IMPROVED CHIMERIC TOXINS

Abstract: A chimeric toxin comprising protein fragments joined together by peptide bonds, the chimeric toxin comprising, in sequential order, beginning at the amino terminal end of the chimeric toxin, (a) the enzymatically active Fragment A of diphtheria toxin, (b) a first fragment including the cleavage domain 11 adjacent the Fragment A of diphtheria toxin, (c) a second fragment comprising at least a portion of the hydrophobic transmembrane region of Fragment B of diphtheria toxin, the second fragment having a deletion of at least 50 diphtheria toxin amino acid residues, the deletion being C-terminal to the portion of the transmembrane region, and the second fragment not including domain 12, and (d) a third fragment comprising a portion of a cell-specific polypeptide ligand, the portion including at least a portion of the binding domain of the polypeptide ligand, the portion of the binding domain being effective to cause the chimeric toxin to bind selectively to a predetermined class of cells to be attacked by the enzymatically active Fragment A.

Description: IMPROVED CHIMERIC TOXINS Background of the Invention This application is a continuation-in-part of USSN 488,608 filed March 2, 1990. This invention relates to the use of recombinant DNA techniques to construct chimeric toxin molecules. The literature contains many examples of fused genes which code for chimeric proteins. For example, Villa-Komaroff et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 7_5:3727-3731, describes a fused gene made up of a eukaryotic structural gene fused to a non-cytoplasmic bacterial gene. The fused gene'codes for a chimeric protein which is transported out of the cytoplasm. Murphy U.S. Patent No. 4,675,382, hereby incorporated by reference, describes the use of recombinant DNA techniques to produce a hybrid, or chimeric, protein, consisting of a portion of the diphtheria toxin (DT) molecule linked via a peptide linkage to a cell-specific ligand such as α-melanocyte stimulating hormone (MSH) . The DT-MSH chimeric toxin was selectively toxic for particular target cells, i.e., α-MSH receptor positive human malignant melanoma cells. A diphtheria toxin-related fusion protein, DAB 486 -IL-2, in which the native receptor binding domain of DT was genetically replaced with a portion of the polypeptide hormone interleukin-2 (IL-2) has been described in Williams et al. (1987) Protein Engineering JL:493-498,hereby incorporated by reference. DAB 48 _-IL-2 is a 68,142 Da fusion protein consisting of, in the following order: Met; DT residues 1-485; and amino acids 2 through 133 of mature human IL-2. DAB4,8 O 6--IL-2 has been shown to bind to the IL-2 receptor and to selectively intoxicate lymphocytes which bear the high affinity form of the IL-2 receptor, Bacha et al . (1988) J. Exp. Med 167: 612-622. Moreover, the cytotoxic action of DAB., 0 ..-ILι-2, like that of native diphtheria toxin, 486 requires receptor-mediated endocytosis, passage through an acidic compartment, and delivery of Fragment A associated ADP-ribosyltransferase to the cytosol of target cells, Bacha et al. (1988) supra. Summary of the Invention In general, the invention features a chimeric toxin including protein fragments joined together by peptide bonds. The chimeric toxin includes, in sequential order, beginning at the amino terminal end of the chimeric toxin: (a) the enzymatically active Fragment A of diphtheria toxin; (b) a first fragment including the cleavage domain 1., adjacent Fragment A of diphtheria toxin; (c) a second fragment including at least a portion of the hydrophobic transmembrane region of Fragment B of diphtheria toxin, the second fragment also having a deletion, C-terminal to the transmembrane region, of at least 50, or more preferably of at least 80, diphtheria toxin amino acid residues, and the second fragment not including domain 1_; and (d) a third fragment including a portion of a cell-specific polypeptide ligand e.g., an interleukin (preferably interleukin 2, or, epidermal growth factor (EGF), including at least a portion of the binding domain of the polypeptide ligand, that portion being effective to cause the chimeric toxin to bind selectively to a predetermined class of cells to be attacked by enzymatically active Fragment A. In preferred embodiments the chimeric toxin possesses at least one of, and more preferably at least two of, and even more preferably at least three of: greater toxicity to receptor-bearing cells than that of an analagous DAB. 8fi -containing-toxin (an analagous DAB. gg -containing toxin is a toxin which is identical to the chimeric toxin of the preferred embodiment except that DAB 4Q6 replaces the fragments of DT recited in (a), (b), and (c) above, i.e., a toxin consisting of DAB 486 fused to the fragment defined in (d) above); a lower K, (i.e., a greater binding affinity) for the receptor (i.e., the sites to which the third fragment (described above) binds on the cells to be attacked) than that of an analagous DAB 486 ~containing-toxin; greater resistance to proteolytic degradation than that of DAB 486 ~containing-toxin; greater resistance to the inhibition of its cytotoxicity by competitive inhibitors, e.g., the polypeptide of (d) above, than that exhibited by an analagous DAB 48 _-containing-toxin; the ability to inhibit protein synthesis in target cells to a given degree by a period of exposure that is shorter than the period of exposure required by an analogous DAB 486 ~containing- toxin to inhibit protein synthesis to the same degree; or the ability to effect a more rapid onset of the inhibition of protein synthesis than that seen in an analagous DAB 48g -containing-toxin. Other preferred embodiments include: chimeric toxins wherein the fragment of Fragment B of diphtheria toxin does not include any diphtheria toxin sequences between the hydrophobic transmembrane region and amino acid residues 484 or 485 of native diphtheria toxin; chimeric toxins lacking diphtheria toxin sequences C-terminal to amino acid residue 386 of native diphtheria toxin; and chimeric toxins including DAB_, 8g fused to the third fragment defined above. Other preferred embodiments include: a chimeric toxin in which the portion of the polypeptide ligand is a portion of interleukin-2 effective to cause the chimeric toxin to bind to IL-2 receptor bearing cells, in particular, T cells; a chimeric toxin in which the portion of the polypeptide ligand is a portion of EGF effective to cause the chimeric toxin to bind to cells bearing the EGF receptor; the chimeric toxin DAB 38g -IL-2; and the chimeric toxin DAB 3gg -EGF. In other preferred embodiments in which the ligand is IL-2 or a portion thereof, the chimeric toxin possesses at least one of: greater toxicity to IL-2 receptor-bearing cells than that exhibited by DAB -IL-2, a lower K, for the IL-2 high affinity receptor than that of DAB 48g -IL-2, or a greater resistance to proteolytic degradation than that exhibited by • * DAB4.8 0 6_-IL-2. In other preferred embodiments in which the ligand is EGF or a portion thereof, the chimeric toxin posseses at least one of: greater toxicity to EGF-receptor-bearing cells than that exhibited by DAB 486 EGF; a lower K d for the EGF receptor than that of DAB 48g EGF, greater resistance to the inhibition of its cytotoxicity by competetive inhibitors, e.g., EGF, than that of DAB 48g -EGF; the ability to inhibit protein synthesis in EGF receptor bearing cells to a given degree by a period of exposure that is shorter than the period of exposure required by DAB 48 _EGF to inhibit protein synthesis to the same degree; or the ability to effect a more rapid onset of the inhibition of protein synthesis in EGF-receptor-bearing cells than that seen in DAB 4g6 EGF. The chimeric toxins of the invention are preferably encoded by fused genes which include regions encoding the protein fragments of the chimeric toxin, DNA sequences encoding the chimeric toxins of the invention, expression vectors encoding those DNA sequences, cells transformed with those expression vectors, and methods of producing the chimeric toxins including culturing cells transformed with expression vectors containing DNA encoding the chimeric toxins and isolating the chimeric toxins from the cells or their supernatants. Native diphtheria toxin, as used herein, means the 535 amino acid diphtheria toxin protein secreted by Corynebacteriuro diphtheriae. The sequence of an allele of the gene which encodes native diphtheria toxin can be found in Greenfield et al. (1983) Proc. Natl. Acad. Sci. USA 8_0:6853-6857, hereby incorporated by reference. Enzymatically active Fragment A, as used herein, means amino acid residues Gly 1 through Arg 193 of native DT, or an enzymatically active derivative or analog of the natural sequence. Cleavage domain 1^ , as used herein, means the protease sensitive domain within the region spanning Cys 186 and Cys 201 of native DT. Fragment B, as used herein, means the region from Ser 194 through Ser 535 of native DT. The hydrophobic transmembrane region of Fragment B, as used herein, means the amino acid sequence bearing a structural similarity to the bilayer-spanning helices of integral membrane proteins and located approximately at or derived from amino acid residue 346 through amino acid residue 371 of native diphtheria toxin. Domain 1 2 , as used herein, means the region spanning Cys 461 and Cys 471 of native DT. The generalized eukaryotic binding site of Fragment B, as used herein, means a region within the C-terminal 50 amino acid residues of native DT responsible for binding DT to its native receptor on the surface of eukaryotic cells. The chimeric toxins of the inventions do not include the generalized eukaryotic binding site of Fragment B. Toxic or cytotoxic, as used herein, means capable of inhibiting protein synthesis in a cell, inhibiting cell growth or division, or killing a cell. DAB 48 _ consists of, in the following order, methionine, and amino acid residues 1-485 of native DT. DAB_ 89 consists of, in the following order, methionine, amino acid residues 1- 386 of native DT, and amino acid residues 484 - 485 of native DT. is a fusion protein consisting of, in the following order, methionine, amino acid residues 1-485 of native DT, and amino acid residues 2-133 of IL-2. DAB 485 ~IL-2 is identical except that it lacks the initial methionine residue. DAB_ 8g -IL-2 consists of DAB 38g fused to amino acid residues 2-133 of IL-2. DAB_ 8g EGF consists of DAB 38g fused to EGF. Receptor means the site to which the cell-specific polypeptide ligand (described in (d) above) binds. Chimeric toxins of the invention display one or more of the following advantages: greater toxicity than that of an analagous DAB 48g -containing toxin; a greater affinity for the receptor than that of an analagous DAB 4g -containing toxin; when expressed in the cytoplasm of E.coli, greater resistance to proteolytic degradation than that exhibited by an analagous DAB 48 _-containing toxin; greater resistance to the inhibition of its cytotoxicity by competitive inhibitors, e.g., the polypeptide of (d) above, than that exhibited by an analagous DAB 48g -containing toxin; the ability to inhibit protein synthesis in target cells to a given degree by a period of exposure that is shorter than the period of exposure required by an analogous DAB 48g -containing- toxin to inhibit protein synthesis to the same degree; or the ability to effect a more rapid onset of the inhibition of protein synthesis than that seen in an analagous DAB 48g -containing-toxin. Aberrant expression of the epidermal growth factor receptor is a characteristic of several malignancies including those of the breast, bladder, prostate, lung and neuroglia. Chimeric toxins of the invention allow therapeutic targeting the cytotoxic action of diptheria toxin to EGF receptor positive tumor cells. In these chimeric toxins the sequences for the binding domain of diptheria toxin have been replaced by those for human EGF. These chimeric toxins inhibit protein synthesis by the same mechanism as diptheria toxin and are specifically cytotoxic for human tumor cells which express elevated levels of EGF receptors. The uptake of these chimeric toxins occur with kinetics which permit use of this molecule as a powerful therapeutic agent for treatment of malignancies characterized by EGF receptor expression. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims. Description of the Preferred Embodiments The drawings will first be briefly described. Drawings Fig. 1 is a diagram of the DT molecule and of various fusion proteins. Fig. 2 is a depiction of the construction of the plasmids of a preferred embodiment. Fig. 3 is a restriction map of DNA sequences encoding various chimeric toxins. Fig. 4 is a graph of the effects of varying doses of chimeric toxins on cultured cells. Fig. 5 is a graph of the ability of chimeric toxins to competitively displace [ 12513-labeled IL-2 from the high affinity IL-2 receptor. Fig. 6 is the sequence of a synthetic EGF gene. Fig. 7 is a diagramatic representation of DAB 486 EGF and DAB-, Qg EGF. Fig. 8 is a graph showing the effect of EGF on DAB 48 _EGF cytotoxicity. Fig. 9 is a graph showing the effect of EGF on DAB 3Qg EGF cytotoxicity. Fig. 10 is a graph showing the effect of EGF and DAB 38g EGF on the EGF binding capacity of A431 cells. Fig. 11 is a graph showing the ability of EGF or DAB 38g EGF to displace [ 125 I] EGF from EGF receptors. Fig. 12 is a graph of the effect of length of exposure to DAB. 8g EGF on the inhibition of protein synthesis. Fig. 13 is a graph of the effect of length of exposure to DAB 3gg EGF on the inhibition of protein synthesis. Fig. 14 is a graph of the kinetics of the inhibition of protein synthesis on cells incubated with DAB 48g EGF or DAB 3gg EGF. Structure and Synthesis of chimeric toxin DAB 4 -.--IL-2 DAB. Q --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 DAB4.8-6.-IL-2 extends past the disulfide bridge linking Cys 461 with Cys 471. See Fig. la for the structure of DT. (The nomenclature adopted for IL-2-toxin is DAB 48g -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 48g -IL-2 was constructed as follows. pUClδ (New England BioLabs) was digested with PstI and Bgll and the Pstl-Bgll fragment carrying the E.coli origin of replication, the polylinker region, and the 3' portion of the β-lacatamase gene (amp r ) was recovered. Plasmid pKK-233-2 (Pharmacia) was digested with PstI and Bgll and the Pstl-Bgll fragment carrying, two transcription terminators and the 5' 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-Sall fragment encoding human IL-2 from plasmid pDW15 (Williams et al . (1988) Nucleic Acids Res. 16:10453- 10467) and ligating it to BamHI/Sall digested pDW22 (described above) . pDW24 was constructed as follows. A BamHI-NcoI fragment carrying the trc promoter and trans1ational initiation codon (ATG) was isolated from plasmid pKK233-2 (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 Haell and recovering the Haell-SphI fragment containing the sequence encoding amino acid residues 1 through 485 of DT. A Ncol/Haell linker (5'CCATGGGCGC 3') was ligated to the Haell-SphI fragment and that contruction was then ligated to the previously isolated BamHI-NcoI fragment carrying the trc promoter. This results in a Bam Hl-SphI fragment bearing, in the following order, the trc promoter, the Ncol 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 48g -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. JL :10453-10467, hereby incorporated by reference. The sequence of IL-2 is found in Williams et al. (1988) Nucleic Acids Res. 35:10453-10467. 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 Fig. 2. The insert corresponding to DAB 48g -IL-2 is shown as a heavy line. In Fig. 2 filled circles indicate Ncol sites, open circles indicate Nsil sites, open diamonds indicate Clal sites, filled squares indicate Hpall sites, open squares indicate SphI sites, and filled triangles indicate Sail 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, CA) . Following synthesis, oligonucleotides were purified by chro atography on Oligonucleotide Purification Cartridges (Applied Biosystems Inc., Foster City, CA) 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 & Sons, N.Y. DNA was digested with restriction endonucleases as recommended by the manufacturer (New England Biolabs, Beverly, MA 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 ___ 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'1 Acad. Sci USA l :5463-5467, as modified by Kraft et al. (1988) Bio Techniques 6 :544-547, using Sequenase (United States Biochemicals, Cleveland, OH). Structure of Improved Diphtheria-IL-2 Chimeric Toxins 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, MI), 50 μg/ml ampicillin, and 0.5 ng/ l 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 5 L/min. When the absorbance (A-. go 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-,P0 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 x 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 x 600 mm G4000PW column (TosoHass). Prior to use, fusion toxins were exhaustively dialysed against HEPES buffered Hank's balanced salt solution (Gibco), pH 7.4. Purified diphtheria toxin was purchased from List Biological Laboratories (Campbell, CA) . For the production of the non-toxic CRM1001, C7(βtox-1001) was grown in 100 ml volumes of C-Y meduim (Rappuoli et al. (1983) J. Bact. 153:1201-1210) in 2-liter Erlen eyer flasks at 35°C for 20 hrs with shaking (240 rpm) . Bacteria were removed by centrifugation at 20,000 x g for 15 min. CRM1001 was precipitated from the culture medium by the addition of NH 4 S0 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, IL) . DAB ' 001) 48g -IL-2 is a chimeric toxin identical to DAB 4gg -IL-2 except for the disruption of the disulfide bridge between Cys462 and Cys472 in DAB(1001) 486 -IL-2. DAB(1001) 48g -IL-2 was constructed by replacing the 587 basepair (bp) Clal-Sphl restriction fragment which encodes most of fragment B of DT) of plasmid pDW24 (which carries DAB 48g -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. Fig. 3 depicts the restriction maps of DNA encoding DAB 4gg -IL-2 and the corresponding fusion protein encoded by DAB 4g -IL-2. (In Fig. 3 stippled boxes between the Nsil and Hpall restriction endonuclease sites designate the diphtheria toxin fragment B-related sequences which encode the membrane associating domains. The amphipathic domain is encoded between the Nsil and Clal ^ sites, and the putative membrane spanning domains are encoded between the Clal and Hpall 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 Fig. 2. Following ligation and transformation, the DNA sequence of the tox-1001 portion of the gene fusion DAB (1001) 4gg -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) 4gg -IL-2, was purified by immunoaffinity chromatography and HPLC. The dose response capacity of DAB 4gg -IL-2, CRM1001, and DAB(1001). o _-IL-2 to block C 14C]-leucine incorporation by high affinity IL-2 receptor bearing HUT 102/6TG cells was determined. As anticipated, DAB. oc -IL-2 was highly toxic for these cells (IC 50 = 4 x 10 -10M); 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) 4gg -IL-2, was found to be as toxic for HUT 102/6TG cells as the wild type DAB4.8 0/ 6.-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 gluta ine, and penicillin and streptomycin to 50 IU and 50 μg/ l, respectively. For cytotoxicity assays, cells were seeded in 96-well V-bottomed plates (Linbro-Flow Laboratories, McLean, VA) at a 4 concentration of 5 x 10 per well m complete medium. Toxins, or toxin-related materials, were added to varying concentrations (10 —12M to 10—6M) and the cultures were incubated for 18 hrs at 37°C in a 5% C0 2 atmosphere. Following incubation, the plates were centrifuged for 5 min. at 170 x g and the medium removed and replaced with 200 μl leucine-free medium (MEM, Gibco) containing 1.0 μCi/ml [ 14C]-leucine (New England Nuclear, Boston, MA). After an additional 90 min. at 37°C, the plates were centrifuged for 5 min. at 170 x g, the medium was removed and the cells were lysed by the addition of 4 M 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 48g -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 48g -IL-2 toxin gene, Figs, lb, 2, and 3. Fig. lb shows the structure of DAB 48g -IL-2 and various mutants derived from DAB 4gg -IL-2. In Fig. lb 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, Asn = asparagine, Asp = aspartic acid, Cys = cysteine, Gly = glycine, His = histidine, lie = isoleucine, Met = methionine, Thr = threonine, Tyr = tyrosine, and Val = valine. The first mutant, DAB_ gg -IL-2 was constructed by removing a 309 bp Hpall - SphI restriction fragment from pDW24 and replacing it with oligonucleotide linker 261/274 (Table 1) to generate plasmid pDW27 (Fig. 1). 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 3gg -IL-2 the 97 amino acids between Thr387 and His485 have been deleted. Table 1. Oligonucleotide linkers construct oligonucleotide linker identification number DAB389-IL-2 274 5'-CG GGT CAC AAA ACG CAT G-3 ' 261 CCA GTG TTT TGC 1/2 Hpall 1/2 SphI DAB295-IL-2 292 5'-C GAT GGT GTG CAT G-3' 293 TA CCA CAC 1/2 Clal 1/2 SphI 10 DAB(Δ205- 337 5'-TA AAT AT-3' 289) 486 -IL-2 338 ACG TAT TTA TAG C 1/2 Nsil 1/2 Clal DAB(Δ205- 337 289) 389 -IL2 338 15 In a similar fashion, a 191 amino acid in-frame deletion was constructed by removing a Clal - SphI restriction fragment from pDW24 and replacing it with the 292/293 oligonucleotide linker (Table 1) to form 20 plasmid pDW28 which encodes DAB 2g _.-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 25 eukaryotic cell cytosol. Purified, DAB 3gg -IL-2 and DAB_ g5 -IL-2 were found to have electrophoretic mobilities of 57 kDa and 47 kDa, respectively. The dose response analysis on HUT 3_0_ 102/6TG cells is shown in Fig. 4. In Fig. 4 DAB4.8 Q 6_- IL-2 is indicated by filled squares; DAB 3gg -IL-2 is indicated by filled circles; DAB 2g -.-IL2 is indicated by open circles; DAB(Δ205-289) 4gg -IL-2 (see below) is indicated by open squares; and DAB(Δ205-289) 3gg - IL-2 (see below) is indicated by open triangles. DAB4.8 0 6-IL-2 and DAB3-.8-9---IL-2 exhibited-an IC5-0- of approximately 4 x 10 M and 1 x 10 M, respectively. In marked contrast, the IC 8 - of DAB 2g _-IL-2 was approximately 1,000-fold lower ( 4 x 10 ~ M) . These results suggest that f agment 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 3gg -IL-2 possessed much greater activity than did DAB 4gg -IL-2. DAB-, gg -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. 8_7:837-840, hereby incorporated by reference, for a characterization of the transmembrane region. DAB 2g5 ~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 2g5 -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 [ 125I]-rIL-2. Fig. 5 shows the competitive displacement of [ 125I.-labeled IL-2 from the high affinity IL-2 receptor by unlabeled rIL-2 depicted by filled circles; DAB 4gg -IL-2 depicted by open triangles; DAB-. gg -IL-2 depicted by closed squares; DAB 2g _.-IL-2 depicted by closed triangles; DAB(Δ205-289) 4gg -IL-2 (see below) depicted by open circles; and DAB(Δ205-289) 38g -IL-2 (see below) depicted by open squares. The concentration of [ 125I]-IL-2 used was 10 pM and the specific activity was approximately 0.7 μCi/pmol. As shown in Table 2, both DAB 38g -IL-2 and DAB 2g _.-IL-2 were found to have an apparent K, that is approximately 3-times lower than that _of o DAB4.8 Q 6,-IL-2 (Kd, = 8 x 10 _9 M vs. Kα, = 2.5 x 10 M) . It is particularly significant that competitive displacement experiments showed that both DAB 38g -IL-2 and DAB 2g _.-IL-2 bind more avidly to the high affinity IL-2 receptor than does DAB 4gg -IL-2(Kd = 8 x 10 ~9 and 8.4 x 10 ~9 M vs. Kd = 2.5 x 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 2g _.-IL-2 binds more avidly to the high affinity IL-2 receptor than DAB 4gg -IL-2, its cytotoxic activity is at least 1,000-fold lower (Fig. 4). 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 fusion proteins to displace [ 125 I]—rIL-2 from high affinity IL-2 receptors on HUT 102/6TG cells unlabeled ligand apparent K<_ K^ DAB-IL-2/r IL-2 rIL-2 1.7 x lO -10 — DAB4 8 g-IL-2 2.5 x 10 ~8 147 DAB3 8 9-IL-2 8.0 x 10 ~9 47 DAB295-IL-2 8.4 x 10 -9 49 DAB(Δ205-289) 486 " -IL- -2 1.0 x 10 -7 588 DAB(Δ205-289) 389 " -IL- -2 2.9 x 10 -8 170 Competitive displacement of [ 125I]-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 x 10 per ml and incubated with [125I]-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 rain, at 37°C under 5% C0 2> The reaction was then overlayed on a mixture of 80% 550 fluid (Accumetric Inc., Elizabethtown, KN) : 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,, 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 4gg -IL-2) plays a role in the intoxication process, in-frame deletions of the 85 amino acid encoding region from Nsil to Clal of both pDW24 and pDW27 to form pDW30 (containing DAB(Δ205-289) 4gg -IL-2) and pDW31 (containing DAB(Δ205-289) 3gg -IL-2), respectively (Figs. 2 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 Figure 4, 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) 3gg - IL-2 was found to displace radiolabeled IL-2 from the high affinity receptor almost as well as DAB 4gg -IL-2; whereas, DAB(Δ205-289) 4gg -IL-2 was found to bind 4-fold less avidly to the receptor (Fig. 5). Increased Resistance to Proteolytic Degradation The chimeric toxin encoded by DAB 3gg -IL-2 is more resistant to proteolytic degradation than is the chimeric toxin encoded by DAB 4gg -IL-2. When purified, as described above, and analysed on SDS-polyacrylamide gels, the DAB 3gg -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. gg -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 4gg -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. Construction of Fusion Genes Encoding DT-EGF Chimeric Toxins DAB 4gg EGF and DAB 3gg EGF can be constructed in a manner analogous to that in which DAB 4g -IL-2 and DAB 3gg -IL-2 were constructed, by methods known to those skilled in the art. To construct a plasmid encoding DAB 48g fused to EGF, plasmid pDW24 (which encodes DAB 48g fused to IL-2) is digested with SphI and HindiII to remove the IL-2 coding sequence. The resulting pDW24 Sphl-Hindlll fragment containing the sequence encoding DT residues 1-485 is ligated to a synthetic Sphl-Hindlll fragment encoding EGF to yield a plasmid encoding DAB 48g fused to EGF. The EGF fragment, shown in Fig. 6, 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' and 3' ends for insertion into the plasmid and for in-frame fusion to the DT coding sequence. To construct a plasmid encoding DAB 3gg fused to EGF a similar protocol can be followed, except that pDW27 (which encodes DAB 3gg 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 38g fused in frame to EGF. The same synthetic EGF fragment used in the construction of the DAB 4gg EGF fusion (Fig. 6) 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 lacl^ gene (Amersham) and the replacement of the ampicillin resistance gene (amp ) 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. The Biological Activity of DT-EGF Chimeric Toxins DAB. gg EGF and DAB 3gg 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 Fig. 7, 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 38g EGF differs from DAB 4gg EGF by the deletion of the 97 amino acids immediately 5' to amino acid residue 484 of DT. The EGF portion of both DAB 4gg EGF and DAB 3gg 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. DT-EGF Chimeric Toxins Are Toxic to EGF-Receptor-Bearing Cells. The cytotoxicity of DAB 4.8-6..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 4gg EGF in assay medium appropriate to the cell type (see Table 3). DAB._ fi EGF concentrations were between 10 -15 and 10-7 M. Following a 20-hour incubation, cells were labeled with C 14Clleucine, trypsinized, harvested onto glass fiber filter mats and counted to determine the percent of control incorporation. Cell lines exhibiting an IC 5Q for DAB 4.θ_O-EGF of less than 0.5 nM were considered sensitive. Table 3: The effect of a DT-EGF chimeric toxin on various cell lines Tumor Cell lines Cell Line Tissue/Type Sensitivity A431 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 Lines Cell Line Tissue Sensitivity WI-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's F12 + 10% FCS; KB (ATCC CCL17), DMEM + NEAA + 10% FCS; BT-20 (ATCC HTB19), MEM + 10% FCS; HeLa S3 (ATCC CCL2.2), Ham'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, MA, see Bacha et al. (1988) J. Exp. Med. i67_:612 for growth conditions), RPMI 1640 + 15% FCS; BeWo (ATTC CCL98), Ham's F12 + 15% FCS; A375 (ATCC CRL 1619), DMEM + 10% FCS; MCF-7 (ATCC TB22) MEM + 10% FCS; SNU-C2B (ATCC CCL250) RPMI 1640 + 10% FCS; WI-38 (ATCC CCL75), Eagle'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. DMEM = Dulbecco's modified Eagles Medium; MEM = Minimum Essential Medium; NEAA = Non-Essential Amino Acids; FCS = Fetal Calf Serum; ATCC = American Type Culture Collection. To demonstrate that the cytotoxic action of DAB 4gg EGF and DAB 3gg EGF are mediated selectively by the EGF receptor, A431 cells were plated in triplicate wells of 96 well plates with DAB 4gg EGF (Fig. 8) or DAB 3gg EGF (Fig. 9) in the presence of the specific competitor of the EGF receptor, human EGF (Upstate Biotechnologies, Inc.) (10 M) , in assay medium (DMEM + 10% FCS). In Fig. 8 solid squares indicate DAB 4gg EGF and solid triang 3 les indicate + EGF. In Fig. 9 solid squares indicate DAB 3gg EGF and solid triangles indicate DABo_8 QQ "EGF + EGF. Following a 20-hour incubation at 37°C, cells were labeled with [ 14C.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, 0 _EGF and DAB---EGF inhibit 486 Jo y —12 protein synthesis with an IC ςn of 3 x 10 M and 3 x 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 48g EGF and DAB 38g EGF while the nonspecific competitors, transferrin (Sigma) anti-transferrin (Dako), and anti-transferrin receptor (Dako), have no effect. These results demonstrate that DAB 4.8 Q 6-EGF and DAB_ 8g EGF are potent and specific cytotoxic agents. Note that DAB 3gg EGF is approximately 10 times more potent than DAB 4gg EGF. DAB 3gg 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 [ 125I]EGF binding capacity (Krupp et al. (1982) J. Biol. Chem. 257:11489). The ability of DAB 3gg 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 Fig. 10. In Fig. 10 open squares indicate EGF and closed diamonds indicate DAB 3gg EGF. A431 cells in triplicate wells of 24 well plates were preincubated with EGF or DAB 3gg 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 0.2 M acetic acid, 0.5 M NaCl) to remove bound, but not internalized, EGF or DAB 3gg EGF. EGF binding capacity was measured by incubating the cells, on ice, with [ 125I]EGF. Following a 90-mmute incubation the cells were washed, solubilized, and counte . An EGF receptor-dependent interaction is also shown by the fact that DAB OQ EGF, like EGF, displaces [ 125I]EGF from the EGF rec J ep ϋ tor, as shown m Fig. 11. In Fig. 11 open squares indicate EGF and solid diamonds indicate DAB 3gg EGF. Results in Fig. 11 are expressed as a percent of control (no competition) cpm. The ability of DAB_ Rq EGF to displace high affinity [ 125I]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 [ 125I]EGF in binding media in the presence of DAB 3gg EGF or EGF. Following incubation, the cells were washed, solubilized and counted. The results are summarized in Table 4. In Table 4 EC, n is the concentration 125 resulting in displacement of 50% of the [ I] EGF. Table 4: Displacement of [ l ' ^ι ] EGF Binding by EGF and DAB 389 EGF fold over fold over Competition EC 50 [ 125 I]EGF EGF EGF 1.0 x 10 "8 M 20 DAB 389 EGF 4.5 x 10 "7 M 900 45 Cytotoxicity of DT-EGF Chimeric Toxins is DT Dependent. 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 4gg EGF and DAB 38g EGF is also dependent upon the same pathway, A431 cells were plated in sextuplicate wells of 96 well plates containing DAB 4gg EGF, DAB 3gg EGF or DMEM + 10% FCS in the absence or presence of chloroquine (10 ~ M) (Sigma). Chloroquine is a lysosomotropic compound which prevents acidification of endosomes (Kim et al. (1965) J. Bacteriol. 9_0:1552). Following a 20-hour incubation at 37°C, the cells were labeled with [ 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 4gg EGF or DAB 3gg 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 Chloroquine Percent of Control Incorporation DAB 486 EGF Concentration No Addition Chloroquine 0 100 86 10-8 M ' 5 60 10 ~9 M 25 96 DAB339EGF Concentration 0 100 73 10 -11 M 4 61 10- 2 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. 2_58:1565). To evaluate the mechanism by which DAB 4βg EGF inhibits protein synthesis, A431 cells were plated in triplicate wells of 24 well plates containing DT, DAB 4gg EGF, or complete medium. Following a 20-hour incubation at 37°C, the cells were washed and incubated in lysis buffer (lOmM Tris, lOmM NaCl, 3mM Mg Cl 2 , lOmM thymidine, ImM 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 counted to quantitate the percent of control EF-2 available for ADP-ribosylation. The results of these experiments are shown in Table 6. DAB 4gg 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 486 EGF Percent of Control Level of EF-2 Avai lable for Toxin Concentration ADP-r ibosylat ion DT 10-8 M < 1 10 ~9 M 17 DAB 486 EGF 10 ~8 M 13 10 ~9 M 20 DAB..-EGF Is An Improved Chimeric Toxin. DAB_0 Qn 5EGF is far more toxic than is shown in Figs. 8 and 9, DAB 3gg EGF exhibits an IC-. Q for the inhibition of protein synthesis in A431 cells approximately 10 times lower than that of DAB 4gg EGF (DAB 3gg EGF IC 50 = 3 x lθ "13 M; DAB 4gg EGF IC 5Q = 3 x 10~ 12 M). The greater potency of DAB 3gg EGF is also shown in experiments measuring the rapidity with which DAB 3gg EGF and DAB 4gg EGF kill A431 cells. Figs. 12 and 12 show the exposure time (of A431 cells to DAB 48g EGF or DAB 3gg EGF) required to induce maximal inhibition of protein synthesis. Cells were exposed to DAB.g g EGF (5 X lθ "9 M) (Fig. 12) or DAB 3gg EGF (5 x 10 ~ M) (Fig. 13) for the indicated times and then washed of unbound DAB 4gg EGF or DAB 3gg EGF. Following an overnight incubation in complete media (DMEM + 10% FCS), the cells were labeled with [ C.leucine. The results show that near maximal inhibition of protein synthesis occurs following a 15-minute exposure to DABj,o OQ yEGF while a greater than 75-minute exposure is required for DAB EGF. The kinetics of protein synthesis inhibition in DAB-. OQ EGF or DAB._-EGF treated A431 cells is shown in Fig. 14. To examine the kinetics of protein synthesis inhibition A431 cells were incubated with DAB 4gg EGF (5xl0 ~9 ) or DAB 3gg EGF (5 x 10 ~9 M) in complete medium at 37°C. At the indicated times, DAB 4.„8b-EGF or DAB_,fc Q i-.E.G.F was removed and the cells were labeled with [ C.leucine for 1 hour. The results indicate that there is a 50% reduction in protein synthesis following a 1-hour incubation with DAB_ o u c .EGF while maximal inhibition occurs by 4 hours. Maximal inhibition of protein synthesis occurs more than 6 hours following incubation with DAB 4gg EGF. Use 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 admnistered 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 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 3gg and DAB 4gg have been fused to interleukin 4 (IL-4). DAB 38g -IL-4 is about 10 times more cytotoxic than is DAB 4gg -IL-4. DAB 3gg has also been fused to interleukin 6. DAB 4gg and DAB 3gg 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 polyepeptide synthesis enable those skilled in this technolgoy 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 moeity expressed on the surface of the target cell membrane. What is claimed is:

Claims: 1. A chimeric toxin comprising protein fragments joined together by peptide bonds, said chimeric toxin comprising, in sequential order, beginning at the amino terminal end of said chimeric toxin, (a) the enzymatically active Fragment A of diphtheria toxin, (b) a first fragment including the cleavage domain 1 adjacent said Fragment A of diphtheria toxin, (c) a second fragment comprising at least- a portion of the hydrophobic transmembrane region of Fragment B of diphtheria toxin, said second fragment having a deletion of at least 50 diphtheria toxin amino acid residues, said deletion being C-terminal to said portion of the transmembrane region, and said second fragment not including domain 1 2 , and (d) a third fragment comprising a portion of a cell-specific polypeptide ligand, said portion including at least a portion of the binding domain of said polypeptide ligand, said portion of said binding domain being effective to cause said chimeric toxin to bind selectively to a predetermined class of cells to be attacked by said enzymatically active Fragment A, said chimeric toxin possessing any of, greater toxicity than that of a toxin comprised of DAB 4gg fused to said third fragment, a lower K, for the sites on cells of said predetermined class to which said chimeric toxin binds than that of a toxin comprised of DAB 4gg fused to said third fragment, greater resistance to proteolytic degradation than that exhibited by a toxin comprised of DAB 4gg fused to said third fragment, greater resistance to the inhibition of its cytotoxicity by said cell-specific polypeptide ligand than that exhibited by DAB 48g fused to said third fragment, the ability to inhibit protein synthesis to a given degree by a period of exposure that is shorter than the period of exposure required by DAB 4gg fused to said third fragment to inhibit protein synthesis to the same degree, or the ability to effect a more rapid onset of the inhibition of protein synthesis than that exhibited by DAB 486 fused to said third fragment. 2. The chimeric toxin of claim 1, wherein said deletion is at least 80 diphtheria toxin amino acid residues in length. 3. The chimeric toxin of claim 1, wherein said Fragment B of diphtheria toxin does not include any diphtheria toxin sequences C-terminal to amino acid residue 386 of native diphtheria toxin. 4. The chimeric toxins of claim 1, wherein (a), (b) , and (c) comprise DAB 3gg . 5. The chimeric toxin of claim 1, wherein said portion of said polypeptide ligand is a portion of epidermal growth factor effective to cause said chimeric toxin to bind to cells bearing the epidermal growth factor receptor. 6. The chimeric toxin of claim 5, wherein (a), (b), and (c) comprise DAB 3gg and (d) comprises EGF. 7. The chimeric toxin of claim 5, wherein said chimeric toxin possessing any of, greater toxicity than that of a toxin comprised of DAB 4gg fused to EGF, a lower K, for the sites on cells of said predetermined class to which said chimeric toxin binds than that of a toxin comprised of DAB 4g fused to EGF, greater resistance to the inhibition of its cytotoxicity by EGF than that exhibited by DAB. Q - fused to EGF, the ability to inhibit protein synthesis to a given degree by a period of exposure that is shorter than the period of exposure required by DAB 4gg fused to EGF to inhibit protein synthesis to the same degree, or the ability to effect a more rapid onset of the inhibition of protein synthesis than that exhibited by DAB 4gg fused to EGF. 8. The chimeric toxin of claim 1, wherein said portion of said polypeptide ligand is a portion of interleukin 2 effective to cause said chimeric toxin to find to T cells. 9. The chimeric toxin of claim 8, wherein (a), (b), and (c) comprise DAB 3gg and (d) comprises amino acid residues 2-133 of IL-2. 10. The chimeric toxin of claim 8, wherein said chimeric toxin possessing any of, greater toxicity than that of a toxin comprised of DAB 4gg fused amino acid residues 2-133 of IL-2, a lower K, for the sites on cells of said predetermined class to which said chimeric toxin binds than that of a toxin comprised of DAB 4gg fused to amino acid residues 2-133 of IL-2, or greater resistance to proteolytic degradation than that exhibited by a toxin comprised of DAB 4gg fused to amino acid residues 2-133 of IL-2. 11. The chimeric toxin of claim 1, wherein said chimeric toxin is encoded by a fused gene comprising regions coding for said protein fragments. 12. A DNA sequence encoding the chimeric toxin of claim 1 . 13. An expression vector containing the DNA sequence of claim 12. 14. A cell transformed with the vector of claim 13. 15. A method of producing the chimeric toxin of claim 1 comprising culturing the cell of claim 14, and isolating said chimeric toxin from said cultured cell or supernatant.

Inventors: WILLIAMS DIANE, WILLIAMS, DIANE
Applicants: WILLIAMS DIANE, WILLIAMS, DIANE
Application year: 1991
Main classification: C07K13
IPC codes: A61K9, A61K38, A61P35, C07H21, C07K14, C07K19, C12N1, C12N15, C12P21, C12R1