WO1991018018A1

WO1991018018A1 - Improved pseudomonas chimeric protein cytotoxic to human cells bearing il2 receptors

Info

Publication number: WO1991018018A1
Application number: PCT/US1991/003264
Authority: WO
Inventors: Ira Pastan; David Fitzgerald; Vijay Chaudhary; Maurice Gately
Original assignee: The United States Of America, Represented By The Secretary, United States Department Of Commerce
Priority date: 1990-05-14
Filing date: 1991-05-09
Publication date: 1991-11-28
Also published as: AU7886491A

Abstract

New chimeric proteins kill human cells expressing IL2 receptors with specificity and toxicity greater than heretofore known chimeric toxins.

Description

IMPROVED PSEUDOMONAS CHIMERIC PROTEIN CYTOTOXIC TO HUMAN CELLS BEARING IL2 RECEPTORS

The present invention is generally related to making recombinant chimeric toxins. More particularly, the present invention is related to devising improved forms of recombinant Pseudomonas exotoxins (rPE) of low animal toxicity and high cytocidal specificity when attached to suitable targeting agents. Active chimeric toxins of the nature and properties as described herein have not heretofore been known or reported.

BACKGROUND OF THE INVENTION It is widely recognized that the specificity of antibodies for cell surface antigens and of growth factors and hormones for cell surface receptors offers the possi- bility of selectively delivering therapeutic agents to target cells. In this regard, the interleukin 2 (IL2) receptor is an important target for the selective immuno- therapy of some autoimmune diseases, and for the preven¬ tion of allograft rejection. In these diseases the pivotal role of T-cell immunity has been made abundantly clear (Shapiro et al, 1987, Transplant Proc. 19:594-598; Heidecke et al, 1984, J. Immunol. 133:582-588; Wall et al, 1983, J. Clin. Endicrinol. Metab. 56:164-168; Bottazzo et al, 1985, N. Encfl. J. Med. 313:353-360; Lemm and Warmatz, 1986, Clin. Exp. Immunol. 64:71-79; Selby et al, 1984, Gut 25:32-40; Gery et al, 1986, in Progress in Retinal Research, eds. Osborne, N. , and Chader, Y. , Perga on, Oxford and New York, pp. 75-109; and Waldmann, 1989, J. Natl. Cancer Inst. 81:914-923) The scientific basis for therapeutic trials using agents that eliminate IL2 receptor-expressing cells comes from the observation that adult T-cell leukemia and activated T cells express high affinity IL2 receptors, whereas normal resting T cells and their precursors do not. Such agents are, therefore, expected to eliminate IL2 receptor-expressing leukemia cells or activated T cells involved in various disease states while not destroying IL2-receptor negative mature normal T cells and their precursors that are still able to express the full repertoire of antigen receptors required for T-cell immune responses.

Targeting and killing activated T cells using a chimeric protein IL2-PE40 has been reported (Lorberboum- Galski et al, 1988, Proc. Natl. Acad. Sci. USA 85:1922- 1929) . In this chimeric protein, human IL2 is fused to the truncated form of Pseudomonas exotoxin (PE) that lacks the cell binding domain of the bacterial toxin. Native PE is produced by Pseudomonas aeru inosa. X-ray diffraction analysis has shown that PE is structurally composed of three different domains. Functional analysis of proteins produced by various deletion mutations in the PE gene has shown that domain I is responsible for cell recognition, domain II for translocation of the toxin across a cell membrane and domain III for ADP-ribosylation of elongation factor 2. Once accessing the cytoplasm, PE irreversibly inhibits protein synthesis by its ADP-ribosylation activity and causes cell death.

In the chimeric protein, the IL2 moiety targets the molecules to cells expressing any of the three forms of the IL2 receptor (Lorberboum-Galski et al, 1988, J. Biol. Chem, 283:18650-18656). Because de novo expression of the IL2 receptor by activated lymphocytes is critical to the initiation of the immune response, the targeted delivery to the IL2 receptor bearing T cells results in a potent and specific cytotoxic effect. IL2-PE40 was shown to be extremely cytotoxic to IL2 receptor-expressing cell lines of human, ape and murine origin (Lorberboum-Galski et al, supra) . It was also extremely cytotoxic to ConA- stimulated mouse and rat spleen cells and had a suppressive effect against antigen activated mouse cells inhibiting the generation of cytotoxic T cells in mixed lymphocyte culture (Ogata et al, 1988, J. Immunol. 141:4224-4228) . Using a highly purified IL2-PE40 preparation

(Bailon et al, 1988, BioTechnolocry 6:1326-1329), it was shown that targeting IL2 receptor positive cells provides an effective and selective immunosuppression. IL2-PE40 was shown to (a) delay and mitigate adjuvant induced arthritis in rats (Case et al, 1989, Proc. Natl. Acad. Sci. USA 86:287-291); (b) significantly prolong the survival of vascularized heart allograft in mice (Lorberboum-Galski et al, 1989, Proc. Natl. Acad. Sci USA 86:1008-1012) ; (c) reduce the incidence and severity of experimental autoimmune uveoretinitis (EAU) in rats [Roberge et al, 1989, J. Immunol, (in press)]; and (d) suppress the growth of a T cell lymphoma in mice. Thus, IL2-PE40 has been shown to be a therapeutically effective agent in both mice and rats. However, when IL2-PE40 was tested on i-r vitro activated human and primate T-cellε expressing IL2 receptors, it was found that it had much lower activity than on the corresponding activated mouse and rat T-cells. Hence, a new, improved recombinant chimeric protein was needed which had specificity as well as greater toxicity to human activated T lymphocytes than heretofore known chimeric proteins.

SUMMARY OF THE INVENTION It is, therefore, an object of the present inven¬ tion to provide a new, improved recombinant chimeric protein of higher but specific cytocidal efficacy to human cells expressing IL2 receptors, compared to IL2-PE40, without substantial effect on other cells. Other objects and advantages will become evident from the following detailed description of the invention.

ABBREVIATIONS Various abbreviations, symbols and terminologies used herein are now set forth. IL2-PE66^4Glu means a chimeric protein comprising an

IL2 and a PE molecule of about 66000 Mr in which 4 posi¬ tively charged amino acids have been replaced with glutamic acid (Glu) , the 4 amino acids being those at positions 57, 246, 247 and 249 of the native PE sequence. Similarly, _IL2-PE^Glu57Gly246'²⁴⁷-²⁴⁹ means a chimeric protein comprising an IL2 and a PE molecule of about 66,000 Mr in which amino acid at position 57 has been replaced with glutamic acid (Glu) and amino acids at positions 246, 247 and 249 have been replaced with glycine (Gly) .

BRIEF DESCRIPTION OF THE DRAWINGS These and other 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 accom¬ panying drawings wherein:

Figure 1 schematically shows the construction of expression plasmid pHL823 which encodes the IL2-PE66^4GIU fusion protein. B = Bglll, A = Aval, E = EcoRI, H = Hind III. The "^*" indicates a mutated PE66. Mutations were introduced that changed amino acids 57, 246, 247, 249 to glutamic acids to create IL2-PE66^4Glu. Figures 2A and 2B show the inhibition of DNA or protein synthesis of human (A) and monkey (B) PHA blasts by IL2-PE66^4Glu. Lymphocytes (1 x 10⁶/ml) from human donors (A) or from monkeys (B) were stimulated with PHA-P (5 μg/ml) for three days. The cells were then washed and incubated in the culture medium containing IL2 (lOU/ l) in the absence or presence of IL2-PE40 (A) and IL2-PE66^4Glu (•) . After 40-48 hr incubation (³H) thymidine incorporation (A) or (³H) leucine incorporation (B) was measured. Results are expressed as the percent of control activated lymphocytes, not exposed to chimeric cytotoxins. Data shown represent one out of five experiments done with different donors.

Figures 3A and 3B show the inhibition of mixed leukocyte reactions of human (A) and monkey lymphocytes (B) by IL2-PE66^4GIU. For mixed lymphocyte reactions, IL2-

PE66^4Glu (•) and IL2-PE40 (A) were added to cells derived from a five day culture for 48 hr incubation. Protein synthesis inhibition or DNA synthesis inhibition was then measured as mentioned elsewhere herein. Data represent one out of three experiments performed with different donors.

Figure 4A shows the levels of IL2-PE66^4Glu in mouse serum after I.P. administration. Mice were injected with 50 μg of IL2-PE66^4Glu in PBS I.P. and serum specimens were obtained by eye bleeding at the indicated time points. Serum levels of IL2-PE66^4Glu were then determined. Figure 4B shows the levels of IL2-PE66^4Glu in a monkey injected I.V. A cynomolgus monkey was injected I.V. with 2.78 mg (0.46 mg/kg) of IL2-PE66^4Glu in PBS and serum specimens collected. Serum levels of IL2-PE66^Glu were then determined. No adverse reaction to the adminis- tration of this amount of IL2-PE66^4GIu were then determined. No adverse reaction to the administration of this amount of IL2-PE66^4Glu was noted.

DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by a new, recombi¬ nant chimeric protein of improved toxicity, specific to human cells expressing IL2 receptors, without substantial effect on other cells.

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 are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

The term "substantially pure" as used herein means the product is as pure as can be obtained by standard isolation and purification techniques. The term "without substantial effect" as used herein means without affecting the normal functioning of the cells to any detectable degree. MATERIALS AND METHODS CYTOTOXICITY OF NEW CHIMERIC PROTEINS: STUDIES WITH IL2- PE66^4Glu

For toxicity and in vivo survival studies with IL2-PE66^4Glu, 6-8 week old mice weighing 17-20 g were used (strains C57BL/6 or BALB/C; Charles River Breeding Labora¬ tories) . All cell lines were maintained in RPMI-1640 medium containing 10% heat inactivated fetal calf serum(FCS) . The CTLL-2 mouse cell line was maintained in the same medium containing 10 units/ml of rat rIL2 (Col¬ laborative Research Inc.). LA3 and LA4 are rat retinal S- Antigen specific T-helper cells that were grown in RPMI- 1640 containing 5 x 10"⁵ M 2-mercaptoethanol and activated by stimulation with antigen (10 μg/ml) presented by thymic APC (20 x 10⁶ cells/ml) . The activated cells were har¬ vested after 48 hrs by purification on a density gradient (Isolymph Gallard Schlesinger N.Y.). EL4J3.4 is a murine lymphoma line transfected with the murine p55 IL2 receptor subunit. The EL4J parental line does not express the IL2 receptor whereas the EL4J3.4 transfectant expresses the high affinity IL2 receptors (Saragovi and Molek, 1987, J. Immunol. 139:1518-1926). EL4J and EL4J3.4 cell lines were grown in RPMI-1640 medium containing a 5 x 10^"5 M 2-mercap- toethanol. For the EL4J3.4 cells mycophenolic acid (1 μg/ml) , xanthine (250 μg/ml) and hypoxanthine (15 μg/ml) were added to maintain the plasmids expressing the p55 receptor subunit. ELT5 is a murine lymphoma line trans¬ fected with the human p55 receptor subunit and expresses the high affinity IL2 receptors. Kit-225, an IL-2 depen- dent human T cell line which expresses p55 and p75 chains of the IL2-R (Hori et al, 1987, Blood 70:1069-1072) was obtained from T. Uchiyama (Kyoto, Japan) and maintained in 10% FCS, RPMI-1640 supplemented with 25U/ml IL-2. Cells were washed once in medium lacking IL-2 prior to addition of the chimeric proteins. Assays were performed in the presence of 25U/ml of IL2. 21-N-5, a murine adherent non- lymphoid cell line transfected with multiple copies of cDNA coding for the full length human p55 receptor IL2 protein (Tac) was obtained from D. Nelson (NIH, Metabolism Branch, NCI, Bethesda) . A subclone expressing high levels of surface Tac and secreting high levels of soluble IL2 receptor was used. Cells were trypsinized, washed once and plated in 10% FCS RPMI-1640. Cryopreserved cynomolguε monkey spenocytes were obtained from healthy animals serving as donors of other solid organs. Reagents

Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs or from Bethesda Research Laboratories and used under conditions recom¬ mended by the supplier. (³H) Leucine and (³H) thymidine were purchased from DuPont-New England Nuclear. Human rIL2 was provided by Hoffman La Roche, Nutley, NJ. Bacterial strains, plasmids and olicronucleotide synthesis

E. coli strain HB101 was used for transformation and preparation of plasmids. BL21 (λDE3) which carries a T7 RNA polymerase gene in lysogenic and inducible form was used as the host for the synthesis of the chimeric pro- teins. pHL310 which encodes IL2-PE40 has been described previously (Lorberboum-Galski et al, supra) . The con¬ structions of pJY3A1136-l.3, which carries the gene for the full length mutated PE is described by Chaudhary et al, 1990 (submitted). Plasmid DNAs were prepared by the standard alka¬ line lysis method and purified on cesium chloride/ethidium bromide gradients [Maniatis et al, 1982, Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory, Cold Spring Harbor, N.Y.)]. Restriction fragments were analyzed by electrophoresis on horizontal agarose gels in

TBE buffer (0.08 g Tris-borate, pH 8.0 2 mM EDTA) . Synthetic oligonucleotides were produced on an applied Biosystems DNA synthesizer and purified on oligonucleotide purification cartridges (Applied Biosystems) . Plasmid construction

A plasmid for the expressing of iL2-PE66^Glu57-²⁴⁶-²⁴⁷-²⁴⁹ (IL2-PE66^4Glu) under the T7 promoter was constructed as summarized in Fig. 1. pHL310 (4.4kb) which carries the IL2-PE40 gene fusion (Lorberboum-Galski et al, supra) was cut with Bglll and EcoRI and a 1.8 kb DNA fragment was eluted and cut with Aval. The largest DNA fragment -0.5 kb in length containing the IL2 gene was eluted and saved. PJY3A1136-1.3 carrying the mutated full length PE gene was cut with Bglll and Hindlll and the vector DNA fragment was eluted and ligated to the above IL2-contain¬ ing DNA fragment and to a synthetic oligonucleotide (Fig. 1) . The resulting plasmid pHL823 was examined for its size and for expression of the IL2-PE66^4Glu fusion protein in BL21 (DE3) cells. IL2-PE66⁴⁰¹" purification

IL2-PE66^4Glu and other chimeric proteins were extracted from the insoluble fraction (inclusion bodies) of expressed E. coli cells using 7M-guanidine hydrochlo¬ ride and purified to apparent homogeneity by using Tac (p55 subunit of human IL2 receptor) employing receptor affinity chromatography (Bailon et al, 1987, BioTechnology 5:1195-1198) followed by ion exchange and size exclusion chromatography (Bailon et al, supra) .

Protein synthesis inhibition assay

Protein synthesis inhibition assays on cell lines were performed as mentioned herein above.

Protein synthesis inhibition and cell proliferation inhibition on PHA blasts

Monkey or human peripheral blood lymphocytes were separated using a ficoll gradient (Lymphocyte separation medium, LSM, Organon Teknika Corp.). Lymphocytes were cultured in 5% C0₂, in air, in DME medium supplemented with 5% heat inactivated (56°C, 30 min) fetal bovine serum, 200 μg/ml L-glutamate, 50 u/ml penicillin, 50 μg/ml streptomycin, 50 μg/ml glutamine and 5 x 10"⁵M 2-mercap- toethanol. PHA-P (5 μg/ml) was added at the start of the culture. Usually cells were harvested and used for assays (2 x 10⁴ cells/200 μl/well) after three days. rIL2 (10

U/ml) was added after 3 days to the culture to maintain growth and viability of PHA blasts during the assay period. At the same time, different concentrations of IL2-PE66^4Glu, IL2-PE40 and other proteins, diluted in PBS/0.2% BSA were added. After a 48 hr incubation, 2 μCi of (³H)leucine or 1 μCi of (³H)thymidine were added to each well for 13 to 20 hr of labeling. Cells were harvested on filters and incorporated radioactivity measured. Mixed leucocyte reaction (MLR)

Monkey peripheral blood lymphocytes were prepared and cultured with thawed monkey spleen mononuclear cells that were inactivated by irradiation (300 rads) at a ratio of 40/60% responder to stimulator cells (10⁶ cells/ml) . After five days of incubation, the cells were harvested, washed and plated into a 96 well plate (2 x 10⁴ cells/200 μl/well) . rIL2 10 U/ml was added to the monkey MLR-blasts and to the human MLR blasts as indicated. At the same time IL2-PE66^4Glu or other proteins were added to the 5 day MLR-blasts harvested cells for 48 hours. Protein syn¬ thesis and cell proliferation inhibition was measured as described above for the PHA blasts. For the human MLR- blast experiments both responder and stimulatory lympho- cytes were prepared from peripheral blood. IL2-PE66^4Glu toxicity

Toxicity was assessed in groups of 3-9 mice, to which IL2-PE66^4GIU was administered I.P. in a single does. Animals were observed for at least 72 hr. In vivo survival of IL2-PE66^4Glu in mice and monkeys

The in vivo survival of biologically active IL2- PE66^4Glu was determined by injecting I.P. 50 μg per mouse of the chimeric protein to 8 mice. Blood samples were obtained by eye bleeding at different times and the serum was immediately separated and frozen for later analysis. Bioactivity was measured by testing for cytotoxicity of the samples on HUT 102 cells (Lorberboum-Galski et al, supra) . Concentrations were estimated by comparison of the ID₅₀ of each sample with a standard inhibition curve generated by dilutions of authentic IL2-PE66^4Glu. The in vivo survival of IL2-PE66^4Glu was also determined by inject¬ ing I.V. 2.78 mg (0.46 g/Kg) of the chimeric protein into a cynomolgus monkey. Concentrations were estimated as described above for mice.

It has been reported that IL2-PE40 killed mouse and rat activated T cells that were stimulated to grow in mixed leukocyte culture or by ConA (Okata et al, supra) . IL2-PE40 was also effective in killing cytotoxic T-cellε prepared from mice. However, when IL2-PE40 was tested against activated human and monkey lymphocytes, it was found to have a much lower activity than against mouse cells (Table I) . Hence, the cytotoxicity of a new chimeric protein, IL2-PE66^4Glu, was tested. Construction of IL2-PE66^4Glu

The expression plasmid pHL823 contains a T7 promoter and directs the synthesis of a fusion protein IL2-PE66^4Glu in which a human IL2 sequence of 131 amino acids (amino acids 2-131 of IL2) is preceded by 3 amino acids met ala asp. The threonine residue at position 131 of IL2 is connected to the amino acid terminus of PE (amino acid 3 of PE) by a 4 amino acid linker: lie, Pro, Glu, Gly. The mutated PE molecule has four basic amino acids at positions 57, 246, 247 and 249 converted to glutamate. The calculated molecular weight of the fusion protein is 80 kd. Fractionation studies revealed that the insoluble fraction prepared from E. coli cells contained most of the fusion protein (results not shown) . There¬ fore, IL2-PE66^4Glu was extracted from the insoluble fraction (inclusion bodies) as described for IL2-PE40 (Bailon et al, supra) . Substantially purified chimeric toxins were used in all experiments. Effects of IL2-PE40 and IL2-PE66^4Glu on cell lines express¬ ing the low, intermediate and high affinity IL2 receptor forms

It has been shown previously (Lorberboum-Galski et al, 1988, Proc. Natl. Acad. Sci USA 85:1922-1929) that IL2-PE40 is extremely cytotoxic to IL2 receptor positive cell lines of human, ape and murine origin expressing either the p55 (Tac) or the p75 receptor subunit as well as on cell lines expressing both p55 and p75 subunits. In Table II, the activity of IL2-PE66^4Glu has been compared with that of IL2-PE40 against a number of human, rat and mouse cell lines having a range of type and numbers of IL2 receptors. Cytotoxicity was assessed by measuring inhibi- tion of protein synthesis on target cells. As shown in Table II, IL2-PE66^4Glu was cytotoxic to many cell lines expressing the IL2 receptor but had no effect on cell lines lacking the IL2 receptor. Cell lines expressing the high affinity IL2 receptor form are sensitive to IL2- PE66^4Glu with ID₅₀ values similar to IL2-PE40 for HUT102 and EL4J3.4 cells. IL2-PE66^4Glu was 2-fold more cytotoxic to the cell lines LA3, LA4, Kit-225 and 21-N-5 than IL2-PE40. LA3 and LA4 cell lines are T helper cells known to induce experimental autoimmune uveoretinitis (EAU) in rats. IL2- PE66^4Glu was less toxic (-2 Fold) for human MT-1 cells expressing the p55 subunit only and much less toxic (27 Fold less) than IL2-PE40 for mouse CTLL-2 cells that express high affinity IL2 receptors. The cytotoxic effect of IL2-PE66^4Glu or IL2-PE40 was completely blocked by excess human rIL2 (results not shown) demonstrating the specificity of the response.

Human and monkey PHA activated lymphocytes are extremely sensitive to IL2-PE66^4G|U cytotoxicity

As demonstrated in Table II, IL2-PE66^4Glu is cyto- toxic for lymphoid cell lines of all species expressing any of the three forms of IL2-receptor and its specific cytotoxicity is dependent on an initial receptor mediated event. Next, it was tested whether activated human and monkey T lymphocytes are also sensitive to IL2-PE66^4Glu. Peripheral blood lymphocytes from humans or monkeys were prepared and stimulated to grow by the addition of the mitogen PHA. These mitogen stimulated cells (PHA blasts) express functional IL2 receptors on their cell surface and require IL2 for growth in vitro (Yamamoto et al, 1985, J. Biochem. 98:49-56) . To test for cytotoxicity, IL2-PE66^4Glu was added for 48 hours to lympho¬ cytes that have been activated for three days and then ³H-thymidine or ³H-leucine incorporation was measured. As shown in Fig. 2 and Table III, human and monkey lympho¬ cytes that are mitogen activated are sensitive to IL2- PE66^4Glu mediated toxicity in a dose-dependent manner. ID₅₀ values ranged between 4 to 32 ng/ l in the individual experiments performed. These ID₃₀ values are in the range of sensitivity of various established cell lines express¬ ing high numbers of IL2 receptors (Table II) . The sensi¬ tivity of activated lymphocytes to IL2-PE66^4Glu was observed either by measuring the decline in protein synthesis or DNA synthesis. In contrast, human and monkey PHA-activat- ed lymphocytes were only minimally inhibited by concentrations of IL2-PE40 as high as 500 ng/ml. This insensitivity was observed regardless of the length of mitogenic activation; even preincubation with IL2-PE40 did not increase their sensitivity (results not shown) . All preparations of IL2-PE40 used in these experiments were active on HUT102 cells with ID₅₀ PF 1-3 ng/ml. Immunosuppressive effect of IL2-PE66^4Glu in a mixed leuko- cyte reaction (MLR blasts)

IL2-PE66^4GIU was next tested for its effect on human and monkey lymphocytes activated in a mixed leukocyte reaction (MLR) . Monkey peripheral blood lymphocytes were separated and cultured with allogenic monkey splenic mononuclear cells that were inactivated by irradiation. IL2-PE66^4Glu and all other proteins tested were added to five day cultures and either protein synthesis or DNA synthesis was measured. As shown in Fig. 3 and Table III, treatment with IL2-PE66^Glu specifically removed the acti- vated cell population from mixed lymphocyte cultures.

Experiments were performed in the absence and presence of suboptimal amounts of rIL2 and a reduction of protein synthesis and cell proliferation was observed under both conditions, suggesting that these effects are not due merely to prevention of IL2 binding to its receptor but rather are the result of receptor-mediated events. Adding excess rIL2 to the cultures reversed the cytotoxicity of IL2-PE66^4Glu. As shown in Fig. 3 and in Table III, human and monkey MLR blasts were quite insensitive to IL2-PE40.

Serum levels of IL2-PE66^4Glu in mice and monkeys To learn about the behavior of IL2-PE66^4Glu in animals, the chimeric toxin was injected I.P. into mice and I.V. into a monkey. First, 50 μg of IL2-PE66^4Glu was injected I.P. into mice and the levels of biologically active IL2-PE66^4Glu were determined by measuring the cyto- toxic activity contained in mouse serum specimens on HUT102 cells (Lorberboum-Galski et al, supra) . As shown in Fig. 4A, when 50 μg of IL2-PE66^4Glu was injected I.P. into mice and the levels of biologically active IL2-PE66^4Glu were determined by measuring the cytotoxic activity contained in mouse serum specimens on HUT102 cells

(Lorberboum-Galski et al. supra) . As shown in Fig. 4A, when 50 μg of IL2-PE66^4Glu was injected I.P., it was rapidly transferred to the blood and serum levels of 2.5 μg/ml were reached 30 to 60 min after injecting the fusion protein. Six hours after injection, serum levels had fallen to less than 100 ng/ml. The pharmacokinetics of IL2-PE66^4GIU injected I.P. into mice are very similar to that of IL2-PE40 (Lorberboum-Galski, supra) .

IL2-PE66^4Glu was also injected I.V. into a cynomol- gus monkey. As shown in Fig. 4B, serum levels of 10 μg/ml were measured 5 to 10 min after the injection. The serum levels then began to fall reaching a 50% of maximum at about 1 hr. Detectable amounts (12.5 ng/ml) of biologi¬ cally active toxin were measured at 24 hr. When IL2-PE40 was injected I.V. into a monkey, it decayed somewhat more rapidly than IL2-PE66^4Glu reaching a value that was 50% of maximum peak levels 20-25 min after injection (data not shown) .

Toxicity of IL2-PE66^4Glu in mice Groups of three mice were injected I.P. with single doses of IL2-PE66^Glu ranging from 1 to 50 μg per animal. Animals were observed for mortality. No animals died at the four lowest doses (Table IV) . One animal died at a dose of 15 μg. All animals receiving either 25 μg or 50 μg died. The LD₅₀ of IL2-PE66^4Glu is -20 μg per animal which is very similar to the LD₅₀ of PE66^4Glu alone. IL2-PE-Glu57Glv246.247 ,249

The methodology for the preparation of IL2-PE- Glu57Gly246,247,249 is similar to the methodology described herein for ILS2-PE66^Glu and various tests are conducted similar to those described for IL2-PE66^4Glu. In summary, the data presented herein clearly demonstrate that a new, improved Pseudomonas chimeric toxin with efficacious cytocidal activity to activated human T lymphocytes has been obtained. A target-specific cytocidal composition, in accordance with the present invention, comprises a cytocidal amount of the chimeric toxin of the present invention in a sterile, non-toxic carrier. A method for killing target cells comprises contacting cells desired to be killed, without substantial effect on other cells, with a cytocidal amount of the chimeric toxin of the present invention in a single dose or repeated doses. It is noted that although the pre¬ ferred and best mode of practicing the invention have been described herein, other methods to achieve the same results including biologically active products (chimeric proteins) and the like, will be easily suggested to one of ordinary skill in the art and are included within the teachings of the present invention. DEPOSIT

A deposit of plasmid pHL823 which can be used to prepare various chimeric toxins in accordance with the present invention, has been made at the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852, on April 19, 1990 under acces¬ sion number 68311. The deposit shall be viably ain- tained, replacing if it becomes nonviable during the life of the patent, for a period of thirty years from the date of the deposit or for five years from the last date for a request of the deposit, whichever is longer, and made available to the public upon issuance of the patent without restriction in accordance with the provisions of the law. The Commissioner of Patents & Trademarks, upon request, shall have access to the deposit. It is understood that the examples and 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 within the spirit and purview of this applica- tion-- and scope of the appended claims.

Table I: Activity of IL2-PE40 on Mouse, Monkey and Human Activated Lymphocytes

Source of activated lymphocytes ID₅₀ ng/ml Mouse 10

Monkey >1000

Human >1000

Mouse lymphocytes were activated by concanavalin A (ConA) (2μg/ml) . Monkey and human lymphocytes were activated by PHA (5 μg/ml) as described in Methods.

Table II: Effect of IL2-PE40 and IL2-PE66^4Glu on various cell lines expressing p55, p75 or both IL2 receptor subunits

*contain human p55

Table III : Effect of IL2-PE66 Glu on

Human and Monkey activated lymphocytes

PHA activation of monkey and human lymphocytes and the MLR experiments were performed as described in Methods. Data represent the range of average values from five experi¬ ments with PHA activation and three experiments with MLR blasts.

Table IV: Toxicity of IL2-PE66^4Glu in mice protein u% per mouse No. died/No. injected

Single dose

IL2-PE66^4Glu 1 0/3 5 0/3

10 0/3

12 0/3

15 1/3

25 3/3 50 3/3

PE66^4GIu 20 0/3

PE40 50 3/5

IL2-PE40 50 5/9

PE 0.3 3/3

Mice were injected I.P. with a single dose of the indicat¬ ed amounts of IL2-PE66^4Glu and other protein. Number of dead mice for each group was determined 72 hr. after administration of IL2-PE66^4Glu. Mice surviving 72 hr. were still alive at 2 months.

Claims

WHAT IS CLAIMED IS:

1. Plasmid pHL823 deposited at ATCC under number 68311.

2. A method of preparing substantially pure IL2- PE66-4Glu, comprising the steps of functionally inserting the plasmid of claim 1 in a suitable expression vector and expressing IL2-PE66-4Glu and then recovering IL2-PE66-4G1U in a substantially pure form.

3. An improved Pseudomonas chimeric toxin having specificity and greater toxicity to human cells expressing

IL2 receptors, compared to IL2-PE40, without substantial effect on cells other than IL2-expressing cells.

4. The chimeric toxin of claim 3, selected from the group consisting of IL2-PE66-4Glu and IL2-PE- Glu57Gly246,247,249.

5. The chimeric toxin of claim 4, being IL2- PE66-4G1U.

6. The chimeric toxin of claim 4, being IL2-PE- Glu57Gly246,247,249.

7. IL2-PE66-4Glu prepared by the method of claim

2.

8. A composition comprising an effective amount of the chimeric toxin of claim 3 to kill human cells expressing IL2 receptors, and a pharmaceutically accept- able carrier.

9. A method for killing human cells expressing IL2 receptors, comprising contacting human cells express¬ ing IL2 receptors with a sufficient amount of the composi¬ tion of claim 8 to kill said cells.

10. The method of claim 9, wherein said human cells are those of adult T-cell leukemia and activated T-cells.

11. The composition of claim 8 for use in killing human cells expressing IL2 receptors.

12. The compositin of claim 11, wherein the human cells are those of adult T-cell leukemia and activated T cells.

13. The use of the composition of claim 8 in the manufacture of a medicament for killing human cells ex¬ pressing IL2 receptors.

14. The use according to claim 13, wherein the human cells are those of adult T-cell leukemia and acti- vated T-cells.