Patent Application: US-58611105-A

Abstract:
a synthetic , soluble , endogenous complex formed from at least one component a and at least one component b , whereby component a comprises a binding domain for extra - cellular surface structures that internalize upon binding of component a of said complex , and component b has a constitutive catalytic kinase activity and effects cell biosynthesis / signalling including cell death after internalization . the complex allows to influence the growth and the physiology of cells . in particular said complex , nucleic acid molecules encoding it , cells transfected or transformed with these nucleic acid molecules are usable for the preparation of medicaments for the treatment of proliferative diseases , inflammatory diseases , allergies and autoimmune diseases .

Description:
the complex according to the invention is a recombinant heterologous complex comprising at least two domains , i . e . one effector domain and at least one cell - specific binding domain . the complex according to the invention is usable for diagnosis and therapy of diseases . the invention described herein draws on previously published work and pending patent applications . by way of example , such work consists of scientific papers , patents or pending patent applications . all of these publications and applications , cited previously or below are hereby incorporated by reference . as used herein , the term “ immunotoxin ” refers to chimeric molecules in which a cell - binding monoclonal antibody or fragments thereof are chemically coupled or genetically fused to toxins or their subunits . the toxin portion of the immunotoxin can be derived form various sources , such as plants , animals , higher and lower microorganisms such as bacteria and fungi , and in particular if the toxin is a catalytic enzyme , the enzyme can be of human origin . the toxin can also be a synthetic drug . immunotoxins as well their constructions are reviewed above and are well known to the person skilled in the art . as used herein , the term “ immunokinase ” refers to chimeric molecules in which a cell - binding monoclonal antibody or fragments thereof are coupled or fused to kinases or their subunits harboring the kinase activity . the term immunokinase is a synonym for the complex of the present invention . as used herein , the term “ component a ” of the complex represents the actively binding structure of the complex of present invention . the component a is selected from the group of actively binding structures consisting of antibodies or their derivatives or fragments thereof , synthetic peptides such as scfv , mimotopes , etc . or chemical molecules such as carbohydrates , lipids , nucleic acids , peptides , vitamins , etc ., and / or small molecules with up to 100 atoms with receptor - binding activity like ligands , in particular single atoms , peptidic molecules , non - peptidic molecules , etc ., and / or cell surface carbohydrate binding proteins and their ligands such as lectins , in particular calnexins , o - type lectins , l - type lectins , m - type lectins , p - type lectins , r - type lectins , galectins and their derivatives , and / or receptor binding molecules such as natural ligands to the cluster of differentiation ( cd ) antigens , like cd30 , cd40 , etc ., cytokines such as chemokines , colony stimulating factors , type - 1 cytokines , type - 2 cytokines , interferons , interleukins , lymphokines , monokines , etc ., and / or adhesion molecules including their derivatives and mutants , and / or derivatives or combinations of any of the above listed of actively binding structures , which bind to cd antigens , cytokine receptors , hormone receptors , growth factor receptors , ion pumps , channel - forming proteins . the component a may also be selected from the group of passively binding structures consisting of allergens , peptidic allergens , recombinant allergens , allergen - idiotypical antibodies , autoimmune - provoking structures , tissue - rejection - inducing structures , immunoglobulin constant regions and their derivatives , mutants or combinations thereof . a component a with higher valency may be generated by combining at least two identical or different binding structures selected from the above mentioned groups . as used herein , the term “ antibody ” refers to polyclonal antibodies , monoclonal antibodies , humanized antibodies , single - chain antibodies , and fragments thereof such as fab , f ( ab ′) 2 , fv , and other fragments which retain the antigen binding function and specificity of the parent antibody . as used herein , the term “ monoclonal antibody ” refers to an antibody composition having a homogeneous antibody population . the term is not limited regarding the species or source of the antibody , nor is it intended to be limited by the manner in which it is made . the term encompasses whole immunoglobulins as well as fragments such as fab , f ( ab ′) 2 , fv , and others which retain the antigen , binding function and specificity of the antibody . monoclonal antibodies of any mammalian species can be used in this invention . in practice , however , the antibodies will typically be of rat , or murine origin because of the availability of rat or murine cell lines for use in making the required hybrid cell lines or hybridomas to produce monoclonal antibodies . as used herein , the term “ human antibodies ” means that the framework regions of an immunoglobulin are derived from human immunoglobulin sequences . as used herein , the term “ single chain antibody fragments ” ( scfv ) refers to antibodies prepared by determining the binding domains ( both heavy and light chains ) of a binding antibody , and supplying a linking moiety , which permits preservation of the binding function . this forms , in essence , a radically abbreviated antibody , having only that part of the variable domain necessary for binding to the antigen . determination and construction of single chain antibodies are described in u . s . pat . no . 4 , 946 , 778 to ladner et al . the “ component b ” of present invention represents the “ targeted kinase ” moiety of the immunokinase of the present invention and may be selected from any kinase known in the art . presently , over 5 , 000 kinase - like sequences from diverse species are available for analysis in public databases . the human genome appears to encode 510 protein kinases in addition to many pseudo - protein kinase genes , and these have been subclassified into over 57 families . there may well be additional protein kinases that remain to be identified ( http :// www . kinexus . ca / kinases . htm ). however , preferably component b is chosen from the following three classes of kinases , which are all known to be active in humans and to retain their kinase activity in a soluble complex . 1 . the eukaryotic protein kinase ( epk ) superfamily , 2 . the histidine protein kinase ( hpk ) superfamily , or 3 . the atypical protein kinase ( apk ) superfamily . if component b is chosen from the epk superfamily , it is selected from the group of calcium / calmodulin - regulated ( cam ) death - promoting kinases , consisting of death - associated protein kinase ( dap - kinase , dapk ), dap kinase - related protein kinase 1 ( drp - 1 ), also named dap - kinase 2 ( dapk2 ), dap like kinase / zipper interacting protein kinase ( dlk / zip - kinase ), also named dap - kinase 3 ( dapk3 ) and dap kinase related apoptosis - inducing kinase ( drak1 and drak2 ) families , the group of calcium / calmodulin - regulated ( cam ) death - promoting kinases - like ( camkl ) family , consisting of at least 49 subfamilies , protein kinase amp - activated alpha 1 catalytic subunit ( prkaa1 ), protein kinase amp - activated alpha 2 catalytic subunit ( prkaa2 ), brsk1 and brsk2 , chk1 checkpoint homologue ( chek1 ), hormonally upregulated neu - associated kinase ( hunk ), serine / threonine kinase 11 ( peutz - jeghers syndrome ) ( stk11 ), map / microtubule affinity - regulating kinase ( mark ) 1 - 4 , markps 01 - 30 , likely ortholog of maternal embryonic leucine zipper kinase ( kiaa0175 ), pas domain containing serine / threonine kinase ( pask ), nim1 , qik and snrk , the group of death - domain receptor interacting protein kinase ( rip - kinase ) family , consisting of at least six subfamilies , rip - kinase 1 , rip - kinase 2 , rip - kinase 3 and rip - kinase 4 , ankyrin repeat domain 3 ( ankrd3 ) and sqk288 , the group of multifunctional cam kinase family , consisting of cam kinases i , ii , including the microtubule affinity - regulating kinases ( mark ) and microtubule affinity - regulating kinases - like 1 ( markl1 ), cam kinase iv and cam kinase kinase subfamilies , the group of dedicated cam kinases , consisting of myosin light chain kinase ( mlck ), phosphorylase kinase and cam kinase iii ( eef - 2k ), the group of mitogen - activated protein kinase ( mapk ) family , consisting of extracellular signal - regulated kinases ( erk ), c - jun nh2 - terminal protein kinases ( jnk ), nemo - like kinase ( nlk ) and p38 kinase subfamilies , the group of cyclin - dependent kinase ( cdk ) family , consisting of the subfamilies , cell cycle related kinase ( ccrk ), cell division cycle 2 ( cdc2 ), cyclin - dependent kinases ( cdk ) 1 - 11 , pctaire protein kinase ( pctk ) 1 - 3 , pftaire protein kinase ( pftk ) 1 - 2 and cell division cycle 2 - like 1 ( pitslre proteins ), the group of eukaryotic translation initiation factor 2 - alpha kinase 3 ( eif2ak3 ) family , also named ( pek ), consisting of the protein kinase interferon - inducible double stranded rna ( dsrna ) dependent ( pkr ) subfamily . if component b is chosen from the hpk superfamily , it is selected from the group of at least eleven families hpk 1 - 11 . if component b is chosen from the apk superfamily , it is selected from the group of alpha protein kinase family , consisting of eukaryotic elongation factor - 2 kinase ( eef - 2k ), myosin heavy chain kinase ( mhc - kinase ), eukaryotic translation initiation factor 2 alpha kinase 1 ( e2k1 ) and channel kinase ( chak1 and chak2 ) subfamilies , the group of fas - activated s / t kinase ( fastk ) family , consisting of the fastk subfamily , the group of protein tyrosine kinase 9 ( a6 ) family , consisting of a6 and protein tyrosine kinase 9 - like ( a6r ) subfamilies , the group of p21 - activated protein kinases ( pak ) family , consisting of the three highly conserved isoforms : alpha - pak ( pak1 ), beta - pak ( pak3 ) and gamma - pak ( pak2 , paki ), the group of interleukin - 1 ( il - 1 )- receptor - associated kinase ( irak ) family , consisting of irak - 1 , irak - 2 , irak - 3 and irak - 4 subfamilies . the term “ target cell ” and or “ target tissue ” refers to cells or tissues carrying an extracellular surface structure to which the component a of the complex actively or passively binds . target cells and target tissues are thus cells and tissues to which the component a of the complex can bind . the target cells and target tissues are further characterized by their ability to internalize the complex according to the present invention upon binding of component a . the term “ soluble ” refers to the ability of the complex to stay in solution when recombinantly expressed , in particular during protein purification , enabling high yields . the term “ soluble ” also refers to the state of the complex in fluidic systems inside an organism , until specifically attached to the target cell / tissue . the term also refers to the state of the complex inside a cell upon release from any kind of incorporation vesicles . the term “ endogenous ” refers to the localization of the complex in the surrounding / environment of a given target cell / tissue . the term synthetic refers to a man - made complex , not found in nature . the term also comprises the meaning of “ recombinant ”. the term “ recombinant ” refers to the preparation of molecules , in particular the covalent joining of molecules from different sources , by any one of the known methods of molecular biology . as used in the present invention , the term “ recombinant ” refers in particular to the fusion of the antibody part to the toxin part by any one of the known methods of molecular biology , such as through production of single chain antibodies . the recombinant dna molecule encoding the recombinant fusion protein comprising the antibody part and the toxin part are recombinantly expressed . recombinant immunotoxin produced in this way may be isolated by any technique known in the field of recombinant dna expression technology suitable for this purpose . the term “ derivative ” refers to a mutated or modified protein which has retained its characterizing activity , i . e . binding activity or kinase activity . particular preferred are constitutively active derivatives . the term derivative comprises proteins which carry at least one amino acid substitution , deletion , addition , a swapping of a single domain or at least one modification of at least one amino acid . preferred are derivatives which carry 20 such changes , more preferred are those with 10 such changes and most preferred are those with 1 to 5 such changes . modifications , which can occur , are phosphorylation , acetylation , methylation , prenylation and sulfation . as used herein , the term “ vector ” comprises dna and rna forms of a plasmid , a cosmid , a phage , phagemid , derivatives of them , or a virus . a vector comprises control sequences and coding sequences . the term “ expression of the recombinant genes encoding the recombinant complex ”, wherein the recombinant complex is a single chain antibody - toxin moiety fusion polypeptide , also called recombinant immunokinase , refers to the transformation and / or transfection of a host cell with a nucleic acid or vector encoding such a complex , and culturing said host cells selected from the group of bacteria , such as e . coli , and / or in yeast , such as in s . cerevisiae , and / or in established mammalian or insect cell lines , such as cho , cos , bhk , 293t and mdck cells , and / or in primary cells , such as human cells , non - human vertebrate cells , and / or in invertebrate cells such as insect cells , and the synthesis and translation of the corresponding mrna , finally giving rise to the recombinant protein , the recombinant complex . in more detail , the term “ expression of the recombinant genes encoding the recombinant complex ”, comprises the following steps : transformation of an appropriate cellular host with a recombinant vector , in which a nucleotide sequence coding for the fusion protein had been inserted under the control of the appropriate regulatory elements , particularly a promoter recognized by the polymerases of the cellular host . in the case of a prokaryotic host , an appropriate ribosome binding site ( rbs ) also precedes the nucleotide sequence coding for the fusion protein , enabling the translation in said cellular host . in the case of an eukaryotic host any artificial signal sequence or pre / pro sequence may be provided , or the natural signal sequence may be employed . the transformed cellular host is cultured under conditions enabling the expression of said insert . as used herein , the expression “ killing of antigen - expressing cells ” refers to the inhibition of protein synthesis or induction of apoptosis , resulting in elimination or death of these cells . the term “ supplementary components s ”, refers to an additional component of the complex comprising a and b . the supplementary component s contributes features and properties to the complex which allow efficient preparation and / or modify the effectiveness of the complex : the inducible regulation of transcription / translation ( e . g ., inducible promoters ); control of protein biosynthesis ( e . g ., leader sequences ); purification / detection of the complex or its components ( e . g ., his tag , affinity tags ); translocation of the apoptotic agents into the target cells ( e . g ., translocation domain , amphiphatic sequences ); intracellular activation / separation of component b ( synthetic pro - granzyme b , amphiphatic sequences ). thus the component s is selected from the group of inducible promoters , leader sequences , affinity tags , his tags , translocation domain , amphiphatic sequences and synthetic pro - granzyme b . the invention also relates to nucleic acid molecules , such as dna and / or rna , or vectors , which code for the complex of the present invention or for individual components for preparing the complex . the feasability of the expression of the nucleic acids encoding a recombinant complex in eukaryotic cells of human origin is successfully documented here , as well as the feasibility to use the complex as an specific apoptotic agents in eukaryotic cells of human origin . this suggests the suitability of nucleic acids coding for a complex according to the invention also for non germ line gene - therapeutic approaches . a person skilled in the art is capable of recognizing the various aspects and possibilities of gene - therapeutic interventions in connection with the various diseases to be treated . in addition to the local application of relatively non - specific vectors ( e . g ., cationic lipids , non - viral , adenoviral and retroviral vectors ), a systemic application with modified target - cell - specific vectors will also become possible in the near future . complexes and nucleic acid molecules and / or vectors coding for the complexes of present invention , are used for the preparation of medicaments for non - germ line gene therapeutic interventions , for the local or systemic application . an interesting alternative to systemic application are the well - aimed ex vivo transfection of defined cell populations and their return into the organism , or the use of the ex vivo transfected defined cell populations for the preparation of a medicament for the treatment of diseases associated with these cell populations . also claimed are cells or in vitro translation systems , which synthesize complete complexes according to the invention or individual components thereof , after transformation and / or transfection with , or addition of the nucleic acid molecules or vectors according to the invention . cells or organisms according to the invention are either of prokaryotic origin , especially from e . coli , b . subtills , s . carnosus , s . coelicolor , marinococcus sp ., or eukaryotic origin , especially from saccharomyces sp ., aspergillus sp ., spodoptera sp ., p . pastoris , primary or cultivated mammalian cells , eukaryotic cell lines ( e . g ., cho , cos or 293 ) or plants ( e . g . n . tabacum ). the invention also relates to medicaments comprising the complex according to the present invention and / or the nucleic acid or vectors encoding the complex of present invention . typically , the complexes according to the invention are administered in physiologically acceptable dosage forms . these include , for example , tris , nacl , phosphate buffers and all approved buffer systems , especially including buffer systems , which are characterized by the addition of approved protein stabilizers . the administration is effected , in particular , by parenteral , intravenous , subcutaneous , intramuscular , intratumoral , transnasal administrations , and by transmucosal application . the dosage of the complexes according to the invention to be administered must be established for each application in each disease to be newly treated by clinical phase i studies ( dose - escalation studies ). nucleic acids or vectors , which code for a complex according to the invention , are advantageously administered in physiologically acceptable dosage forms . these include , for example , tris , nacl , phosphate buffers and all approved buffer systems , especially including buffer systems , which are characterized by the addition of approved stabilizers for the nucleic acids and / or vectors to be used . the administration is effected , in particular , by parenteral , intravenous , subcutaneous , intramuscular , intratumoral , transnasal administrations , and by transmucosal application . the complex according to the invention , nucleic acid molecules coding therefore and / or cells or in vitro translation systems can be used for the preparation of a medicament for treating tumor diseases , allergies , autoimmune diseases , and chronic / acute inflammation reactions . following the construction of three types of recombinant complexes ( immunokinases ), first results obtained demonstrate their superior quality with regard to binding specificity as well as cytoxicity . pcr - amplified dapk2 ′ dna ( fig1 ) was directionally cloned into the ampicillin - resistant pms -( l - ang - ki - 4 )- iii / g eukaryotic expression vector containing a lgk - leader ( l ) sequence at the n - terminus , ki - 4 ( scfv ) ( component a ) and a tandem myc - and his - tag epitope at the c - terminus of the expression cassette ( fig2 ) successful cloning was verified by dna sequence analysis . three days after transfection of 293t - cells , the appropriate sized expected recombinant complex ( immuno - kinase ) pms -( l - dapk2 - ki - 4 )- ii / g ( m r ˜ 66 , 000 ) was detected by western blot analysis of protein mini - preparations . transfected producer - cells were further cultivated under zeocin selection pressure in medium culture flasks and were used for larger scale production of the recombinant complex ( immunokinase ) pms -( l - dapk2 - ki - 4 )- iii / g . under normal culture conditions , between 0 . 1 and 0 . 5 μg of the recombinant protein were purified from 1 ml cell culture supernatant by a one step ni - nta purification procedure . the intact recombinant complex ( immunokinase ) was secreted into the supernatant of transfected 293t - cells , as visualized by immunoblot using mouse - anti - penta - his monoclonal antibody . pcr - amplified eef - 2k dna encoding component b ( fig1 , 4 a - e ) was directionally cloned into the pet - derived kanamycin - resistant pbm - ki - 4 ( scfv ) prokaryotic expression vector containing an iptg - inducible lac operator , a pelb signal peptide followed by an enterokinase - cleavable his 10 tag , and ki - 4 ( scfv ) ( component a ) ( fig2 ). successful cloning of the recombinant complex construct pmt - ki - 4 ( scfv )- eef - 2k was verified by dna sequence analysis . after transformation , recombinant e . coli bl21 star ™ ( de3 ) clones were cultivated under osmotic stress conditions in the presence of compatible solutes . the recombinant complex ( immunokinase ) was directed into the periplasmic space and the functional pmt - ki - 4 ( scfv )- eef - 2k ( m ° r ˜ 113 , 000 ) protein directly purified by combination of imac and sec to & gt ; 90 % purity . at least 1 mg of purified pmt - ki - 4 ( scfv )- eef - 2k protein was routinely prepared from 1 liter of bacterial shaking cultures . the intact recombinant complex ( immunokinase ) was secreted to the periplasmic compartment , as visualized by immunoblot using mouse - anti - penta - his monoclonal antibody . fusing the ki - 4 ( scfv ) coding regions , component a of the complex , to the kinase coding sequences , component b of the complex , did not affect the binding activity of the v h / v l antibody format of component a . component a conferred specificity against the cd30 molecule . the purified recombinant complex ( immunokinase ) comprising the anti - cd30 component a always bound to the hodgkin - derived cell line l540cy as measured by flow cytometry ( fig3 ). to characterize the cytotoxic activity of the recombinant complex comprising anti - cd30 ′ ( as component a ) and kinases ( component b ) in vitro , the proliferation of different target cells was evaluated after incubation with different amounts of the recombinant complexes ( immunokinases ) pms -( l - dapk2 - ki - 4 )- iii / g and pmt - ki - 4 ( scfv )- eef - 2k , respectively . growth inhibition of the cd30 - positive cell lines l540cy and hl60 were documented by a xtt - based colorimetric assay . toxic effects were observed only against cd30 - positive cells with a calculated median ic 50 of between 4 and 35 ng / ml on l540cy cells ( fig4 ) the cd30 - negative ramos and 8701 - bc cell lines were not affected by recombinant immunokinase concentrations of up to 10 μg / ml . thus the component a ( anti - cd30 scfv ) of the complex conferred specificity to the recombinant complex , limiting the cytotoxic effects of the kinase domain to the selected target cells . e . coli xl1 - blue ( supe44 hsdr17 reca1 enda1 gyr a46 thi rela1 lacf ′[ pro ab + lacl q lacz ? m15 tn10 ( tetr )]) were used for the propagation of plasmids , and e . coli bl21 star ™ ( de3 ) ( f − ompt hsds b ( r b − m b − ) gal dcm rne131 de3 ) as host for synthesis of recombinant immunokinases . synthetic oligonucleotides were synthesized by mwg biotech ( ebersberg , germany ). the bacterial expression vector pbm - ki - 4 is derived from the pet27b plasmid ( novagen , madison , usa ), and is used for the expression of the c - terminal fusion of not i / blp i - kinase domains to the anti - cd30 scfv ( klimka , a . et al ., 1999 ). the eukaryotic expression vectors pmskangii and pmslangkiii are derived from the psectag plasmid ( invitrogen , carlsbad , usa ) and are used for n - or c - terminal fusion of xbai / blpi - kinase domains to the ki - 4 ( scfv ) ( stöcker , m . et al ., 2003 ). plasmids were prepared by the alkaline lysis method and purified using plasmid preparation kits from qiagen ( hilden , germany ). restriction fragments or pcr products were separated by horizontal agarose gel electrophoresis and extracted with qiaquick ( qiagen ). all standard cloning procedures were carried out as described by sambrook , j . et al ., 1989 . all cell lines , including the cd30 - positive cell lines l540cy ( kapp , u . et al ., 1992 ) and hl - 60 ( thepen , t . utrecht , the netherlands ) the cd30 - negative cell lines ramos ( atcc , va , usa ) and 8701 - bc ( minafra , s . et al ., 1989 ) and the producer cell line 293t ( atcc ) were cultivated in complex medium ( rpmi 1640 ) supplemented with 10 % ( v / v ) heat - inactivated fetal calf serum , 50 μg / ml penicillin , 100 μg / ml streptomycin and 2 m m l - glutamine . all cells were cultured at 37 ° c . in a 5 % co 2 in air atmosphere . for the selection of transfected cells , zeocin ( invitrogen ) was added to a final concentration of 100 μg / ml . cloning and expression of pms -( l - dapk2 - ki - 4 )- iii / g ( seq id no 1 ) and pms -( ki - 4 - dapk2 )- ii / g ( seq id no 3 ) for the construction of a vector encoding a recombinant complex with n - or c - terminal dap - kinase 2 ( dapk2 )- fusions , dapk2 was pcr amplified to introduce the restriction sites xbai and blpi . after xbai / blpi - digestion , the pcr - product was cloned into the eukaryotic expression vector pms -( l - ang - ki - 4 )- iii / g and pms -( ki - 4 - ang )- ii / g respectively , digested with the same restriction enzymes . the resulting recombinant constructs pms -( l - dapk2 - ki - 4 )- iii / g ( seq id no : 1 ) and pms -( ki - 4 - dapk2 )- ii / g ( seq id no : 3 ) encoding the immukinase proteins l - dapk2 - ki - 4 - mh ( seq id no 2 ) and l - ki - 4 - dapk2 - mh ( seq id no 4 ) were verified by sequence analysis . after transfast - mediated ( promega , mannhein , germany ) transformation into 293t - cells , the recombinant immunokinase was expressed as described by stöcker m . et al ., 2003 . briefly , one μg plasmid - dna and 3 μl transfast have been used according to the manufactures protocol for 12 well cell culture plates . transfection efficiency was between 75 and 95 % determined by counting green fluorescent cells . 3 days after initial transfection , cell culture supernatants were analyzed for recombinant protein . subsequently , transfected cells were transferred into medium - sized cell culture flasks ( nunc ; 85 m 2 ) and grown in rpmi complex medium supplemented with 100 μg / ml zeocin . one to two weeks productively transfected clones were green fluorescing and hence could be detected by fluorescence microscopy . transfected cell populations were established by subcultivation of these clones . purifications of the his - tagged proteins were accomplished by the ni - nta metal - affinity method ( hochuli , v ., 1989 , porath , j . et al ., 1975 ) ( qiagen ). the protein purification followed a modified protocol for the purification of native protein from qiagen ( the expressionist 07 / 97 ). for protein mini - preparation , 900 μl centrifugation - cleared cell culture supernatant was supplemented with 300 μl of 4 × incubation buffer ( 200 mm nah 2 po 4 , ph 8 . 0 ; 1 . 2m nacl ; 40 mm imidazol ) and 30 μl 50 % ni - nta . following 1 h incubation , the ni - nta resin was pelleted by centrifugation . after washing the sediment twice in 175 μl 1 × incubation buffer , bound protein was eluted with 30 μl of elution buffer ( 50 mm nah 2 po 4 , ph 8 . 0 ; 1 . 2m nacl ; and 40 mm imidazol ) and 30 μl 50 % ni - nta . following an 1 h incubation , the ni - nta resin was pelleted by centrifugation . after washing the sediment twice in 175 μl 1 × incubation buffer , bound protein was eluted with 30 μl of elution buffer ( 50 mm nah 2 po 4 , ph8 . 0 ; 300 mm nacl ; 250 mm imidazol ) for 20 min at rt . larger scale purification of eukaryotically - expressed proteins up to 500 ml cell culture supernatant was performed on a biologic workstation ( bio - rad , usa ). cell culture supernatants were loaded onto a ni - nta column and following elution of the his - tagged proteins were made under the conditions described above . the eukaryotic elongation factor - 2 kinase ( eef - 2k ) was amplified by pcr to introduce the restriction sites noti and blpi . after noti / blpi - digestion , the pcr - fragment was cloned into the bacterial expression vector pbm - ki - 4 , digested with the same restriction enzymes . the resulting recombinant construct pmt - ki - 4 ( scfv )- eef - 2k ( seq id no : 5 ) was verified by dna sequence analysis . after transformation into bl21 star ™ ( de3 ), the immunokinase ki - 4 ( scfv )- eef - 2k ( seq id no 6 ) were periplasmically expressed under osmotic stress in the presence of compatible solutes as described by barth , s . et al . 2000 . briefly , transformed bacteria were harvested 15 h after iptg induction . the bacterial pellet was resuspended in sonication - buffer ( 75 mm tris / hcl ( ph 8 ), 300 mm nacl , 1 capsule of protease inhibitors / 50 ml ( complete ™, roche diagnostics , mannheim , germany ), 5 mm dtt , 10 mm edta , 10 % ( v / v ) glycerol ) at 4 ° c . and sonicated 6 times for 30 s at 200 w . the m22 ( scfv )- eta ′ fusion proteins were enriched by imac ( immobilized metal - ion affinity chromatography ) using nickel - nitriloacetic chelating sepharose ( qiagen ) and sec ( size exclusion chromatography ) with bio - prep se - 100 / 17 ( biorad , münchen , germany ) columns according to the manufacturer &# 39 ; s instructions . recombinant protein was eluted with pbs ( ph 7 . 4 ) and 1 m nacl , analyzed by sodium dodecyl sulfate / polyacrylamide gel electrophoresis ( sds - page ), quantified by densitometry ( gs - 700 imaging densitometer ; biorad ) after coomassie staining in comparison with bsa standards and verified by bradford assays ( biorad ). sds - page , coomassie staining , and western blotting were performed as described by barth , s . et al ., 1998 . briefly , recombinant his - tagged immunokinases were detected by mouse - anti - penta - his moab ( qiagen ). bound antibody was detected by a horseradish - conjugated donkey - anti - mouse - igg moab ( dianova , hamburg , germany ), followed by ecl - mediated ( amersham biosciences , freiburg , germany ), chemilumlnescence reaction and exposition to appropriate x - ray film ( roche , penzberg , germany ) or alkaline - phosphatase - conjugated anti - mouse - igg moab ( sigma chemical co ., deisenhofen , germany ) and a solution of tris - hcl ( ph 8 . 0 ) and 0 . 2 mg / ml naphtol - as - bi - phosphate ( sigma chemical co .) supplemented with 1 mg / ml fast - red ( serva , heidelberg , germany ). the binding activity of recombinant complexes ( immunokinases ) were determined by cm - elisa using biological active membranes of tumor cells as described recently by tur , m k . et al ., 2003 . briefly , elisa maxisorp - plates ( nalge nunc international , roskilde , denmark ) were coated with 100 μl (˜ 0 . 9 mg protein / ml ) freshly prepared membrane fractions of cd30 - positive l540cy / hl60 cells and ramos / 8701 - bc as control in 0 . 02 m bicarbonate buffer , ph 9 . 6 , overnight at 4 ° c . plates were washed five times with pbs ( ph 7 . 4 ) containing 0 . 2 % tween 20 ( tpbs ) and blocked with 200 μl 2 % bsa in pbs . after overnight incubation at 4 ° c ., plates were washed five times with tpbs and 1 - 10 μg / ml of recombinant immunokinases diluted with 0 . 5 % bsa ( w / v ) and 0 . 05 % tween 20 ( v / v ) in pbs was added to the plates and incubated at rt ( 23 ° c .) for 1 h . peroxidase labeled anti - his igg conjugate ( qiagen ) were added diluted with 0 . 5 % bsa and 0 . 05 % tween 20 in pbs according to manufactures instructions . bound antibodies were visualized after addition of 100 μl 2 ′, 2 ′- azino - bis ( 3 - ethylbenzthiazoline - 6 - sulphonic acid ) ( abts ) solution ( roche molecular biochemical &# 39 ; s , mannheim , germany ) by measuring the extinction at 415 nm with an elisa - reader ( mwg biotech ). cell binding activity of the recombinant complexes ( immunokinases ) expressed in e . coli bl21 star ™ ( de3 ) was evaluated using a facscalibur flow cytometry instrument and cellquest software ( becton dickinson , heidelberg , germany ). cells were stained with recombinant protein as described ( 25 ). briefly , ten thousand events were collected for each sample , and analyses of intact cells were performed using appropriate scatter gates to exclude cellular debris and aggregates . 5 × 10 5 cells were incubated for 1 h on ice with 50 μl of bacterial protein extract at a concentration of 30 - 40 μg / ml or 100 μl of the immunokuinase containing supernatants respectively . the cells were washed with pbs buffer containing 0 . 2 % w / v bsa and 0 . 05 % w / v sodium azide ( pba ) and then incubated for 30 min with anti - penta - his moab ( qiagen ) diluted 1 : 2 in pba buffer . cells were washed and incubated with fluorescein - iso - thiocyanate ( fitc )- labeled goat - anti - mouse igg ( dako diagnostica , hamburg , germany ) for 1 h at 4 ° c . after a final wash , the cells were treated with 2 μl 6 . 25 mg / ml propidiumiodide and subsequently analyzed on a facscalibur ( becton dickison , heidelberg , germany ). the cytotoxic effect of the recombinant complexes ( immunokinases ) on target cells was determined by measurement of metabolization of yellow tetrazolium salt ( xtt ) to a water soluble orange formazan dye was determined as published by barth , s . et al . 2000 . various dilutions of the recombinant immunokinase were distributed in 100 μl - aliquots in 96 - well plates . two - four × 10 4 target cells in 100 μl aliquots of complete medium were added and the plates were incubated for 48 h at 37 ° c . afterwards , the cell cultures were pulsed with 100 μl fresh culture medium supplemented with xtt / pms ( final concentrations of 0 . 3 mg and 0 . 383 ng respectively ) for 4 h . the spectrophotometrical absorbances of the samples were measured at 450 and 650 nm ( reference wavelength ) with an elisa reader ( mwg biotech ). the concentration required to achieve a 50 % reduction of protein synthesis ( ic 50 ) relative to untreated control cells and to 1 % triton x treated positive controls was calculated graphically via excel generated diagrams . all measurements were done in triplicate . 1 . kaminski , m . s ., zasadny , k . r ., francis , i . r ., fenner , m . c ., ross , c . w ., milik , a . w ., estes , j ., tuck , m ., regan , d ., fisher , s ., glenn , s . d ., and wahl , r . l . iodine - 131 - anti - b1 radioimmunotherapy for b - cell lymphoma . j clin oncol , 14 : 1974 - 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