 0 . 1 and 2 . 5 kb . if mbp - vp3 displays sequence specificity , it is likely to bind certain fragments with a relatively high affinity . mbp - vp3 was incubated with the ldna / rsai fragment collection , bound to amylose and eluted with a nacl gradient . subsequently , the fractions were separated on agarose gel . clearly , there is no obvious specificity for one or more fragments . mbp - vp3 displays a higher for large fragments , but this is expected to stem from a cooperatibe binding effect . therefore , we conclude that mbp - vp3 is a general dna - binding protein without significant sequence - specificity . furthermore , both the mbp - vp3 / dna and dna / mbpvp3 elution profiles show that a fraction of the mbp - vp3 / dna complexes are resistant to treatment with 2 m nacl . apparently , mbp - apoptin is able to form particularly heterogeneous complexes with dna , some of which are effectively irreversible . to assay the ability of apoptin protein to specifically induce apoptosis in tumor cells a micro - injection system was set up . human osteosarcoma - derived saos - 2 tumor cells , jurkat t cells and normal human diploid vh10 primary cells were cultured on glass cover slips . jurkat t cells are suspension cells , so they were cultured on glass surfaces pre - coated with the lectin wheat germ agglutinin to cause adherence 1 day prior to microinjection . in addition , we obtained human normal primary mesenchymal stem cells and human normal primary hepatocytes from biowhittaker , which were cultured no more than 1 - 3 passages in medium and under conditions recommended by the manufacturer . for the cells from biowhittaker , we included the following control : microinjection into the nucleus of a dna plasmid , cmv - fadd , which is a positive control for apoptosis induction . the cells were micro - injected in the cytoplasm with protein mbp - vp3 , vp3h6 , or mbp alone at 3 mg / ml using an eppendorf micro - injector with the injection - pressure condition of 0 . 5 psi , or in the nucleus in the case of cmv - fadd dna ( 50 ng / ul ). the cells were co - injected with dextran - rhodamine ( mw : 70 kda ; molecular probes , leiden , nl ) to be able to later identify injected cells . the cells were incubated at 37 ° c . after injection until the cells were fixed with formaldehyde - methanol - acetone . presence of mbp - apoptin or his - tagged apoptin protein was determined with immuno - histochemistry with antibodies directed against mbp ( monoclonal mouse anti - mbp - clone r29 ; zymed laboratories , inc . and polyclonal rabbit anti - mbp - clone c18 / sc - 808 ; santa cruz biotechnology , inc .) and / or against apoptin ( vp3 ; anti - vp3c ). presence of fadd was determined with a monoclonal fadd antibody ( transduction laboratories ). apoptosis was counted as abnormal nuclear morphology after counter - staining with dapi and examination with an immunofluorescence microscope ( telford et al ., 1992 ). the results show that both mbp - vp3 and vp3h6 protein were able to induce rapid apoptosis in human tumor cells ( within 3 - 6 hours , but did not induce apoptosis in normal human cells . the mbp control protein did not induce apoptosis in any of the cell lines under these conditions . in contrast , fadd induced rapid apoptosis in normal cells , confirming that the cells were at least competent to undergo apoptosis , and highlighting the fact that they were strongly resistant to proteinaceous apoptin - induced apoptosis . the fact that both proteinaceous apoptin ( vp3 ) substances can induce apoptosis in human tumor cells lacking p53 implies that both proteinaceous apoptin substances induce apoptosis where known anti - cancer therapies fail . in addition , the fact that apoptin was completely harmless in the notoriously chemotherapeutically sensitive primary liver and stem cells underscores that proteinaceous apoptin should not be toxic in human patients . furthermore , the apoptin - characteristic tumor - specific cellular localization was observed for both mbp - vp3 as well as for vp3h6 protein . in the human tumorigenic saos - 2 cells , both mbp - vp3 and vp3h6 apoptin proteinaceous substance was predominantly located within the nucleus of pre - apoptotic cells . somewhat later , these mbp - vp3 - and vp3h6 - positive cells underwent apoptosis . in normal non - transformed human vh 10 cells , mesenchymal stem cells and hepatocytes , both mbp - vp3 and vp3h6 are mainly localized in cytoplasmic structures as has been described for apoptin by danen - van oorschot et al . ( 1997 ). in conclusion , exogenous produced proteinaceous apoptin substances , comprising a mbp fusion and / or a ( his ) 6 tag , harbor a tumor - specific apoptosis activity , which is non - toxic for primary human cells and that can be the base of a novel anti - cancer therapy . 4 . 2 apoptosis induction in the absence of de novo protein production . the following experiment shows an example of an application of the proteinaceous apoptin substance for studying the tumor - specific apoptosis pathway , which can be induced by apoptin . saos - 2 cells were incubated with transcription inhibitors cycloheximide ( 20 ug / mil or actinomycin d ( 10 ug / mil ) or translation inhibitors puromycin ( 10 ug / ml ) and emetin ( 10 ug / ml ), or without these inhibitors . the transcription - as well as the translation - inhibitors were obtained from sigma , mo , usa . the inhibition efficacy of the various transcription - and / or translation - inhibitors was proven to be accurate in saos - 2 cells by micro - injecting saos - 2 cells with a plasmid expressing the green - fluorescence protein ( gfp ). in all cases , no gfp protein could be produced by the saos - 2 cells when they were treated with any of the 4 translation - or transcription - inhibitors . in contrast , the saos - 2 cells micro - injected with the gfp - encoding plasmid produced the gfp in clearly detectable amounts as visualized by direct fluorescence techniques . for each type of inhibitor , 2 dishes with saos - 2 cells were used . as positive control , saos - 2 cells without inhibitors were also grown . all cell cultures were micro - injected with mbp - vp3 protein ( produced and purified as described above ) or mbp protein . in all cases when mbp - vp3 was micro - injected the majority of the saos - 2 cells underwent apoptosis 6 hours after micro - injection , whereas cells treated with one of the inhibitors did not undergo apoptosis , even at later time points . independent of the presence of inhibitors , cells injected with mbp protein did not go into apoptosis . these results indicate that apoptin - induced apoptosis is not inhibited by the transcription - inhibitors cycloheximide or actinomycin d and not by translation - inhibitors emetine or puromycin . therefore , one can conclude that apoptin protein can induce apoptosis in human tumor cells without de novo synthesis of cellular proteins . fluorescein - labelled mbp - vp3 was prepared as described under point 3 . 2 of the present application . in order to determine whether chemical coupling of a substance to proteinaceous apoptin was possible without altering its tumor - specific death characteristics , we directly labeled mbp - vp3 with a fluorescein moiety and performed microinjection experiments on saos - 2 and vh10 cells as described above . the only difference was in this case , the mbp - vp3 did not need to be stained with antibodies to observe it under fluorescence microscopy . these experiments show that fluorescein - labeled apoptin translocates to the nucleus of tumor cells and induces apoptosis with similar kinetics to that of unlabelled apoptin . furthermore , fluorescein - labeled apoptin remained in the cytoplasm of normal cells and did not induce apoptosis . these results show that proteinaceous apoptin can be readily coupled to a chemical sidegroup and that this coupling does not have to interfere with the tumor - specific functioning of apoptin . it is likely that other compounds or peptides can be similarly attached to proteinaceous apoptin , or functional fragments thereof , without loss of function or specificity ; such technology could allow a novel means for e . g . tumor - specific nuclear delivery or tumor - specific toxin delivery . the avialabilty of a sidegroup , which does not affect the properties of the apoptin protein , is also used as a diagnostic or research tool . furthermore , this sidegroup can also be a protein or a peptide , for example , tat , tumor - specific ( single - chain ) antibodies or egf as a delivery means . 4 . 5 apoptosis induction of the his - tagged nls - apoptin fragment containing a . a . 1 - 69 . in the following experiments , we have examined whether a bacterially produced chimeric protein consisting of the n - terminal half of apoptin ( amino acids 1 - 69 ) with at its n - terminus the nuclear localization signal ( amino acids n - terminal - proline - proline - lysine - lysine - lysine - arginine - lysine - valine - c - terminal ( seq id no : ______ )) of sv40 large t antigen and at its c - terminus a histidine tag , also reveals apoptotic activity in human tumor cells . the used expression plasmid encoding the apoptin protein fragment nls - vp3 / 1 - 69 - h6 is shown in fig4 b . the proteinaceous substance nls - vp3 / 1 - 69 - h6was purified and produced as has been described for the above mentioned histidine - tagged apoptin ( vp3 ) proteins . human saos - 2 cells were micro - injected with nls - vp3 / 1 - 69 - h6 protein as described for the above - mentioned micro - injected proteinaceous substances . as negative control , the human tumor cells were micro - injected with the non - apoptotic protein mbp and as positive control mbp - vp3 protein was micro - injected . within 6 hours after micro - injection , the majority of the saos - 2 cells containing both mbp - vp3 and nls - vp3 / 1 - 69 - h6 protein became apoptotic , whereas the cells containing mbp protein did not . comparable results were obtained with nls - vp3 / 1 - 80h6 . therefore , we conclude that proteinaceous substances containing the complete apoptin protein sequence or a specific apoptin protein fragment can induce apoptosis in human tumor cells . pmaltbvp3dn66 , mbp fusion of c - terminal 55 residues of apoptin ( mbp - apoptin ( 66 - 121 ). the c - terminal domain of apoptin ( orf bp 196 - 363 ) was cloned in pmaltb at bamhi and sa / l . the protein was expressed and purified as described for the mbp - vp3 protein . the protein is called mbp - vp3 - 66 - 121 . in order to determine whether additional tumor - specific fragments of apoptin could be generated , we made and tested mbp - vp3 - 66 - 121 in microinjection / immunofluorescence experiments in saos - 2 tumor cells and low - passage cd 31 - human normal dermal fibroblast cells exactly as described previously . mbp - vp3 , as a control , induced tumor - specific nuclear localization and death in saos - 2 but not cd31 - fibroblasts . interestingly , the mbp - vp3 - 66 - 121 protein behaved very similarly to the full - length protein both in function as well as in specificity . these results indicate that a c - terminal fragment of apoptin behaves as a functional fragment of the full length apoptin : the fragment is capable of killing tumor cells but does not induce apoptosis in normal cells . these results suggest that protein fragments smaller than full - length apoptin , and even peptides thereof , can be generated and used to achieve tumor - specific killing with no side effects in normal cells . a dna was constructed that encodes in frame for respectively a 6xhis - tag , a tat - transduction domain , an ha - tag , followed by the coding sequence for apoptin and a stop codon . this construct was cloned in frame in a pet16b ( novagen ) expression vector , which encodes for a 10 × his - tag followed by the insert . the resulting construct is referred to as petxnvp3 , and the resulting protein will be referred to as xnvp3 . see for the dna sequence , fig7 and for the protein sequence , fig8 . [ 0125 ] 5 . 1 . 2 description of expression and purification petxnvp3 plasmid dna was transformed into bl21 ( de3 ) plyss bacteria ( novagen ) on lb plates containing the appropriate antibiotics , and an antibiotic resistant colony was grown to od 600 of 0 . 6 in 1 liter lb plus antibiotics in a shaker flask . iptg was added to 1 mm , and the cells were grown for an additional 3 hrs . a bacterial pellet was obtained by centrifugation and sonicated on ice in 30 ml lysis buffer containing 150 mm nacl and 0 . 5 % triton . after centrifugation the pellet was again sonicated as above , and inclusion bodies were centrifuged as a pellet . expression of xnvp3 was confirmed by sds - page followed by coomassie - blue staining and western - blot analysis with 111 . 3 , a vp3 - protein specific monoclonal antibody . the inclusion bodies were re - suspended in nickel loading buffer ( nib ; 8m urea , 1m nacl , 50 mm phosphate buffer ph 7 . 5 , 40 mm imidazole ) and loaded onto a 4 ml ni - nta column ( qiagen ) equilibrated in the same buffer . the column was washed with 30 ml nlb , and washed with 10 ml monos loading buffer ( mslb ; 8m urea , 250 mm nacl , 50 mm phosphatebuffer ph 7 . 5 ) supplemented with 40 mm imidazole . the bound proteins were eluted with mslb supplemented with 250 mm imidazole . the protein containing fractions were pooled and subsequently loaded on a 5 ml mono - s chromatography column ( pharmacia ) and washed with 2 column volumes mslb . refolding and elution was performed by washing the mono - s column with 2m nacl , 50 mm phosphate buffer ph 7 . 5 . the protein containing fractions were pooled and the buffer was exchanged on a pd10 column ( pharmacia ) against dmem or pbs . the protein was aliquoted and frozen at − 80 ° c . until further use . to show that xnvp3 was able to penetrate into cells , and to assess its intracellular localization , the following experiment was performed . xnvp3 protein was added to cultured saos - 2 cells and vh10 cells at 50 nm concentration in medium . after one hour the cells were fixed with 80 % acetone , and the intracellular presence of xnvp3 protein was tested using antibodies 12ca5 ( directed against the ha - tag ) or 111 . 3 antibodies ( directed against vp3 protein ). in both tumor and normal cells xnvp3 was located in the nucleus , as seen with confocal laser microscopy . this shows that xnvp3 can transduce into cells . next , we examined whether xnvp3 protein can bind to apoptin associated proteins as has been reported for apoptin protein ( noteborn and danen - van oorschot , 1999 ). to that end , the following experiment was performed . xnvp3 protein was incubated with cos cells that had been transfected 48 hours earlier with expression vectors encoding myc - tagged aap1 , which is one of the apoptin - associating proteins , or lacz . subsequently , the cells were lysed and an immuno - precipitation ( ip ) was performed with 12ca5 . as a negative control , cos cells transfected with the same expression vectors but not incubated with xnvp3 were treated the same way . as a positive control , cos cells transfected with the same expression vectors together with an expression vector for ha - tagged vp3 were treated the same way . the ip &# 39 ; s were analyzed for the presence of the myctagged proteins by sds - page and western blotting with 9e10 ( against the myc - tag ). the results show that xnvp3 can bind to an intracellular aap in a similar fashion as apoptin ( vp3 ) encoded by a transfected plasmid . therefore , one can conclude that apoptin proteinaceous substances reveal essential biological activities of apoptin i . e . binding to its cellular counterparts . to show that xnvp3 can induce apoptosis in tumor cells , saos - 2 and vhsv - 40 cells were cultured in the presence of 10 or 50 nm xnvp3 . to correct for possible degradation of xnvp3 , the medium was replaced twice a day with fresh medium containing freshly thawed xnvp3 . after four days the cells were fixed and the intracellular presence of xnvp3 and apoptosis were assessed with immuno - histochemistry as described before ( danen - van oorschot et al ., 1997 ). the results show that xnvp3 can induce apoptosis in these cells at concentrations of around 10 to 50 nm . a similar experiment was performed with non - transformed vh10 cells to establish the specificity of xnvp3 . vh10 cells were cultured in medium containing 10 or 50 nm xnvp3 , but also in a five time higher concentration at 250 nm . after four days , no significant apoptosis could be detected in the vh10 cells . the same experiment was also performed with non - transformed keratinocytes and with primary mouse lymfocytes , and no apoptosis above background ( medium alone ) could be detected here either . to extend the observation that xnvp3 can cause apoptosis specifically in tumor cells , saos - 2 cells , u2 - os cells , and vh10 cells were split 1 : 10 and cultured for two weeks in the presence of 50 nm xnvp3 ( replaced twice daily as described above ) or in normal medium . the cells were then fixed with methanol / acetic acid , and stained with coomassie blue . although the cells in the control medium had all grown to confluency , the saos - 2 and u20s cells treated with xnvp3 had all disappeared from the dish , indicating that they had undergone apoptosis . the vh 10 cells treated with xnvp3 had grown to the same density as control treated vh10 , showing that xnvp3 does not inhibit growth of non - transformed cells . mbpvp3 was purified as described for the above - mentioned micro - injection experiments . the protein was then chemically conjugated to synthetic tat - peptide ( aa 37 - 72 , cfitkalgisygrkkrrqrrppqgsqthqvslskq ( seq id no : ______ )) as described by fawell et al . ( 1994 ). in short , the purified mbp - vp3 protein was activated with iodoacetamide , and desalted and concentrated . 4 -( maleimidomethyl )- cyclohexanecarboxylic acid n - hydroxysuccinimide ester ( smcc ) was added and after 30 minutes at room temperature the reaction was terminated by desalting on a g - 25 column in 100 mm na2hpo4 ( ph 7 . 5 ). tat peptide was added to the mbpbvp3 - smcc adduct and stored overnight at 4 ° c . the cross - linked conjugate was purified by size exclusion gel filtration and frozen at − 80 ° c . in small aliquots in the presence of 10 % glycerol . a sample was analyzed by sds page using coomassie - blue staining or western blotting with antibodies raised against the tat - peptide , or against vp3 . the preparation was estimated to contain more than 50 % conjugated mbp - vp3 protein , based on the relative intensity of the protein band with an apparent higher molecular weight on sds - page or the western blot using anti - vp3 antibodies . the conjugated protein is referred to as mbp - vp3 - tat . 5 . 2 . 2 . description of in vitro transduction and specific killing of tumor cells to show that mbp - vp3 - tat was able to penetrate into cells , and to assess its intracellular localization , the following experiment was performed . mbp - vp3 - tat protein was added to cultured saos - 2 cells and vh10 cells at 5 microgram / ml concentration in medium . after 16 hours the cells were fixed by acetone fixation , and the intracellular presence of mbp - vp3 - tat was tested with antibodies directed against mbp ( monoclonal mouse anti - mbp - clone r29 ; zymed laboratories , inc . and polyclonal rabbit anti - mbp - clone c18 / sc - 808 ; santa cruz biotechnology , inc .) or 111 . 3 ( reactive with vp3 ). in both tumor and normal cells mbp - vp3 - tat was located in the nucleus , as seen with confocal laser microscopy . this shows that mbp - vp3 - tat can transduce into cells . to show that mbp - vp3 - tat can bind to apoptin associated proteins as well as transfected vp3 in cells , the following experiment was performed . mbp - vp3 - tat was incubated with cos cells that had been transfected 48 hours earlier with an expression vector encoding myc - tagged aap - 1 or lacz . after 16 hours , the cells were lysed and anti mbp - vp3 - tat immunoprecipitation ( ip ) was performed with anti - mbp antibodies . as a negative control , cos cells transfected with the same expression vectors but not incubated with mbp - vp3 - tat were treated the same way . as a positive control , cos cells transfected with the same expression vectors together with an expression vector for ha - tagged vp3 were lysed and an ip was performed with anti - ha antibodies . the ip &# 39 ; s were analyzed for the presence of the myc - tagged proteins by sds - page and western blotting with 9e10 ( against the myc - tag ). the results show that mbp - vp3 - tat can bind to intracellular aap &# 39 ; s proving again that the apoptin proteinaceous substance contains the biological activity as seen for apoptin produced by transcription and translation of its dna . to show that mbp - vp3 - tat can induce apoptosis in tumor cells , saos - 2 and vhsv - 40 cells were cultured in the presence of 1 or 5 microgram / ml mbp - vp3 - tat . to correct for possible degradation of mbp - vp3 - tat , the medium was replaced twice a day with fresh medium containing freshly thawed mbp - vp3 - tat . after four days the cells were fixed and the intracellular presence of mbp - vp3 - tat and apoptosis were assessed with immunohistochemistry as described before ( danen - van oorschot , 1997 ). the results show that mbp - vp3 - tat can induce apoptosis in these cells at concentrations of around 1 or 5 microgram / ml . a similar experiment was performed with non - transformed vh10 cells to establish the specificity of mbp - vp3 - tat . vh10 cells were cultured in medium containing 1 or 5 microgram / ml mbp - vp3 - tat , but also in a five time higher concentration at 25 microgram / ml . after four days , no significant apoptosis could be detected in the vh10 cells . the same experiment was also performed with non - transformed keratinocytes and with primary mouse lymphocytes , and no apoptosis above background ( medium alone ) could be detected here either . to extend the observation that mbp - vp3 - tat can cause apoptosis specifically in tumor cells , saos - 2 cells , u2 - os cells , and vh10 cells were split 1 : 10 and cultured for two weeks in the presence of 5 microgram / ml mbp - vp3 - tat ( replaced twice daily as described above ) or in normal medium . the cells were then fixed with methanol / acetic acid , and stained with coomassie blue . although the cells in the control medium had all grown to confluency , the saos - 2 and u2 - os cells treated with mbp - vp3 - tat had all disappeared from the dish , indicating that they had undergone apoptosis . the vh10 cells treated with mbp - vp3 - tat had grown to the same density as control treated vh10 , showing that mbp - vp3 - tat does not inhibit growth of non - transformed cells . 6 . 1 delivery of mbp - apoptin by chemically coupling to anti - prostate - specific membrane antibodies . prostate - specific membrane antigen ( psma ) is a membrane - bound glycoprotein that is highly restricted to prostatic epithelial cells . psma is increased in association with prostatic cancer , particularly in hormone refractory disease . for instance , the lncap prostate cancer cell line has an estimated 180 , 000 molecules of psma per cell on its surface . devitt et al . ( 2000 ) developed the j591 monoclonal , which internalises the prostate cancer cell upon binding to psma . therefore , we have examined the effect of chemically coupling of purified j591 monoclonal antibody to purified bacterially produced mbp - apoptin . the coupling was carried out as described for the conjugation of tat peptide to mbp - apoptin ( section 5 . 2 . 1 ). next , we studied the cell killing effect of the addition of the covalently linked j591 - mbp - apoptin protein product into the medium ( 5 microgram per milliliter ) of psma - positive lncap cells and , as control , in the medium of psma - negative saos - 2 cells . to correct for possible degradation of j591 - mbp - apoptin , the medium was replaced twice a day with fresh medium containing freshly thawed j591 - mbp - apoptin . after four days the cells were fixed and the intracellular presence of j591 - mbp - apoptin and apoptosis were assessed with immunohistochemistry as described before ( danen - van oorschot , 1997 ). the results show that j591 - mbp - apoptin can induce apoptosis in lncap cells , but not in saos - 2 cells at concentrations of around 5 microgram / ml . these results show that linkage of mbp - apoptin to a specific antibody , which can become internalized , results in a specific receptor - mediated uptake of ( mbp )- apoptin and consequently in induction of apoptosis . a similar experiment was performed with non - transformed normal human primary prostate epithelial cells to establish the tumor - specificity of j591 - mbp - apoptin . the primary human prostate cells contain psma at their surface . they were cultured in medium containing 5 microgram / ml j591 - mbp - apoptin , but also in a five times higher concentration at 25 microgram / ml . after four days , no significant apoptosis could be detected in these primary prostatic epithelial cells . immunofluorescence analysis clearly showed that the primary prostate epithelial had taken up significant amounts of j591 - mbp - apoptin protein . these results show that although the primary prostate cells have taken up the j591 - mbp - apoptin product , the apoptin part does not induce apoptosis as has been described for apoptin as well as for mbp - apoptin protein products . with other words , delivery to a cell via surface antigens of a protein product containing apoptin will result in induction of tumor - specific apoptosis . similar results were obtained with chemically coupling of single - chain fv antibodies directed against her2 / neu ( scfvher ; wang et al ., 2001 )). her2 / neu has been implicated in the oncogenesis of human prostate cancer . clinical studies have suggested that over expression of her2 is one of the indicators of poor prognosis in prostate cancer treatment . the above - described lncap cells express besides psma also high levels of her2 protein . therefore , we incubated lncap cells with mbp - apoptin coupled chemically with scfvher . exposure of lncap cells to scfvher - mbp - apoptin caused remarkable cell death . pc3m cells , lacking her2 protein , did not undergo apoptosis upon treatment with scfvher - mbp - apoptin protein . addition of the chemically coupled scfvher / mbp - apoptin products to the medium of primary human prostate cells also did not result in induction of apoptosis , which shows the safety of the approach of delivery of apoptin protein chemically coupled to a scfv molecule , which can deliver apoptin into the cell . in conclusion , the data obtained with recombinant mbp - apoptin coupled to specific ( single - chain ) antibodies confirm the observation that coupling of a fluorescein moiety to ( mbp )- apoptin does not negatively influence the tumor - specific activity of apoptin . therefore , specific delivery of biological active ( tumor - specific induction of apoptosis ) ( mbp -) apoptin protein to human tumor cells via coupling to specific antibody protein molecules forms the base for a novel anti - tumor therapy . a dna plasmid was constructed that encodes in frame for scfvher coding sequences followed by the coding sequence for mbp - apoptin . to that end , scfvher sequences were cloned into the plasmid pmbp - vp3 . the antibody moiety of the fusion protein was fused to the n - terminal part of mbp - apoptin via the linker peptide ( gly ( 4 ) ser ( 6 )). the final plasmid was called pscfvmbp - apoptin . recombinant scfvmbp - apoptin was produced in bl21 ( de3 ) bacteria and purified as described for mbp - apoptin ( see above ). western - blot analysis using apoptin - specific polyclonal antibodies revealed the production of the expected fusion protein product . to test the apoptosis activity of the recombinant scfvher - mbp - apoptin product the following tissue culture experiments were carried out . to that end , the bacterially produced recombinant scfvher - mbp - apoptin protein product was added into the medium ( 1 - 5 microgram per milliliter ) of her2 - positive lncap cells and , as control , into the medium of her2 - negative pc3m cells . to correct for possible degradation of scfvher - mbp - apoptin , the medium was replaced twice a day with fresh medium containing scfvher - mbp - apoptin fusion protein . after four days the cells were fixed and the intracellular presence of scfvher - mbp - apoptin and apoptosis were assessed with immunohistochemistry as described before ( danen - van oorschot , 1997 ). the results show that scfvher - mbp - apoptin can induce apoptosis in lncap cells , but not in pc3m cells . addition of the scfvher / mbp - apoptin fusion products into the medium of primary human prostate cells did not result in induction of apoptosis , which shows the safety of this apoptin fusion protein . in conclusion , these results show that a bacterially produced recombinant scfv - mbp - apoptin fusion product can be obtained in a soluble and biologically active form without loss of apoptin &# 39 ; 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