fig1 a is a schematic diagram of the diphtheria toxin ( dt ) molecule , and fig1 b is a schematic diagram of various dt / interleukin ( il )- 2 fusion proteins . fig2 is a depiction of the construction of the plasmids of a preferred embodiment . fig3 is a restriction map of dna services encoding various chimeric toxins . fig4 is a graph of the effects of varying doses of chimeric toxins on cultured cells . fig5 is a graph of the ability of chimeric toxins to competitively displace 125 i !- labeled il - 2 from the high affinity il - 2 receptor . fig7 a and 7b are diagrammatic representations of dab 486 egf , respectively . fig8 is a graph showing the effect of egf on dab 486 egf cytotoxicity , fig9 is a graph showing the effect of egf on dab 389 egf cytotoxicity , fig1 is a graph showing the effect of egf and dab 389 egf on the egf binding capacity of a431 cells . fig1 is a graph showing the ability of egf or dab 389 egf to displace 125 i ! egf from egf receptors . fig1 is a graph of the effect of length of exposure to dab 486 egf on the inhibition of protein synthesis . fig1 is a graph of the effect of length of exposure to dab 389 egf on the inhibition of protein synthesis . fig1 is a graph of the kinetics of the inhibition of protein synthesis on cells incubated with dab 486 egf or dab 389 egf . dab 486 - il - 2 is a chimeric toxin consisting of met followed by amino acid residues 1 through 485 of mature dt fused to amino acid residues 2 through 133 of il - 2 . the dt portion of the chimeric toxin dab 486 - il - 2 includes all of dt fragment a and the portion of dt fragment b extending to residue 485 of mature native dt . thus dab 486 - il - 2 extends past the disulfide bridge linking cys 461 with cys 471 . see fig1 a for the structure of dt . ( the nomenclature adopted for il - 2 - toxin is dab 486 - il - 2 , where d indicates diphtheria toxin , a and b indicate wild type sequences for these fragments , and il - 2 indicates human interleukin - 2 sequences . mutant alleles are indicated by a number in parentheses following dab . the numerical subscript indicates the number of dt - related amino acids in the fusion protein . since the deletion of the tox signal sequence and expression from the trc promoter results in the addition of a methionine residue to the n - terminus , the numbering of dab - il - 2 fusion toxins is + 1 out of phase with that of native diphtheria toxin .) pdw24 , which carries dab 486 - il - 2 was constructed as follows . puc18 ( new england biolabs ) was digested with psti and bgli and the psti - bgli fragment carrying the e . coli origin of replication , the polylinker region , and the 3 &# 39 ; portion of the β - lacatamase gene ( amp r ) was recovered . plasmid pkk - 233 - 2 ( pharmacia ) was digested with psti and bgli and the psti - bgli fragment carrying , two transcription terminators and the 5 &# 39 ; portion of the β - lactamase gene was recovered . pdw22 was constructed by ligating these two recovered fragments together . pdw23 was constructed by isolating a bamhi - sali fragment encoding human il - 2 from plasmid pdw15 ( williams et al . ( 1988 ) nucleic acids res . 16 : 10453 - 10467 ) and ligating it to bamhi / sali digested pdw22 ( described above ). pdw24 was constructed as follows . a bamhi - ncoi fragment carrying the trc promoter and translational initiation codon ( atg ) was isolated from plasmid pfk233 - 2 ( pharmacia ). the dna sequence encoding amino acid residues 1 through 485 of dt was obtained by digesting pabc508 ( williams et al . ( 1987 ) protein engineering 1 : 493 - 498 ) with sphi and haeii and recovering the haeii - sphi fragment containing the sequence encoding amino acid residues 1 through 485 of dta . a ncoi / haeii linker ( 5 &# 39 ; ccatgggcgc 3 &# 39 ;) was ligated to the haeii - sphi fragment and that contruction was then ligated to the previously isolated bamhi - ncoi fragment carrying the trc promoter . this results in a bam hi - sphi fragment bearing , in the following order , the trc promoter , the ncoi site ( which supplies the atg initiator codon for met ), and the sequence encoding residues 1 through 485 of native dt . this fragment was inserted into pdw23 that had been digested with bam hi and sphi . the resulting plasmid was desigated pdw24 . the fusion protein ( dab 486 - il - 2 ) encoded by pdw24 is expressed from the trc promoter and consists of met followed by amino acids 1 through 485 of mature dt fused to amino acids 2 through 133 of human il - 2 . the sequence of dt is given in greenfield et al . ( 1983 ) supra . the sequence encoding il - 2 was synthesized on an applied biosystems dna - synthesizer , as described in williams et al . ( 1988 ) nucleic acids res . 16 : 10453 - 10467 , hereby incorporated by reference . the sequence of il - 2 is found in williams et al . ( 1988 ) nucleic acids res . 16 : 10453 - 10467 . fusion of the sequence encoding mature dt to atg using an oligonucleotide linker is described in bishai et al . ( 1987 ) j . bact . 169 : 5140 - 5151 , hereby incorporated by reference . pdw24 is shown in fig2 . the insert corresponding to dab 486 - il - 2 is shown as a heavy line . in fig2 filled circles indicate ncoi sites , open circles indicate nsii sites , open diamonds indicate clai sites , filled squares indicate hpaii sites , open squares indicate sphi sites , and filled triangles indicate sali sites . oligonucleotides and nucleic acids were manipulated as follows . oligonucleotides were synthesized using cyanoethyl phosphoramidite chemistry on an applied biosystems 380a dna synthesizer ( applied biosystems inc ., foster city , calif .). following synthesis , oligonucleotides were purified by chromatography on oligonucleotide purification cartridges ( applied biosystems inc ., foster city , calif .) as directed by the manufacturer . purified oligonucleotides were resuspended in te buffer ( 10 mm tris base , 1 mm edta , ph 8 . 0 ). to anneal complementary strands , equimolar concentrations of each strand were mixed in the presence of 100 mm nacl , heated to 90 ° c . for 10 min , and allowed to cool slowly to room temperature . plasmid dna was purified by the alkaline lysis / cesium chloride gradient method of ausebel et al . ( 1989 ) current protocols in molecular biology , john wiley & amp ; sons , n . y . dna was digested with restriction endonucleases as recommended by the manufacturer ( new england biolabs , beverly , mass . and bethesda research laboratories , gaithersburg , md .). restriction fragments for plasmid construction were extracted from agarose - tbe gels , ligated together ( with or without oligonucleotide linkers ) and used to transform e . coli using standard methods . ausebel et al ( 1989 ) supra and maniatis et al . ( 1982 ), molecular cloning laboratory manual , cold spring harbor laboratory , cold spring harbor , n . y . plasmid dna sequencing was performed according to the dideoxy chain termination method of sanger et al . ( 1987 ) proc . nat &# 39 ; l acad . sci usa 74 : 5463 - 5467 , as modified by kraft et al . ( 1988 ) bio techniques 6 : 544 - 547 , using sequenase ( united states biochemicals , cleveland , ohio ). expression and purification of chimeric toxins was as follows . all dt - related il - 2 fusion proteins used herein were expressed in the cytoplasm of e . coli strain jm101 from the trc promoter , amann et al . ( 1985 ), gene 40 : 183 - 190 , hereby incorporated by reference . recombinant e . coli were grown in m9 minimal medium ( maniatis et al . ( 1982 ) supra ) supplemented with 10 mg / ml casamino acids ( difco , detroit , mich . ), 50 μg / ml ampicillin , and 0 . 5 ng / ml thymine in 10 liter volumes in a microgen fermentor ( new brunswick scienctific , edison , n . j .). bacterial cultures were grown at 30 ° c ., and sparged with air at 5l / min . when the absorbance ( a 590 nm ) of the culture reached 0 . 3 , expression of chimeric tox gene was induced by the addition of isopropyl - β - d - thiogalactopyranoside . two hours after induction , bacteria were harvested by centrifugation , resuspended in buffer · 101 ( 50 mm kh 2 po 4 , 10 mm edta , 750 mm nacl , 0 . 1 % tween 20 , ph 8 . 0 ), and lysed by sonication ( branson sonifier ). whole cells and debris were removed by centrifugation at 27 , 000 × g , and the clarified extract was then filter sterilized and applied to an anti - diphtheria toxin immunoaffinity column . bound proteins were eluted with 4m guanidine hydrochloride , reduced by the addition of β - mercaptoethanol to 1 % and then sized by high pressure liquid chromatography on a 7 . 5 × 600 mm g4000pw column ( tosohass ). prior to use , fusion toxins were exhaustively dialysed against hepes buffered hank &# 39 ; s balanced salt solution ( gibco ), ph 7 . 4 . purified diphtheria toxin was purchased from list biological laboratories ( campbell , calif .). for the production of the non - toxic crm1001 , c7 ( βtox - 1001 ) was grown in 100 ml volumes of c - y medium ( rappuoli et al . ( 1983 ) j . bact . 153 : 1201 - 1210 ) in 2 - liter erlenmeyer flasks at 35 ° c . for 20 hrs with shaking ( 240 rpm ). bacteria were removed by centrifugation at 20 , 000 × g for 15 min . crm1001 was precipitated from the culture medium by the addition of nh 4 so 4 to 70 % saturation , and collected by centrifugation . following dialysis against 10 mm phosphate buffer , ph 7 . 2 , crm1001 was purified by ion exchange chromatography on de - 52 cellulose as previously described by pappenheimer et al . ( 1972 ), immunochem . 9 : 891 - 906 . the concentration of all purified proteins was determined by using pierce protein assay reagent ( pierce chemical co ., rockford , ill .). dab ( 1001 ) 486 - il - 2 is a chimeric toxin identical to dab 486 - il - 2 except for the disruption of the disulfide bridge between cys462 and cys472 in dab ( 1001 ) 486 - il - 2 . dab ( 1001 ) 486 - il - 2 was constructed by replacing the 587 basepair ( bp ) clai - sphi restriction fragment which encodes most of fragment b of dt ) of plasmid pdw24 ( which carries dab 486 - il - 2 ) with the analogous fragment from dna encoding the tox - 1001 mutant allele of dt . tox - 1001 encodes non - toxic diphtheria toxin - related protein crm1001 and has been shown to result from a single point mutation which changes cys471 to tyr471 , rappuoli et al . ( 1986 ) in protein carbohydrate interactions in biological systems , academic press , inc ., london , pp . 295 - 296 , hereby incorporated by reference . fig3 depicts the restriction maps of dna encoding dab 486 - il - 2 and the corresponding fusion protein encoded by dab 486 - il - 2 . ( in fig3 stippled boxes between the nsii and hpaii restriction endonuclease sites designate the diphtheria toxin fragment b - related sequences which encode the membrane associating domains . the amphipathic domain is encoded between the nsii and clai sites , and the putative membrane spanning domains are encoded between the clai and hpaii sites . hatched boxes indicate the relative position of internal in - frame deletion mutations .) the construction of pdw26 , which encodes the chimeric toxin with the cys 472 to tyr 472 mutation , is shown in fig2 . following ligation and transformation , the dna sequence of the tox - 1001 portion of the gene fusion dab ( 1001 ) 486 - il - 2 was determined in order to insure that the cys471 to tyr471 mutation was recloned . e . coli ( pdw26 ), was grown in m9 minimal media , cells were harvested , lysed and the fusion toxin , designated dab ( 1001 ) 486 - il - 2 , was purified by immunoaffinity chromatography and hplc . the dose response capacity of dab 486 - il - 2 , crm1001 , and dab ( 1001 ) 486 - il - 2 to block 14 c !- leucine incorporation by high affinity il - 2 receptor bearing hut 102 / 6tg cells was determined . as anticipated , dab 486 - il - 2 was highly toxic for these cells ( ic 50 = 4 × 10 - 10 m ); whereas , crm1001 was found to be non - toxic . in marked contrast to crm1001 , however , the fusion toxin which carries the cys472 to tyr472 mutation , dab ( 1001 ) 486 - il - 2 , was found to be as toxic for hut 102 / 6tg cells as the wild type dab 486 - il - 2 . these results demonstrate that the fragment b disulfide bond is not required for biological activity of the fusion toxin . hut 102 / 6tg cytotoxicity assays were performed as follows . cultured hut 102 / 6tg cells were maintained in rpmi 1640 medium ( gibco , grand island , n . y .) supplemented with 10 % fetal bovine serum ( cellect , gibco ), 2 mm glutamine , and penicillin and streptomycin to 50 iu and 50 μg / ml , respectively . for cytotoxicity assays , cells were seeded in 96 - well v - bottomed plates ( linbro - flow laboratories , mclean , va .) at a concentration of 5 × 10 4 per well in complete medium . toxins , or toxin - related materials , were added to varying concentrations ( 10 - 12 m to 10 - 6 m ) and the cultures were incubated for 18 hrs at 37 ° c . in a 5 % co 2 atmosphere . following incubation , the plates were centrifuged for 5 min . at 170 × g and the medium removed and replaced with 200 μl leucine - free medium ( mem , gibco ) containing 1 . 0 μci / ml 14 c !- leucine ( new england nuclear , boston , mass .). after an additional 90 min . at 37 ° c ., the plates were centrifuged for 5 min . at 170 × g , the medium was removed and the cells were lysed by the addition of 4m koh . protein was precipitated by the addition of 10 % trichloroacetic acid and the insoluble material was then collected on glass fiber filters using a cell harvester ( skatron , sterling , va .). filters were washed , dried , and counted according to standard methods . cells cultured with medium alone served as the control . all assays were performed in quadruplicate . since the disulfide bond between cys462 - cys472 was not required for the cytotoxic action of dab 486 - il - 2 , it was of interest to determine what dt fragment b sequences were essential for the delivery of fragment a to the cytosol . several in - frame deletion mutations were introduced into the fragment b encoding portion of the dab 486 - il - 2 toxin gene , fig1 b , 2 , and 3 . fig1 b shows the structure of dab 486 - il - 2 and various mutants derived from dab 486 - il - 2 . in fig1 b a wide bar indicates the fusion protein , narrow connecting lines represent deletions , numbers above the bars are amino acid residue numbers in the dab nomenclature , numbers below the bars correspond to the amino acid residue numbering of native dt , cross hatching indicated amphipathic regions , darkened areas correspond to the transmembrane region , il - 2 - 2 - 133 indicates amino acid residues 2 - 133 of il - 2 , ala = alanine , ash = asparagine , asp = aspartic acid , cys = cysteine , gly = glycine , his = histidine , ile = isoleucine , met = methionine , thr = threonine , tyr = tyrosine , and val = valine . the first mutant , dab 389 - il - 2 was constructed by removing a 309 bp hpaii - sphi restriction fragment from pdw24 and replacing it with oligonucleotide linker 261 / 274 ( table 1 ) to generate plasmid pdw27 ( fig1 ). this linker restores fragment b sequences from pro383 to thr387 , and allows for in - frame fusion to il - 2 sequences at this position . thus , in dab 389 - il - 2 the 97 amino acids between thr387 and his485 have been deleted . table 1______________________________________oligonucleotide linkers . oligonucleotide identificationconstruct number linker______________________________________dab . sub . 389 - il - 2 274 5 &# 39 ;- cg ggt cac aaa acg cat g - 3 &# 39 ; 261 cca gtg ttt tgc 1 / 2 hpaii 1 / 2 sphidab . sub . 295 - il - 2 292 5 &# 39 ;- c gat ggt gtg cat g - 3 &# 39 ; 293 ta cca cac 1 / 2 clai 1 / 2 sphidab ( δ205 - 337 5 &# 39 ;- ta aat at - 3 &# 39 ; 289 ). sub . 486 - il - 2 338 acg tat tta tag c 1 / 2 nsii 1 / 2 claidab ( δ205 - 337289 ). sub . 389 - 1l2 338______________________________________ in a similar fashion , a 191 amino acid in - frame deletion was constructed by removing a clai - sphi restriction fragment from pdw24 and replacing it with the 292 / 293 oligonucleotide linker ( table 1 ) to form plasmid pdw28 which encodes dab 295 - il - 2 . in this case , the in - frame deletion encompasses the putative membrane - spanning helices that have been predicted by lambotte et al . ( 1980 ) j . cell . biol . 87 : 837 - 840 , to play a role in the delivery of fragment a to the eukaryotic cell cytosol . purified , dab 389 - il - 2 and dab 295 - il - 2 were found to have electrophoretic mobilities of 57 kda and 47 kda , respectively . the dose response analysis on hut 102 / 6tg cells is shown in fig4 . in fig4 dab 486 - il - 2 is indicated by filled squares ; dab 389 - il - 2 is indicated by filled circles ; dab 295 - il2 is indicated by open circles ; dab ( δ205 - 289 ) 486 - il - 2 ( see below ) is indicated by open squares ; and dab ( δ205 - 289 ) 389 - il - 2 ( see below ) is indicated by open triangles . dab 486 - il - 2 and dab 389 - il - 2 exhibited an ic 50 of approximately 4 × 10 - 10 m and 1 × 10 - 10 m , respectively . in marked contrast , the ic 50 of dab 295 - il - 2 was approximately 1 , 000 - fold lower ( 4 × 10 - 7 m ). these results suggest that fragment b sequences between thr 387 and his 486 do not play a major role in the delivery of fragment a to the cytosol . sequences between ser292 and thr387 on the other hand are essential for the efficient delivery of fragment a . surprisingly , dab 389 - il - 2 possessed much greater activity than did dab 486 - il - 2 . dab 389 - il - 2 , which lacks native dt residues 387 through 483 , and which has increased toxic activity , leaves the hydrophobic transmembrane segment located approximately between native dt residues 346 and 371 intact . see lambotte et al . ( 1980 ) j . cell biol . 87 : 837 - 840 , hereby incorporated by reference , for a characterization of the transmembrane region . dab 295 - il - 2 , which removes native dt residues 291 through 481 , and which has greatly reduced toxicity , removes the transmembrane region ( 346 - 371 ). in order to rule out the possibility that the reason for the low potency of dab 295 - il - 2 for hut 102 / 6tg cells was related to altered binding to the high affinity il - 2 receptor , we have conducted a series of competitive displacement experiments using 125 i !- ril - 2 . fig5 shows the competitive displacement of 125 i !- labeled il - 2 from the high affinity il - 2 receptor by unlabeled ril - 2 depicted by filled circles ; dab 486 - il - 2 depicted by open triangles ; dab 389 - il - 2 depicted by closed squares ; dab 295 - il - 2 depicted by closed triangles ; dab ( δ205 - 289 ) 486 - il - 2 ( see below ) depicted by open circles ; and dab ( δ205 - 289 ) 389 - il - 2 ( see below ) depicted by open squares . the concentration of 125 i !- il - 2 used was 10 pm and the specific activity was approximately 0 . 7 μci / pmol . as shown in table 2 , both dab 389 - il - 2 and dab 295 - il - 2 were found to have an apparent k d that is approximately 3 - times lower than that of dab 486 - il - 2 ( k d = 8 × 10 - 9 m vs . k d = 2 . 5 × 10 - 8 m ). it is particularly significant that competitive displacement experiments showed that both dab 389 - il - 2 and dab 295 - il - 2 bind more avidly to the high affinity il - 2 receptor than does dab 486 - il - 2 ( kd = 8 × 10 - 9 and 8 . 4 × 10 - 9 m vs . kd = 2 . 5 × 10 - 8 m ). these results provide evidence that fusion of il - 2 sequences to toxophores of smaller mass may serve to position the il - 2 binding domain for more favorable receptor interaction . it is of interest to note that while dab 295 - il - 2 binds more avidly to the high affinity il - 2 receptor than dab 486 - il - 2 , its cytotoxic activity is at least 1 , 000 - fold lower ( fig4 ). these results indicated that avid binding to the target receptor is not in itself sufficient for the biologic activity of the dt - related il - 2 fusion toxins , and that fragment b sequences between ser292 and thr387 are essential for a post - receptor binding event in the intoxication process . table 2______________________________________relative ability of ril - 2 and dab - il - 2 related fusionproteins to displace . sup . 125 i !-- ril - 2 from highaffinity il - 2 receptors on hut 102 / 6tg cellsunlabeled ligand apparent k . sub . d k . sub . d dab - il - 2 / ril - 2______________________________________ril - 2 . sup . 1 . 7 × 10 . sup .- 10 -- dab . sub . 486 - il - 2 2 . 5 × 10 . sup .- 8 147dab . sub . 389 - il - 2 8 . 0 × 10 . sup .- 9 47dab . sub . 295 - il - 2 8 . 4 × 10 . sup .- 9 49dab ( δ205 - 289 ). sub . 486 - il - 2 1 . 0 × 10 . sup .- 7 588dab ( δ205 - 289 ). sub . 389 - il - 2 2 . 9 × 10 . sup .- 8 170______________________________________ competitive displacement of 125 i !- ril - 2 by ril - 2 and dab - il - 2 fusion toxins was determined as follows . the radiolabeled il - 2 binding assay was performed essentially as described by wang et al . ( 1987 ) j . exp . med . 166 : 1055 - 1069 . cells were harvested and washed with cell culture medium . hut 102 / 6tg cells were resuspended to 5 × 10 6 per ml and incubated with 125 i !- ril - 2 ( 0 . 7 μci / pmol ) in the presence or absence of increasing concentrations of unlabeled ril - 2 or the dab - il - 2 fusion toxins for 30 min . at 37 ° c . under 5 % co 2 . the reaction was then overlayed on a mixture of 80 % 550 fluid ( accumetric inc ., elizabethtown , kans .) : 20 % parafin oil ( d = 1 . 03 g / ml ) and microcentrifuged . the aqueous phase and the pellet of each sample , representing free and bound ligand , respectively , was then counted in a nuclear chicago gamma counter . apparent dissociation constants , k d , were determined from the concentrations of unlabeled ligand required to displace 50 % of radiolabeled ril - 2 binding to receptors . in order to test the hypothesis that an amphipathic region ( amino acids 210 - 252 in dab 486 - il - 2 ) plays a role in the intoxication process , in - frame deletions of the 85 amino acid encoding region from nsii to clai of both pdw24 and pdw27 to form pdw30 ( containing dab ( δ205 - 289 ) 486 - il - 2 ) and pdw31 ( containing dab ( δ205 - 289 ) 389 - il - 2 ), respectively ( fig2 and 3 ; table 1 ) were constructed . following ligation and transformation , the dab - il - 2 related fusion proteins were expressed and purified , as described above . as shown in fig4 the deletion of fragment b sequences which include the amphipathic region result in a marked loss of cytotoxic activity against high affinity il - 2 receptor positive cells in vitro . it is of interest to note that dab ( δ205 - 289 ) 389 - il - 2 was found to displace radiolabeled il - 2 from the high affinity receptor almost as well as dab 486 - il - 2 ; whereas , dab ( δ205 - 289 ) 486 - il - 2 was found to bind 4 - fold less avidly to the receptor ( fig5 ). the chimeric toxin encoded by dab 389 - il - 2 is more resistant to proteolytic degradation than is the chimeric toxin encoded by dab 486 - il - 2 . when purified , as described above , and analysed on sds - polyacrylamide gels , the dab 389 - il - 2 hybrid toxin is accompanied by very few degradation products ( as evidenced by the relative absence of bands of smaller size than that of the intact chimeric toxin ). purified dab 486 - il - 2 on the other hand is accompanied by numerous dark bands of lower molecular weight than the intact chimeric toxin . these lower molecular weight bands react with anti - dab 486 - il - 2 antibodies , supporting the conclusion that they are degradation products . sodium dodecylsulfate ( sds )- polyacrylamide gel electrophoresis ( page ) was performed according to the method of laemmli ( 1970 ) nature 227 : 680 - 685 using 12 % gels and a mini - protein ii gel apparatus ( biorad ). proteins were fixed in 12 . 5 % trichloroacetic acid for 5 min and stained with coomassie brilliant blue according to the diezal procedure , diezal et al . ( 1972 ) anal . biochem . 48 : 617 - 624 . dab 486 egf and dab 389 egf can be constructed in a manner analogous to that in which dab 486 - il - 2 and dab 389 - il - 2 were constructed , by methods known to those skilled in the art . to construct a plasmid encoding dab 486 fused to egf , plasmid pdw24 ( which encodes dab 486 fused to il - 2 ) is digested with sphi and hindiii to remove the il - 2 coding sequence . the resulting pdw24 sphi - hindiii fragment containing the sequence encoding dt residues 1 - 485 is ligated to a synthetic sphi - hindiii fragment encoding egf to yield a plasmid encoding dab 486 fused to egf . the egf fragment , shown in fig6 was synthesized , as described , using preferred codons for expression in e . coli ( see grosjean et al . ( 1982 ) gene 18 : 199 - 209 , hearby incorporated by reference ). the synthetic fragment includes appropriate linkers at the 5 &# 39 ; and 3 &# 39 ; ends for insertion into the plasmid and for in - frame fusion to the dt coding sequence . to construct a plasmid encoding dab 389 fused to egf a similar protocol can be followed , except that pdw27 ( which encodes dab 389 fused to il - 2 ) is used in place of pdw24 . the il - 2 encoding dna is removed from pdw27 by digestion with sphi and hindiii and egf encoding dna is inserted in its place , resulting in a dab 389 fused in frame to egf . the same synthetic egf fragment used in the construction of the dab 489 egf fusion ( fig6 ) can be used . those skilled in the art will realize that the protocols given above are not the only way of making the chimeric toxins of the invention . refinements include changes in pdw24 , pdw27 , and plasmids derived therefrom directed toward compliance with the good manufacturing practises of the food and drug administration , e . g ., the inclusion of the laci q gene ( amersham ) and the replacement of the ampicillin resistance gene ( amp r ) with the gene for neomycin / kanamycin resistance from tn5 ( pharmacia ) in the plasmids that are used for expression of the chimeric toxins of the invention . these alterations can be performed without undue experimentation by one skilled in the art . dab 486 egf and dab 389 egf are the products of fusion genes in which the receptor binding domain of dt has been removed and replaced with dna encoding human egf . as shown in fig7 the resulting proteins contain the enzymatically active fragment a of dt and the lipid associating domains of fragment b of dt required for translocation of fragment a into the cytosol . dab 389 egf differs from dab 486 egf by the deletion of the 97 amino acids immediately 5 &# 39 ; to amino acid residue 484 of dt . the egf portion of both dab 486 egf and dab 389 egf governs receptor binding . thus , these molecules have the potential to specifically target the cytotoxicity of dt to tumor cells characterized by egf receptor expression . the cytotoxicity of dab 486 egf for a panel of human cell lines was assessed and compared to a431 cells ( atcc crl 1555 ), a human epidermoid carcinoma cell line with a high number of egf receptors . the results are shown in table 3 . included in the study were human tumor cell lines which have been reported to express high numbers of egf receptors ( e . g ., bt - 20 , hela , lncap and u - 87 mg ) as well as human tumor cell lines ( e . g ., c91 / pl , bewo and a375 ) and normal tissue cell lines ( e . g ., wi - 38 , hs 67 and hepm ) expressing few or no egf receptors . cytotoxicity was evaluated as follows . cells were plated in triplicate wells of 96 well plates with dab 486 egf in assay medium appropriate to the cell type ( see table 3 ). dab 486 egf concentrations were between 10 - 15 and 10 - 7 m . following a 20 - hour incubation , cells were labeled with 14 c ! leucine , trypsinized , harvested onto glass fiber filter mats and counted to determine the percent of control incorporation . cell lines exhibiting an ic 50 for dab 486 egf of less than 0 . 5 nm were considered sensitive . table 3______________________________________the effect of a dt - egf chimeric toxin on various cell linescell line tissue / type sensitivity______________________________________tumor cell linesa431 vulval epidermoid carcinoma + a549 lung carcinoma + kb oral epidermoid carcinoma + bt - 20 breast adenocarcinoma + hela s3 cervical carcinoma + t47d breast ductal carcinoma + lncap . fg prostate carcinoma + hos osteosarcoma + u - 87 mg glioblastoma / astrocytoma + c91 / pl htlv - 1 transformed t cell - bewo choriocarcinoma - a375 malignant melanoma - mcf - 7 breast adenocarcinoma - snu - c2b cecum carcinoma - normal cell lineswi - 38 diploid lung fibroblast - hs 67 thymus - ccd - 18co colon fibroblast - hism smooth muscle , jejunum - fh74s int fetal small intestine - hepm embryonic palatal - mesenchyme - ______________________________________ growth conditions and passage schedules used were those defined by atcc ( except as noted below ). culture media were as follows : a431 ( atcc crl1555 ), dmem + 10 % fcs ; a549 ( atcc ccl185 ) ham &# 39 ; s f12 + 10 % fcs ; kb ( atcc ccl17 ), dmem + neaa + 10 % fcs ; bt - 20 ( atcc htb19 ), mem + 10 % fcs ; hela s3 ( atcc ccl2 . 2 ), ham &# 39 ; s f12 + 10 % fcs ; t47d ( atcc htb133 ), rpmi 1640 + 10 % fcs ; lncap . fg ( atcc crl1740 ), rpmi 1640 + 10 % fcs ; hos ( atcc crl1543 ), mem + 10 % fcs ; u - 87 mg ( atcc htb14 ), mem + 10 % fcs ; c91 / pl ( from robert swartz , nemc , boston , mass ., see bacha et al . ( 1988 ) j . exp . med . 167 : 612 for growth conditions ), rpm1 1640 + 15 % fcs bewo ( attc ccl98 ), ham &# 39 ; s f12 + 15 % fcs ; a375 ( atcc crl 1619 ), dmem + 10 % fcs ; mcf - 7 ( atcc tb22 ) mem + 10 % fcs ; snu - c2b ( atcc ccl250 ) rpm1 1640 + 10 % fcs ; wi - 38 ( atcc ccl75 ), eagle &# 39 ; s basal + 10 % fcs ; hs 67 ( atcc htb 163 ), dmem + 10 % fcs ; ccd - 18co ( atcc crl 1459 ), mem + 10 % fcs ; hism ( atcc crl 1692 ), dmem + 10 % fcs ; fhs74int ( atcc ccl241 ), dmem + 10 % fcs ; hepm ( atcc crl 1486 ), mem + 10 % fcs . to demonstrate that the cytotoxic action of dab 486 egf and dab 389 egf are mediated selectively by the egf receptor , a431 cells were plated in triplicate wells of 96 well plates with dab 486 egf ( fig8 ) or dab 389 egf ( fig9 ) in the presence of the specific competitor of the egf receptor , human egf ( upstate biotechnologies , inc .) ( 10 - 7 m ), in assay medium ( dmem 10 % fcs ). in fig8 solid squares indicate dab 486 egf and solid triangles indicate dab 486 egf + egf . in fig9 solid squares indicate dab 389 egf and solid triangles indicate dab 389 egf + egf . following a 20 - hour incubation at 37 ° c ., cells were labeled with 14 c ! leucine , trypsinized , harvested onto glass fiber filter mats and counted to determine the percent of control incorporation . the results show that , in the absence of egf , dab 486 egf and dab 389 egf inhibit protein synthesis with an ic 50 of 3 × 10 - 12 m and 3 × 10 - 13 m , respectively . egf almost completely abolishes this activity . likewise , anti - egf ( biomedical technologies , inc .) and anti - egf receptor ( upstate biotechnologies , inc .) also abolish the cytotoxicity of dab 486 egf and dab 389 egf while the nonspecific competitors , transferrin ( sigma ) anti - transferrin ( dako ), and anti - transferrin receptor ( dako ), have no effect . these results demonstrate that dab 486 egf and dab 389 egf are potent and specific cytotoxic agents . note that dab 389 egf is approximately 10 times more potent than dab 486 egf . dab 389 egf , like egf , induces down regulation of the egf receptor , providing further evidence for the egf receptor - specific nature of dt - egf chimeric toxins . binding and internalization of egf induces down regulation of the egf receptor which can be detected as a decrease in 125 i ! egf binding capacity ( krupp et al . ( 1982 ) j . biol . chem . 257 : 11489 ). the ability of dab 389 egf to induce egf receptor internalization and subsequent down regulation was evaluated and compared to that induced by native egf . the results are shown in fig1 . in fig1 open squares indicate egf and closed diamonds indicate dab 389 egf . a431 cells in triplicate wells of 24 well plates were preincubated with egf or dab 389 egf ( 10 - 8 m ) for the indicated times in dmem + 0 . 1 % bsa ( bovine serum albumin ) at 37 ° c . the cells were then placed on ice and acid stripped ( with 0 . 2m acetic acid , 0 . 5m nacl ) to remove bound , but not internalized , egf or dab 389 egf . egf binding capacity was measured by incubating the cells , on ice , with 125 i ! egf . following a 90 - minute incubation the cells were washed , solubilized , and counted . an egf receptor - dependent interaction is also shown by the fact that dab 389 egf , like egf , displaces 125 i ! egf from the egf receptor , as shown in fig1 . in fig1 open squares indicate egf and solid diamonds indicate dab 389 egf . results in fig1 are expressed as a percent of control ( no competition ) cpm . the ability of dab 389 egf to displace high affinity 125 i ! egf binding to a431 cells was evaluated as follows . a431 cells , plated in triplicate wells of 24 well plates , were preincubated in binding media ( phosphate buffered saline ph 7 . 2 + 0 . 1 % bsa + 15 mm sodium azide + 50 mm 2 - deoxyglucose ) for 1 hour at 37 ° c . and then incubated with 125 i ! egf in binding media in the presence of dab 389 egf or egf . following incubation , the cells were washed , solubilized and counted . the results are summarized in table 4 . in table 4 ec 50 is the concentration resulting in displacement of 50 % of the 125 i ! egf . table 4______________________________________displacement of . sup . 125 i ! egf binding by egf anddab . sub . 389 egf fold over fold overcompetition ec . sub . 50 . sup . 125 i ! egf egf______________________________________egf 1 . 0 × 10 . sup .- 8 m 20 -- dab . sub . 389 egf 4 . 5 × 10 . sup .- 7 m 900 45______________________________________ upon binding to its receptor , the cellular uptake of native dt occurs by endocytosis of clathrin coated vesicles ( middlebrook et al . ( 1978 ) j . biol . chem . 253 : 7325 ). dt is then found in endosomes where the low ph induces a conformational change facilitating the translocation of the enzymatic fragment a portion of dt into the cytosol . to determine if the cytotoxicity of dab 486 egf and dab 389 egf is also dependent upon the same pathway , a431 cells were plated in sextuplicate wells of 96 well plates containing dab 486 egf , dab 389 egf or dmem + 10 % fcs in the absence or presence of chloroquine ( 10 - 5 m ) ( sigma ). chloroquine is a lysosomotropic compound which prevents acidification of endosomes ( kim et al . ( 1965 ) j . bacteriol . 90 : 1552 ). following a 20 - hour incubation at 37 ° c ., the cells were labeled with 3 h ! leucine , trypsinized , harvested onto glass fiber filter mats and counted . the results are shown in table 5 , expressed as the percent of control ( no dab 486 egf or dab 389 egf ) incorporation and represent the mean of three experiments . the results show that chloroquine blocks the cytotoxicity of dt - egf chimeric toxins . table 5______________________________________sensitivity of dab - egf chimeric toxin - cytotoxicity to chloroquinepercent of control incorporation no addition chloroquine______________________________________dab . sub . 486 egf concentration0 100 8610 . sup .- 8 m 5 6010 . sup .- 9 m 25 96dab . sub . 389 egf concentration0 100 7310 . sup .- 11 m 4 6110 . sup .- 12 m 57 100______________________________________ following translocation into the cytosol , fragment a catalyzes the cleavage of nad and the covalent linkage of adp - ribose to elongation factor 2 ( ef - 2 ) resulting in the inhibition of protein synthesis ( bacha et al . ( 1983 ) j . biol . chem . 258 : 1565 ). to evaluate the mechanism by which dab 486 egf inhibits protein synthesis , a431 cells were plated in triplicate wells of 24 well plates containing dt , dab 486 egf , or complete medium . following a 20 - hour incubation at 37 ° c ., the cells were washed and incubated in lysis buffer ( 10 mm tris , 10 mm nacl , 3 mg cl 2 , 10 mm thymidine , 1 mm egta , 1 % triton x - 100 ) with 32 p ! nad in the presence of purified dt fragment a ( calbiochem ). tca precipitable extracts were collected on glass fiber filters and coated to quantitate the percent of control ef - 2 available for adp - ribosylation . the results of these experiments are shown in table 6 . dab 486 egf , like dt , reduced ( in a dosage dependent manner ) the amount of ef - 2 available for adp ribosylation . table 6______________________________________adp - ribosylation of ef - 2 by dab . sub . 486 egf percent of control level of ef - 2 available fortoxin concentration adp - ribosylation______________________________________dt 10 . sup .- 8 m & lt ; 1 10 . sup .- 9 m 17dab . sub . 486 egf 10 . sup .- 8 m 13 10 . sup .- 9 m 20______________________________________ dab 389 egf is far more toxic than is dab 486 egf . as shown in fig8 and 9 , dab 389 egf exhibits an ic 50 for the inhibition of protein synthesis in a431 cells approximately 10 times lower than that of dab 486 egf ( dab 389 egf ic 50 = 3 × 10 - 13 m ; dab 486 egf ic 50 = 3 × 10 - 12 m ). the greater potency of dab 389 egf is also shown in experiments measuring the rapidity with which dab 389 egf and dab 486 egf kill a431 cells . fig1 and 12 show the exposure time ( of a431 cells to dab 486 egf or dab 389 egf ) required to induce maximal inhibition of protein synthesis . cells were exposed to dab 486 egf ( 5 × 10 - 9 m ) ( fig1 ) or dab 389 egf ( 5 × 10 - 9 m ) ( fig1 ) for the indicated times and then washed of unbound dab 486 egf or dab 389 egf . following an overnight incubation in complete media ( dmem + 10 % fcs ), the cells were labeled with 14 c ! leucine . the results show that near maximal inhibition of protein synthesis occurs following a 15 - minute exposure to dab 389 egf while a greater than 75 - minute exposure is required for dab 486 egf . the kinetics of protein synthesis inhibition in dab 389 egf or dab 486 egf treated a431 cells is shown in fig1 . to examine the kinetics of protein synthesis inhibition a431 cells were incubated with dab 486 egf ( 5 × 10 - 9 ) or dab 389 egf ( 5 × 10 - 9 m ) in complete medium at 37 ° c . at the indicated times , dab 486 egf or dab 389 egf was removed and the cells were labeled with 14 c ! leucine for 1 hour . the results indicate that there is a 50 % reduction in protein synthesis following a 1 - hour incubation with dab 389 egf while maximal inhibition occurs by 4 hours . maximal inhibition of protein synthesis occurs more than 6 hours following incubation with dab 486 egf . the improved chimeric toxins of the invention are administered to a mammal , e . g ., a human , suffering from a medical disorder , e . g ., cancer , or other conditions characterized by the presence of a class of unwanted cells to which a polypeptide ligand can selectively bind . the amount of protein administered will vary with the type of disease , extensiveness of the disease , and size of species of the mammal suffering from the disease . generally , amounts will be in the range of those used for other cytotoxic agents used in the treatment of cancer , although in certain instances lower amounts will be needed because of the specificity and increased toxicity of the improved chimeric toxins . the improved chimeric toxins can be administered using any conventional method ; e . g ., via injection , or via a timed - release implant . in the case of msh improved chimeric toxins , topical creams can be used to kill primary cancer cells , and injections or implants can be used to kill metastatic cells . the improved chimeric toxins can be combined with any non - toxic , pharmaceutically - acceptable carrier substance . other embodiments are within the following claims . for example , chimeric toxins have been constructed , by methods known to those skilled in the art , in which dab 389 and dab 486 have been fused to interleukin 4 ( il - 4 ). dab 389 - il - 4 is about 10 times more cytotoxic than is dab 486 - il - 4 . dab 389 has also been fused to interleukin 6 . dab 486 and dab 389 have also been fused to human chorionic gonadotropin . the improved chimeric toxins of the invention include portions of dt fused to any cell - specific polypeptide ligand which has a binding domain specific for the particular class of cells which are to be intoxicated . polypeptide hormones are useful such ligands . chimeric toxins , e . g ., those made using the binding domain of α or β msh , can selectively bind to melanocytes , allowing the construction of improved dt - msh chimeric toxins useful in the treatment of melanoma . other specific - binding ligands which can be used include insulin , somatostatin , interleukins i and iii , and granulocyte colony stimulating factor . other useful polypeptide ligands having cell - specific binding domains are follicle stimulating hormone ( specific for ovarian cells ), luteinizing hormone ( specific for ovarian cells ), thyroid stimulating hormone ( specific for thyroid cells ), vasopressin ( specific for uterine cells , as well as bladder and intestinal cells ), prolactin ( specific for breast cells ), and growth hormone ( specific for certain bone cells ). improved chimeric toxins using these ligands are useful in treating cancers or other diseases of the cell type to which there is specific binding . for a number of cell - specific ligands , the region within each such ligand in which the binding domain is located is now known . furthermore , recent advances in solid phase polypeptide synthesis enable those skilled in this technology to determine the binding domain of practically any such ligand , by synthesizing various fragments of the ligand and testing them for the ability to bind to the class of cells to be labeled . thus , the chimeric toxins of the invention need not include an entire ligand , but rather may include only a fragment of a ligand which exhibits the desired cell - binding capacity . likewise , analogs of the ligand or its cell - binding region having minor sequence variations may be synthesized , tested for their ability to bind to cells , and incorporated into the hybrid molecules of the invention . other potential ligands include monoclonal antibodies or antigen - binding , single - chain analogs of monoclonal antibodies , where the antigen is a receptor or other moiety expressed on the surface of the target cell membrane .