AU636453B2 - An improved toxin for construction of immunotoxins - Google Patents

Description

WO 91/09965 PC/US90/07464 1 AN IMPROVED TOXIN FOR CONSTRUCTION OF IMMUNOTOXINS The parent application (06/911,227) teaches the production of recombinant proteins from modified Pseudomonas exotoxin (PE) gene fused with DNA sequences encoding a recognition protein for which a specific receptor exists on the cells. This was exemplified in the parent application with the construction and expression of an IL-2-PE fusion gene. The present application relates to the production of improved toxins which are useful in constructing more effective immunotoxins. Particularly, the invention provides an altered PE molecule, designated herein as Lys-PE40, that can be easily conjugated to an antibody with effective cytotoxicity toward target cel33.

BRIEF DESCRIPTION OF THE DRAWINGS Various objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: Figure 1 schematically illustrates the structure of plasmid pVC85L showing the presence of a T7 promoter, and OmpA signal sequence and domains II (translocation domain) and III (ADP ribosylation domain) of Pseudomonas exotoxin.

Figure 2A shows the results of SDS-PAGE analysis of LysPE40 pools at various stages of purification. 12.5% gels were stained with Coomassie blue. Lane 1, QMA concentrated culture medium proteins; Lane 2, Q Sepharose pool; Lane 3, Flow through material from Mono S; Lane 4, Mono S pool; Lane 5, TSK 250 pool. The positions of molecular weight standards are indicated. B: SDS-PAGE analysis of anti-TFR-LysPE40. Samples were applied on a 10% reducing polyacrylamide gel. Lane 1, Lane 2, LysPE40; Lane 3, anti-TFR. Gels were stained with Coomassie blue.

Figure 3A demonstrates the inhibition of protein synthesis in A431 cells by anti-TFR-LysPE40. Immunotoxin was added to the cells in the absence and presence (o) WO 91/09965 PCT/US90/07464 2 of excess anti-TFR (tp pg/ml) for 18-24 hrs at 37 0 C. B: Cytotoxic activity of anti-Tac-LysPE40 on HUT102 cells.

was added at various concentrations to the indicated cell lines. For competition experiment pg/ml anti-Tac was added before the addition of the immunotoxin. 3H leucine incorporation was measured as described in the text.

Figure 4 shows the blood levels of anti-TFR- BALB/C mice or nude mice with A431 tumors were injected I.P. with 100 jg or 50 pg of Immunotoxin levels were measured in serum at different time periods. Results are average of two different experiments.

Figure 5 shows the effect of anti-TFR-LysPE40 on the growth of A431 tumors in nude mice. Mice were injected with 3 x 106 A431 cells and treated with the immunotoxin as indicated. Mice were given four doses of pg each on the indicated days: days 2, 4, 6, 8; days 9, 11, 13 and 15; no treatment. B. Mice given four doses on days 5, 7, 9 and 11; no treatment; 5pg; 20 pg; 50 pg; single dose of 150 pg on day Figure 6 shows the gross appearance of subcutaneous A431 tumors in nude mice with and without treatment with HB21-PE40.

Nude mice were injected subcutaneously with 3 x 106 A431 cells on day 0. Without further treatment, the tumors were apparent as small bumps under the skin on day arrows). By day 15, tumors were large, often erupting through the skin surface Mice treated with HB21-PE40 on days 2, 4, 6 and 8 usually showed no development of tumor, demonstrated here as the absence of gross tumor on day 26 When mice with large tumors were treated with HB21-PE40 (on days 9, 11, 13 and 15), the tumors often began to shrink, uullapsing inward on their necrotic centers arrows).

Figure 7 shows the histological appearance of typical treated and control A431 subcutaneous tumors.

WO 91/09965 PCT/US90/07464 3 A431 tumors were removed at necropsy, fixed on formaldehyde and processed for routine paraffin embedding and sectioning, and staining with hematoxylin and eosin.

A section from a tumor removed from a mouse at day 15 is shown in A-4. A similar section of a tumor removed from a mouse on day 19 that had been treated on days 9, 11, 13 and 15 with anti-TFR-LysPE40 is shown in B-B. The crosssection shown in A shows that the majority of the untreated tumor contains viable tumor cells (VT) with only small areas of necrosis (arrow). In contrast, the tumor from a treated mouse shows that a large percentage of the tumor is necrotic with only a small rim of viable tumor (VT) remaining (s=skin). and are higher magnification fields from the margins of tumors demonstrating that the untreated tumor is mostly composed of homogeneous viable tumors whereas the treated tumor shows a rim of viable cells of variable thickness lying djacent to a connective tissue capsule The majority of the treated tumor shows large areas of central necrosis, and in some areas of the tumor margin all tumor cells are necrotic, whereas in other areas a viable tumor rim only a few cells thick remains double arrow).

(Mags: A,B 5, bar 1 mm; B' x26, bar 200 4lm; B" x260; bar nm).

DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by an improved Pseudomonas exotoxin (PE) of the type including a deletion in the receptor binding domain Ia of the native toxin, wherein the improvement comprises a recombinant PE molecule possessing at least one lysine residue in domain Ia of the PE molecule, which otherwise will be devoid of a lysine residue when having a deletion in the receptor binding domain Ia, said lysine residue providing an essential linkage for effective coupling of the recombinant PE to other molecules, such as antibodies and the like.

WO 91/09965 PCY/US90/07464 4 When PE is chemically attached to antibodies or other targeting molecules, lysine residues are required for coupling PE to other molecules. Since all 12 lysine residues of domain I are lost when domain I is deleted from PE, one of the three lysine residues in the other part of the molecule (for instance, domain III), must be used to couple PE40 to an antibody or another targeting molecule. However, when one of these three lysine residues are used, a conjugate with low activity is obtained (Kondo et al, J. Biol. Chem. 263:9470, 1988). In order to overcome this problem an' to obtain a conjugate with high activity, a new PE molecule was created by deleting most of domain I (residues 6-252) but maintaining one of the 12 lysine residues originally present in domain I. This new molecule, in accordance with the present invention, is designated herein as In order to demonstrate the effect of the new molecule on immunotoxin constructs, two novel immunotoxins were made, first with LysPE40 conjugated to antiTac antibody and second conjugated to an antibody specific for the human transferrin receptor (anti-TFR).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials dre now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are only illustrative and not limiting.

The term "antibody" as used herein means a portion of an immunoglobulin molecule (see W.E. Paul, ed., "Fundamental Immunology," Raven Press, 1984, pp. 131-165) capable of binding to an antigen. According to this WO 91/09965 PCT/US90/07464 5 definition, the term "antibody" includes various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fv fragment containing only the light and heavy chain variable regions, an Fab or (Fab)' 2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody (Bird et al., 1988, Science 242, 424-426; Huston et al., 1988, Proc. Natl.

Acad. Sci. USA 85, 5879-5883), and the like. The antibody may be of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al., 1984, Proc. Nat.

Acad. Sci. USA 81, 6851-6855) or humanized (Jones et al., 1986, Nature 321, 522-525, and published UK patent application #8707252). Methods of producing antibodies suitable for use in the present invention are well known to those skilled in the art and can be found described in such publications as Harlow Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. The genes encoding the antibody chains may be cloned in cDNA or in genomic form by any cloning procedure known to those skilled in the art. See for example Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982.

The term "without significant cytotoxicity" as used herein means that the fusion protein of the present invention does not affect the normal functions of the untargeted cells to any appreciable degree or to any abnormal level.

The recombinant antibody-LysPE40 fusion protein may contain one polypeptide chain or two chains. The example disclosed herein relates to the one chain case.

To produce two chains, no amino acid linker would be inserted between the V, and VL sequences. Instead a termination codon would be inserted after the VH sequence, and an initiation codon and ribosome binding sequence would be inserted before the VL sequence. In another embodiment of the invention, the VL and VH sequences will be followed respectively by part or all of the light and heavy chain constant regions, the whole kappa light WO 91/09965 PCT/US90/07464 6 chain constant region and the CHl domain of the heavy chain constant region, with or without the heavy chain hinge domain. The VL, V, and PE40 genes may occur in any order on the plasmid, hence the PE40 gene may be attached to either the 5' or 3' end of either the light or heavy chain gene. Those skilled in the art will realize that additional modifications, deletions, insertions and the like may be made to the antibody and LysPE40 genes.

Especially, deletions or changes may be made in LysPE40 or in the linker connecting the antibody gene to LysPE40, in order to increase cytotoxicity of the fusion protein toward target cells or to decrease cytotoxicity toward cells without antigen for the antibody. All such constructions may be made by methods of genetic engineering well known to those skilled in the art (see, generally, Maniatis et al., supra) and may produce proteins that have differing properties of affinity, specificity, stability and toxicity that make them particularly suitable for various clinical or biological applications.

MATERIALS AND METHODS The following example is offered by way of illustration, not limitation.

Construction of a vector that encodes LysPE40 and secretes it into the medium.

Plasmid pVC4 (derived from pJH4 by treating pJH4 with SphI and Tthllll and relegating the large fragment containing the PE gene) was joined to an OmpA signal sequence as described by Chaudhary et al., PNAS 85, 2929- 2943, 1988, to produce pVC45. Site directed mutagenesis is a convenient way to make deletions and it was used to create plasmid pJY85L which produces LysPE40 as shown in Figure 1. A HindIII/SalI fragment of the PE gene in was cloned into M13, mpl9 and uracil containing single stranded DNA prepared by the method of Kunkel (kunkel, PNAS 82, 488-492, 1985). An oligonucleotide 36 nucleotides in length with the structure CCTTGAAAGCTTGGCGTAATCATG3' was synthesized which generated a large deletion in PE of amino acids 6 through 252 and WO 91/09965 PCT/US90/07464 7 retained a lysine residue between amino acids 5 and 253 to give a molecule with the sequence: 1 2 3 4 5 x 253 254 255 256 Ala-Glu-Glu-Ala-Phe-Lys-Gly-Gly-Ser-Leu A 176 bp HindIII/SalI fragment was excised from the replicative form of the mutant DNA in M13 and ligated with a 3.3 kb HindIII/SalI fragment of pVC45 to give which encodes a protein with the following structure: Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly- Phe Ala-Thr-Val-Ala-Gln-Ala-Ala-Asn-.Leu-Glu-Glu-Ala-Phe-Lys Gly-Gly-Ser This protein is processed at the and the processed product secreted into the medium. When the plasmid was expressed in E. coli BL21 (ADE3) cells, LysPE40 was found in and purified from the medium with a yield of greater than 1 mg per liter. The sequence of amino acids at its amino terminus was found to be that predicted from the DNA sequence: 1 2 3 4 5 253 254 255

H

2 N Ala-Asn-Leu-Ala-Glu-Glu-Ala-Phe-Lys-Gly-Gly-Ser (the numbers indicate the location of the amino acids in the native PE structure).

A deposit of plasmid pJY85L has been made at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, on December 18, 1989 under the accession number 68189. The deposit shall be viably maintained, replacing it if it becomes nonviable, for the life of the patent, for a period of years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public upon issuance of a patent from this application, without restriction, in accordance with the provisions of the law.

The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.

WO 91/09965 PCT/US90/07464 8 Expression of BL21 DE3) cells were transformed with plasmid and grown in LB medium containing 100 pg/ml ampicillin at 37 0 C. At absorbance 0.6 (650 nm), isopropyl-1thio-P-D-galactopyranoside was added to a final concentration of 1 mM. Cells were harvested 90 min later. The culture medium was used as the source of LysPE40 because most of the protein was secreted into the medium.

Purification of Clarified culture medium, 25 liters, containing was diluted four-fold with chilled deionized water and applied on a 10 x 5.5 cm silica-based anion exchange column (QMA, Waters) at a flow rate of 100 ml/min. The column was washed with 0.05 M sodium phosphate buffer, pH7.0, and proteins were eluted with two liters of 0.25 M NaCI in the equilibration buffer. LysPE40 from the QMA column was concentrated further by using Amicon membranes to 150 ml, dialyzed for 12-16 hours against 0.02 M Tris-HCl, pH7.6, centrifuged at 10,000 x g for minutes and applied on a 2.5 x 14 cm Q-Sepharose column equilibrated with 0.02 M Tris-HCl, pH 7.6. Proteins were eluted with a 500 ml linear gradient of 0-0.5 M NaCI; fractions were detected by SUS PAGE, pooled, diluted 10-fold with 0.02 M Mes, pH 5.5, and loaded onto an FPLC Mono S 16/10 column equilibrated with 0.02 M Mes, pH 5.5. LysPE40 bound to the column and was eluted by a 100 ml linear gradient of 0-0.5 M NaC1. It was further purified on TSK250 (22.5 x 600 mM) column (BioRad) using 0.2 M sodium phosphate buffer pH containing 1 mM EDTA.

Construction of immunoconjiuates (2.5 mg/ml) in 0.2 M sodium phosphate buffer pH 7.0 containing 1 mM EDTA was mixed with a 3-fold molar excess of SMCC and incubated at room temperature (about 22 0 -24 0 C) for 30 minutes. Protein was separated from the unreacted cross linker on a PD10 column. MoAb (HB21 or aTac) (5-10 mg/ml) was mixed with a 3-fold molar excess of 2-iminothiolane-HC1 in 0.2 M sodium phosphate WO 91/09965 PCT/US90/07464 9 buffer pH 8.0 containing 1 mM EDTA and incubated at 37°C for 1 hr. The derivatized antibody was separated from the reactants on a PD10 column. To make immunotoxins, derivatized LysPE40 and derivatized antibody were mixed in 4:1 molar ratio and incubated at room temperature for 16hrs. Immunotoxin was then purified by successive chromatography on a mono Q and TSK 250 columns.

In order to determine the efficacy, two different immunotoxins were prepared and used. One is anti-TFR- LytiPE40, which binds to human transferrin receptor (TFR), and the other is anti-Tac-LysPE40, which binds to 55 kD a subunit of human IL2 receptor.

SDS-PAGE and immunoblottinq SDS-PAGE described by (Reference).

Protein synthesis inhibition assay Activities of the conjugates were tested on A431, KB, HUT102, HT29, OVCAR2, OVCAR3, OVCAR4, CEM and MOLT4 cells by measuring 3 H-leucine incorporation (Kondo et al., J. Biol. Cham. 263, 9470, 1988). HUT102 cells were washed two times with RPM1640 and used immediately. Immunotoxins were diluted with 0.2% human serum albumin in PBS prior to addition to cells.

Assay of blood levels of anti-TFR-LysPE40 in Mice BALB/C mice or nude mice bearing A431 subcutaneous tumors were injected with 100 pg or 50 pg of immunotoxin I.P. Blood was drawn at different time points and the level of the IT was assayed by incubating serum with A431 cells and measuring its effect on protein synthesis. A standard curve was made using Antitumor activity of anti-TFR-LysPE40 in nude mice bearing a human epidermoid carcinoma A431 cells c x 106) were injected subcutaneously on day 0 into nude mice: this injection proauced detectable tumors in all mice by day five. Treatment of mice was started either 2 days, 5 days or 9 days after tumor implantation. Each treatment group consisted of 4-6 animals. Tumors were measured using a caliper every fourth day and the volume of the tumor was calculated WO 91/09965 PCT/US90/07464 10 using the formula: tumor volume in mm 3 length x (width) 2 x 0.4. Tests with anti-Tac-LysPE40 were performed similarly.

As mentioned before, the amino end of PE40 was altered so that it contained a lysine residue and an OmpA signal sequence. (The OmpA signal sequence was added primarily to direct the export of LysPE40 to the growth medium.) The structure of LysPE40 and the plasmid encoding LysPE40 which is under the control of a T7 promoter is shown in Figure 1. Similar to PE40, LysPE40 was also secreted in the culture medium in large amounts. The purity of the protein at each purification step is shown in Figure 2. Material from the TSK 250 column, which was used as the final step, was found to be homogeneous when analyzed by SDS PAGE (Fig. 2, lane 5) as well as by immunoblotting with an antibody to PE (data not shown).

Typically, 2 mg of pure LysPE40 was obtained f:;om one liter of culture.

The N-terminal sequence of the purified protein (LysPE40) was found to be Ala-Asn-Leu-Ala-Glu-Ala-Phe-Lys- Gly-Gly-Ser-Leu. The purified protein had the exact sequence expected from the DNA sequence and processing occurred within the OmpA sequence (Figure In contrast, the N-terminal sequence of PE40 is Ala-Asn-Leu-Ala- Glu-Glu-Gly-Gly.

Construction of immunotoxins with was c emically coupled to two different monoclonal antibodies, HB21 which binds to the human transferrin receptor (anti-TFR) (Haynes et al, J. Immunol 127:347, 1981) and anti--,'c which binds to the 55 kDa subunit of human interleukin 2 receptor (Uchiyama et al, J. Immuno., 126:1393, 1981). After the conjugation, the immunotoxins (ITs) were purified on MonoQ and TSK 250 columns. Purified conjugates contain LysPE40 coupled to both the light and heavy chain and do not contain any free (Figure For comparison, immunotoxins were also made with native PE.

WO 91/09965 PCT/US90/07464 11 Activity of immunotoxins made with The activity of anti-TFR- JPE40 was assayed on a variety of human cell lines and it inhibited protein synthesis in all the human cell lines studied (Table I).

Anti-TFR-LysPE40 was most active on A431 cells with an ID 50 of 4.0 ng/ml. Specificity was demonstrated by showing that excess unconjugated antibody prevented the cytotoxic effect of anti-TFR-LysPE40 (Fig. 3A). was not cytotoxic to murine Swiss 3T3 cells even at 2 gg/ml (data not shown).

The cytotoxic activity of anti-Tac-LysPE40 was determined on HUT102 cells, a human T cell leukemia line containing IL2 receptors. As shown in Figure 5, anti-Tacinhibited protein synthesis in HUT102 cells with an IDs 5 of 2.5 ng/ml and excess anti-Tac blocked this effect demonstrating the specificity of the immunotoxin (Fig. 3B). Anti-Tac-LysPE40 did not inhibit protein synthesis on IL2 receptor negative cells, even at 2000 ng/ml, further showing the specificity of the IT.

Blood levels of anti-TFR-LysPE40 in mice Balb/C mice were injected I.P. with a single dose of 100 pg of anti-TRF-LysPE40 and blood was drawn at different times after the injection to assay for immunotoxin activity. As shown in Fig. 4, a peak blood level of 78 pg/ml was obtained 4 hrs after the injection and a level of 10 pg/ml was still present 24 hrs after the injection. A similar experiment was performed in arthymic mice bearing A431 tumors. After injecting 50 pg of antia blood level of 27 pg/ml was detected 4 hrs after injection and 8 gg/ml after 24 hrs (Fig. 4).

Effect of anti-TFR-LysPE40 on A431 tumors in mice was assayed for its ability to inhibit the growth of A431 cells as subcutaneous xenografts in nude mice. To produce tumors, 3 x 106 A431 cells were injected subcutaneously on day 0. In the control group treated only with diluent, the tumors grew very rapidly and the animals were sacrificed on day 19 with very large tumors that were penetrating the skin WO 91/09965 PC/US90/07464 12 (Fig. 5, In a group that received anti-TFR-LysPE40 on days 2, 4, 6, 8, no tumors were evident on day 24 when the experiment was terminated (Fig. 5A and In another group of animals, treatment was delayed until the tumors were about 125 mm3 in volume and given on days 9, 11, 13 and 15. As soon as the treatment was initiated, the tumors stopped increasing in size (Figure 5A and 6) and then developed soft centers. Histological examination showed that almost all of the cells in the center of the tumor were nonviable (Figure However, at the rim of the tumor many viable cells were observed.

To determine if the antitumor effect was dose related, treatment with various amounts of anti-TFRwas begun on day 5 when small tumors were evident (Figure 5B). Even the 5 Vg dose produced a delay in tumor growth but after the treatment was stopped on day 11, the tumors began to grow rapidly. As the dose was increased, or 50 pg), some or all of the tumors became undetectable by day 11, but with time began to reappear and grow.

One group of animals received a single dose on day 5 of 150 pg, which is close to the ID 5 s. Two animals died, but in the other three animals the tumor regressed and did not reappear. When antibody alone or an immunotoxin composed of an antibody that did not react with the tumor (MOPC- LysPE40) was administered at 50 pg doses, no antitumor response was observed.

In summary, the results indicate that LysPE40 can be efficiently coupled to antibodies yielding an immunotoxin with high cytotoxic activity against cultured cell lines bearing the appropriate antigen, and no detectable cytotoxicity against cultured cells to which the antibody does not bind. Furthermore, an immunotoxin composed of an antibody to the human transferrin coupled to could be administered safely in large amounts to mice and caused regression of a rapidly growing human epidermoid carcinoma implanted subcutaneously.

When administered intraperitoneally in mice, antiappeared rapidly in the blood. A dose of WO 91/09965 PCT/US90/07464 13 pg gave a peak blood level 4 hrs post injection of about pg/ml, and blood levels were still 8 pg/ml at 24 hrs (Fig. 3) indicating that this immunotoxin has a relatively long half-life. A single dose of 100 pg gave a peak blood level of 80 pg/ml. Since a 10 g mouse has a blood volume of about 1 ml, almost all the immunotoxin is found in the blood four hours after intraperitoneal administration.

In the treatment protocols described herein, the tumor cells were allowed to grow to form a detectable solid tumor before treatment was initiated. Under these conditions, a treatment consisting of four injections given over eight days caused obvious tumor regression (Figs. At the lower dose levels with small tumors, or even at the high dose level with large tumors, viable tumor cells remained. By continuing the treatment for longer periods, a larger antitumor effect could most likely be achieved. Nevertheless, the protocol caused marked regression of a solid tumor resistant to standard chemotherapy, thereby indicating that LysPE40 when attached to target-specific antibodies acts as a potent immunotoxin.

A therapeutic composition in accordance with the present invention comprises an effective amount of the to a target-specific ligand to kill target cells, and a pharmaceutically acceptable carrier, the target cells being those that are desired to be selectively killed and carry a binding site to which said ligand specifically binds.

Of course, ligands other than antibodies, such as receptors, growth factors and molecules which selectively recognize target cells are coupled to the LysPE40 following the methodology similar to that described herein to obtain target-specific cytotoxic entities.

It is understood that the embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included WO 91/09965 PCT/US90/07464 14 within the spirit and purview of this application and scope of the appended claims.

TABLE I ACTIVITY OF ANTI-TFR-LYSPE40 ON VARIOUS HUMAN CELL LINES CELLS ID 0 a(nc/ml A431 4.0 >2000 KB 14.0 >2000 HT29 6.6 >2000 HUT102 21.0 >2000 CEM 22.5 >2000 OVCAR2 135.0 ND OVCAR3 280.0 ND OVCAR4 30.0 ND MOLT4 32.0 ND aIDs, is described as concentration of the immunotoxin needed for 50% inhibition of protein synthesis.

N.D. not done.

TABLE II INCIDENCE OF TUMOR IN NUDE MICE TREATED WITH Athymic mice were injected subcutaneously with 3 x 106 A431 cells. On days 5, 7, 9 and 11, the animals were injected I.P. with anti-TFR-LysPE40 at the indicated dose.

Number of animals with tumors/total animals is shown on different days after tumor transplantation.

Treatment DAYS POST TUMOR IMPLANTATION 11 15 19 24 29 None 5/5 5/5 5/5 pg 5/6 6/6 6/6 6/6 6/6 p 1/5 1/5 3/5 3/5 3/5 p 0/5 0/5 1/5 1/5 1/5 150 uq* 0/3 0/3 0/3 0/3 0/3 0/3 *Single dose on day

Claims (6)

1. An improved Pseudomonas exotoxin (PE) of the type including a deletion in the receptor binding domain Ia of the native toxin, wherein the improvement comprises a recombinant PE molecule possessing at least one lysine residue in domain Ia of the PE molecule which otherwise will be devoid of a lysine residue when having a deletion in the receptor binding domain la, said lysine residue providing an essential linkage for effective coupling of the recombinant PE to other molecules.

2. A cytotoxic molecule comprising the recombinant PE of claim 1 coupled to a target-specific ligand substantially at said domain Ia lysine.

3. The cytotoxic molecule of claim 2 wherein said ligand is an antibody of a growth factor.

4. A composition comprising an effective amount of the cytotoxic molecule of claim 2 to kill target cells and a pharmaceutically acceptable carrier.

A method for achieving targeted cytotoxicity, comprising contacting cells targeted to be killed with cytotoxic amount of the cytotoxic molecule of claim 2, said target cells being those having binding sites to which the target-specific ligand binds, but the composition being without detectable cytotoxicity to cells which lack said binding sites.

6. A cytotoxic molecule according to claim 1, substantially as hereinbdfore described with reference to the example or any one of the attached figures. DATED: 15 September 1992 PHILLIPS ORMONDE FITZPATRICK Attorneys for: f THE UNITED STATES OF AMERICA, REPRESENTED BY THE SECRETARY, UNITED STATES DEPARTMENT OF COMMERCE NT