Chimeric toxins for targeted therapy

Abstract

The present invention relates particularly to neoplastic cells targeted chimeric toxins comprising of cell targeting moieties and cell killing moieties for recognizing and for destroying the neoplastic cells, wherein the cell targeting moieties consist of gonadotropin releasing hormone homologues and the cell killing moieties consist of Pseudomonas Exotoxin A. The present invention further relates to pharmaceutical compositions containing as an active ingredient these neoplastic cells targeted chimeric toxins and to a method for the production of these chimeric toxins. The said invention also relates to a method for cancer therapy, treating malignant carcinoma cells and benign hyperplasia including uterine leiomyoma cells, extrauterine endometrial island cells, benign hyperplasia of prostate and breast and pituitary tumor adenoma cells, by the use of the above-mentioned chimeric toxins.

Description

FIELD OF THE INVENTION

[0001] The present invention relates generally to therapeutic agents useful particularly in cancer targeted therapy but also in treating malignant adenocarcinomas, such as breast, colon, hepatic, ovarian and renal adenocarcinomas, and treating benign tumors of the uterus, hyperplasia, endometriosis, BPH, polycystic disease of the breast and pituitary adenomas.

[0002] More specifically, the said invention relates to Pseudomonas exotoxin based chimeric toxins aimed at those neoplastic cells bearing gonadotropin-releasing hormone binding sites. The present invention further relates to pharmaceutical compositions comprising as an active ingredient the above-mentioned neoplastic cell-targeting chimeric toxins. Furthermore, the present invention relates to a method for the production of said chimeric toxins. These chimeric proteins, according to the present invention, are comprised of cell-targeting moieties which consist of gonadotropin releasing hormone homologues linked to cell-killing moieties which consist, preferably, of the bacterial toxin Pseudomonas exotoxin A, for recognizing and destroying neoplastic cells bearing gonadotropin releasing hormone binding sites.

[0003] Targeting is a term for the selective delivery of chemotherapeutic agents to specific cell populations. It is possible to create chimeric molecules that possess cell-targeting and cellular toxin domains. These chimeric molecules function as cell selective poisons by virtue of their ability to target selective cells and then kill those cells via their toxin component. Pseudomonas exotoxin A (hereinafter called PE), a bacterial toxin used in construction of such chimeric proteins, acts by irreversibly arresting protein synthesis in eukaryotic cells, resulting in cell death.

[0004] The term "gonadotropin releasing hormone homologues" in this invention relates to the gonadotropin releasing hormone gene itself or its analogues and antagonists. Also included in the scope of the present invention are salts of the described chimeric proteins. The term "salts" includes both salts of carboxy groups as well as acid addition salts of amino groups of the protein molecule. The invention further relates to pharmaceutical compositions comprising the chimeric proteins as defined above together with a pharmaceutically acceptable inert carrier. The proteins of the present invention may be administered by methods known in the art for the administration of proteins.

BACKGROUND OF THE INVENTION

[0005] Gonadotropin releasing hormone (hereinafter called GnRH) participates in the hypothalamic-pituitary gonadal control of human reproduction. The involvement of GnRH has been demonstrated in several carcinomas and GnRH analogue treatment has been applied in breast, prostatic, pancreatic, endometrial and ovarian cancers (Kadar et al. Prostate 12:229-307, 1988). These analogues suppress tumor cell growth in vitro and in vivo. The existence of GnRH binding sites was revealed in the corresponding malignant cells and in well-established cell lines (Emons et al. J. Clin. Endocrinol. Metab. 77:1458-1464, 1993), though preliminary results suggest that the GnRH receptor involved may differ from the previously documented receptor (Kakar et al. Biochem. Biophys. Res. Comm. 189:289-295, 1992).

[0006] Although GnRH binding sites have been demonstrated in a number of solid tumors and various carcinoma cell lines derived mainly from hormone dependent tissues, their existence in colon or renal carcinoma has not been previously documented. The presence of specific GnRH binding sites in colon, breast, prostate, ovarian endometrium, renal and liver carcinomas, is shown here. Surprisingly, the specific GnRH binding sites are not limited to hormone-dependant tissues, as indicated by the marked killing of colon adenocarcinoma, renal cell adenocarcinoma and hepatocarcinoma cells.

[0007] WO93/15751 describes various conjugates of GnRH, a linking group and Pseudomonas exotoxin A, prepared using the techniques of synthetic organic chemistry, used for the sterilization of animals by killing gonadotropin releasing cells of the animals pituitary gland.

[0008] The present invention describes the construction, by the techniques of genetic engineering, of PE based chimeric toxins, aimed at targeting those neoplastic cells bearing GnRH binding sites. The chimeric toxins of the present invention are fusion proteins and, as such, do not contain a chemical linking group (as in the above-mentioned patent). Therefore, they are completely different proteins from the molecules described in WO93/15751.

[0009] Using different kinds of targeting moieties, a large number of immunotoxins have been generated in the last 20 years by chemical linkage techniques or recombinant DNA technology. The size of these targeting moieties varies widely, ranging from large antibodies to small growth factors, cytokines and antibody fragments.

[0010] The ability of large chimeric proteins, as the Met-GnRH-PE constructions described in the present invention, to target cells via a very small portion of the polypeptide (a peptide of ten amino acids, as used as the targeting moiety of the present invention), and yet retain their original functions, namely binding and internalization, open up new possibilities in designing targeted immunotoxins.

[0011] Colon, breast, and prostate cancer--three out of the four major malignancies occurring in humans, together with ovarian, endometrium, renal and liver carcinomas, account for more than 50% of cancer related death. The presence of specific GnRH binding sites in all of these cancers, may suggest a more general role of GnRH and/or GnRH-like peptides in the malignant process.

[0012] Collectively, these results disclose what could be considered the Achilles' heel of these malignant growths, a finding that could open up new vistas in the fight against cancer.

[0013] In view of their efficient growth inhibition of the above-mentioned cancer cells and their specificity regarding the non-target cells, the novel Met-GnRH-PE chimeric toxins are promising candidates for cancer treatment.

SUMMARY OF THE INVENTION

[0014] The present invention relates particularly to neoplastic cell-targeting chimeric toxins comprising cell-targeting moieties and cell-killing moieties for recognizing and for destroying the neoplastic cells, wherein the cell-targeting moieties consist of gonadotropin releasing hormone homologues and the cell-killing moieties consist of Pseudomonas exotoxin A. The present invention further relates to pharmaceutical compositions containing as an active ingredient these neoplastic cell-targeting chimeric toxins and to a method for the production of these chimeric toxins. The said invention also relates to a method for cancer therapy, treating malignant adenocarcinoma and hepatocarcinoma cells and benign hyperplasia including uterine leiomyoma cells, extrauterine endometrial island cells, benign hyperplasia of prostate and breast, and pituitary tumor adenoma cells, by the use of the above-mentioned chimeric toxins.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention describes Pseudomonas exotoxin A (PE) based chimeric toxins constructed by ligating an oligonucleotide encoding ten amino acids of a gonadotropin releasing hormone (GnRH) analog (GnRH coding sequence with tryptophan replacing glycine as the sixth amino acid), and a preceding Met (see FIG. 1C), upstream of a mutated form of PE (domains I (mutated), II and III) thereby generating Met-GnRH-PE66, and a ten amino acid synthetic GnRH oligomer (GnRH coding sequence with tryptophan replacing glycine as the sixth amino acid), with a preceding Met, ligated to domains II and III of the PE, thereby generating Met-GnRH-PE40 protein.

[0016] The applications, potential markets and commercial advantages of the said chimeric proteins according to the present invention are listed:

[0017] There are two main applications:

[0018] 1) Malignant Adenocarcinomas and Hepatocarcinomas:

[0019] Breast, colon, ovarian and renal adenocarcinomas and hepatocarcinoma were all sensitive to Met-GnRH-PE mediated cytotoxicity. Thus, the potential market for this new chimeric protein includes all adenocarcinoma and hepatocarcinoma patients, either as a first line of treatment or for patients in which other modalities of treatment had failed.

[0020] 2) Benign Tumors of the Uterus and Hyperplasia:

[0021] This group of pathologies includes various tissues that are known to be sensitive to GnRH and thus can be targeted by the Met-GnRH-PE chimeric proteins.

[0022] a. Uterine:

[0023] Uterine leiomyoma is the most common benign tumor in women. The uterine myomas are found to carry a large number of GnRH receptors. GnRH analogs are clinically used for down-regulation and shrinkage of these myomas. The disadvantage of GnRH analogs is that these compounds cannot be used for long periods and the myomas return to their original size after cessation of the treatment. The use of Met-GnRH-PE for the destruction of the myomas can help to avoid what was considered to be imminent hysterectomies.

[0024] b. Endometriosis--Endometrioma:

[0025] The existence of endometrial tissue out of the uterus leads to the disease called endometriosis which can cause infertility, abdominal pain and even surgical interventions. The endometrial islands are known to be very sensitive to hormonal changes. One of the therapeutic modalities found to be clinically efficient is the GnRH analog. Using Met-GnRH-PE, these explants of endometrial tissue can be eliminated, thereby helping infertile couples as well as women who are undergoing laparotomy for the resection of these endometrial islands. The treatment of both the leiomyoma and the endometria can be administered systematically or locally by either ultrasonic or laparoscopic guided injection into the peritoneal cavity.

[0026] c. Benign Prostatic Hyperplasia (BPH):

[0027] The prostatic cells are known to express GnRH receptors and prostatic cancer is successfully treated today with GnRH agonists. The BPH causes severe symptoms of dysuria, urinary retention and sometimes can be treated only by prostatectomy. The use of Met-GnRH-PE can therefore replace prostatectomy procedures carried out on prostate hyperplasia that is not malignant.

[0028] d. Polycystic Disease of the Breast:

[0029] The mammary cells are also known to express the GnRH receptors. As in the case of BPH, the polycystic disease of the breast may be symptomatic, cause pain and may mimic breast carcinoma. The use of Met-GnRH-PE may eliminate the need for numerous check-ups and needless mammograms and help woman suffering from breast pains and non-malignant breast tumors.

[0030] e. Pituitary Adenoma:

[0031] Some of the pituitary adenomas are derived from gonadotropic cells. The pituitary adenoma, even though non-malignant, can cause a grave prognosis by causing local pressure on vital organs (eyes, brain stem). The trans-sphenoidal surgery used for the pituitary adenoma has many disadvantages, including recurrency and neurological sequela. Met-GnRH-PE may be aimed directly against the gonadotropic cells without damaging other functions of the pituitary gland. Met-GnRH-PE chimeric toxin may be administered intrathecally.

[0032] Commercial Advantages:

[0033] 1. The wide variety of tumors that respond to the Met-GnRH-PE chimeric protein.

[0034] 2. The high selectivity that allows a large therapeutic range.

[0035] 3. The use of GnRH as a targeting peptide leaving the large population of postmenopausal women in whom the GnRH has no physiological role perfect candidates for the treatment.

[0036] 4. Its high specificity enables systemic administration together with the local effect.

[0037] 5. The ability to eradicate small populations of cells in a tissue that will not itself be harmed.

[0038] The proteins of the present invention may be administered by methods known in the art for the administration of proteins. Also included in the scope of the present invention are salts of the described chimeric proteins. The term "salts" includes both salts of carboxy groups as well as acid addition salts of amino groups of the protein molecule. Salts of the carboxy group may be formed by methods known in the art and include both inorganic salts as well as salts with organic bases. The invention further relates to pharmaceutical compositions comprising the chimeric proteins as defined above together with a pharmaceutically acceptable inert carrier. The pharmaceutical composition may be administered by injection (intravenous, intra-articular, subcutaneous, intramuscular, intrathecal or intraperitoneal) topical application, oral administration, sustained release, or by any other route including the enteral route.

[0039] The said invention will be further described in detail by the following experiments and figures. These experiments and figures do not intend to limit the scope of the invention but to demonstrate and clarify it only.

DESCRIPTION OF THE FIGURES

[0040] FIGS. 1A-1C: Construction and expression of the Met-GnRH-PE66 chimeric toxin. FIG. 1A, SDS-PAGE gel and FIG. 1B, immunoblotting analysis of TGnRH-PE66 plasmid expression. Whole cell extract of the lysed bacteria (lane 1). Soluble fraction (lane 2). Insoluble fraction (lane 3). FIG. 1C, construction of TGnRH-PE66 plasmid.

[0041] T7 promotor. GnRH analogue peptide. Ampicillin.RTM.. PE664Glu. The numbers represent the corresponding amino acids.

[0042] FIGS. 2A and 2B: The effect of increasing concentrations of Met-GnRH-PE66 on various cell lines.

[0043] FIG. 2A: .box-solid. SW-48 colon adenocarcinoma, .circle-solid.HepG2 hepatocarcinoma, .tangle-solidup. Caco2 colon adenocarcinoma.

[0044] FIG. 2B: OVCAR3 ovarian adenocarcinoma, HeLa cervix adenocarcinoma, .largecircle. MDA MB-231 breast adenocarcinoma, HT-29 colon adenocarcinoma.

[0045] FIGS. 3A-3F: The effect of Met-GnRH-PE66 on various primary cultures. FIG. 3A, colon carcinoma primary cultures established from three patients. FIG. 3B, renal cell carcinoma primary culture. FIG. 3C, breast carcinoma primary cultures established from four patients. FIG. 3D, ovarian carcinoma primary cultures established from two patients. FIG. 3E, metastases of primary cultures established from the corresponding patients represented in FIGS. 3A, 3C and 3D by the same symbols. FIG. 3F, control cells: # leukocytes. bone marrow. .circle-solid. fibroblasts. .gradient. colon.

[0046] FIGS. 4A-4B: Histopathological diagnosis of primary cultures. FIG. 4A, anti-keratin positive staining of a colon primary culture. FIG. 4B, anti-desmin negative staining of a colon primary culture.

[0047] FIG. 5: Displacement of [.sup.125I] GnRH bound to membranes of SW-48 cells by: .largecircle. Met-GnRH-PE66. .box-solid.GnRH analogue (des-Gly.sup.10, [d-Ala.sup.6]-LHRH).

[0048] FIG. 6: Purification of Met-GnRH-PE66. Lane 1--protein marker. Lane 2--whole cell extract. Lane 3--soluble fraction. Lane 4--insoluble fraction after refolding. Lane 5--after DEAE-Sepharose column. Lane 6--after Sepharyl S-200 HR column.

[0049] FIG. 7: Purification of Met-GnRH-PE40: Lane1--protein marker. Lane 2--whole cell extract. Lane 3--soluble fraction. Lane 4--insoluble fraction after refolding. Lane 5--after DEAE-Sepharose column. Lane 6--after Sepharyl S-200 HR column.

[0050] FIG. 8: Effects of Met-GnRH-PE chimeric proteins on SW-48 colon adenocarcinoma cell line:

[0051] Met-GnRH-PE66 insoluble fraction after refolding.

[0052] A Met-GnRH-PE66 purified protein.

[0053] Met-GnRH-PE40 purified protein.

EXPERIMENTS

Experiment 1

Met-GnRH-PE66 Chimeric Toxin Construction

[0054] A plasmid vector carrying the mutated full length PE gene (pJY3Al136-1,3) (Chaudhary et al., J. Biol. Chem. 256:16306-16310, 1990) was cut with NdeI and Hind III. The insert was a 36 base pair synthetic oligomer consisting of the GnRH coding sequence with tryptophan replacing glycine as the sixth amino acid, and was flanked by NdeI (5' end) and HindIII (3' end) restriction sites. The resulting TGnRH-PE66 plasmid was confirmed by restriction endonucleases digestion and DNA sequence analysis (FIG. 1C).

Experiment 2

TGnRH-PE40 Plasmid Construction

[0055] To construct the Met-GnRH-PE40 protein (Met-GnRH-domains II and III of the PE), the TGnRH-PE66 plasmid vector (FIG. 1C) was digested with NdeI and BamHI and ligated to a NdeI-BamHI 750 bp fragment from the plasmid PHL-906 (Fishman et al., Biochemistry 33:6235-6243, 1994) along with an insert which is a 36 base pair synthetic oligomer consisting of the GnRH coding sequence with tryptophan replacing glycine as the sixth amino acid, flanked by NdeI (5' end) and HindIII (3' end) restriction sites. The resulting TGnRH-PE40 plasmid was confirmed by restriction endonuclease digestion and DNA sequence analysis.

Experiment 3

Protein Expression

[0056] The protein expression method was the same for Met-GnRH-PE40 and Met-GnRH-PE66, unless mentioned. Escherichia coli strain BL21 (DE3) carrying the plasmid TGnRH-PE66 was grown in LB medium containing ampicillin (100 .mu.g/ml) and Escherichia coli strain BL21 (DE3) carrying the plasmid GnRH-PE40 was grown in Super-LB medium containing ampicillin (50 .mu.g/ml). After reaching an A600 value of 1.5-1.7, the cultures were induced 90 minutes for Met-GnRH-PE66 and overnight for Met-GnRH-PE40, at 37.degree. C. with 1 mM isopropyl-d-thiogalactoside (IPTG). Cells were collected by centrifugation and the pellet was incubated at -70.degree. C. for several hours.

[0057] The frozen pellet was thawed and suspended in lysis buffer (50 mM Tris HCl, pH 8.0, 1 mM EDTA and lysozyme 0.2 mg/ml), followed by sonication (3.times.30 seconds) and centrifugation at 35,000.times.g for 30 minutes. The supernatant (soluble fraction) was removed and the pellet (insoluble fraction) served as the source for the chimeric proteins and for their purification.

[0058] Analysis of the insoluble fraction by SDS/PAGE gel electrophoresis revealed a major band (70%) with an expected molecular mass of 67 kDa, corresponding to the chimeric protein, and two major unrelated bacterial proteins of 42 and 28 kDa (FIG. 1A). Immunoblotting with polyclonal antibodies against PE, confirmed these data (FIG. 1B).

Experiment 4

Effect of the Met-GnRH-PE66 Chimeric Proteins on Various Cell Lines

[0059] In the experiments described below, the insoluble fraction of E. coli expressing cells was used as the source of the Met-GnRH-PE66 chimeric protein.

[0060] The cytotoxic activity of Met-GnRH-PE66 was tested in various established cell lines: SW-48 colon adenocarcinoma, HepG2 hepatocarcinoma, Caco2 colon adenocarcinoma, OVCAR3 ovarian adenocarcinoma, HeLa cervix adenocarcinoma, MDA MB-231 breast adenocarcinoma, HT-29 colon adenocarcinoma. Unless specified, all cell lines were maintained in RPMI 1640 medium, cultured in 100 mm Petri dishes in a humidified atmosphere of 5% CO.sub.2/95% air at 37.degree. C. HepG2 and Caco2 were maintained in Eagle's Minimal Essential Medium, and HeLa cells were maintained in Dulbecco's Modified Eagle's Medium. All media were supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml of penicillin and 100 .mu.g/ml streptomycin. On day 0, cells (10.sup.4 in 0.2 ml culture medium) were seeded in 96 well tissue culture microplates and 24 hours later various concentrations of the Met-GnRH-PE66 were added. After 24 hours incubation [.sup.3H]leucine [5 .mu.Ci per well] was added for an additional 24 hours. At day 3, the plates were stored at -70.degree. C. for several hours, followed by a quick thawing at 37.degree. C. Cells were harvested on filters and the incorporated radioactivity was measured with a beta counter. The chimeric protein was found to kill cells in a dose-dependent manner, with considerable variation between cell lines (Table 1) ranging from the strong response of HepG2 hepatocarcinoma, SW-48 and Caco2 colon adenocarcinoma (FIG. 2A) to the intermediate one of OVCAR3 ovarian adenocarcinoma, MDA MB-231 breast adenocarcinoma, HT-29 colon adenocarcinoma, and HeLa cervix adenocarcinoma (FIG. 2B). Although cytotoxicity was measured by inhibition of amino acid incorporation, cell death was reflected in cell number and/or cell necrosis 24 hours following the addition of the chimeric protein.

[0061] To confirm the specificity of Met-GnRH-PE66 activity, two other PE based recombinant proteins, expressed and extracted under the same conditions, were used as controls. No substantial growth inhibition was exerted by either PE664Glu, encoded by the mutated full length PE gene, or by PIS2, an unrelated 80 bp sequence fused to PE664Glu. When 15 .mu.g/ml of PE664Glu or PIS2 were added, protein synthesis ranged from a slight increase to 20% inhibition in the different cultures. Growth inhibition resulting from treatment with one of the two proteins was considered nonspecific.

[0062] The results are expressed as the percent of the control experiments in which cells were not exposed to any protein (results are summarized in Table 1 and in FIG. 2).

1TABLE 1 Cytotoxic Activity of Met-GnRH-PE66 on Various Cell Lines ID.sub.50 Cell Line Origin (.mu.g Total Protein/Well)* Caco2 Colon adenocarcinoma 0.4 HT-29 Colon adenocarcinoma 1.2 SW-48 Colon adenocarcinoma 0.3 OVCAR3 Ovarian adenocarcinoma 3 MDA MB-231 Breast adenocarcinoma 2.3 HeLa Cervix adenocarcinoma 1.8 HepG2 Hepatocarcinoma 0.3 *The ID.sub.50 values show the effect of the insoluble fraction enriched with the chimeric protein

Experiment 5

The Effect of Met-GnRH-PE66 on Various Primary Cultures

[0063] In order to evaluate the cytotoxic effectiveness of the chimeric proteins on cells resembling the original in vivo tumors as closely as possible and to exclude the possibility that the Met-GnRH-PE66 cytotoxicity was a characteristic developed by cells upon prolonged passages, primary cultures were established.

[0064] Fresh tissue specimens were obtained from various cancer patients undergoing therapeutic debulking procedures. Control specimens were obtained from donors or patients undergoing diagnostic or therapeutic procedures for non-malignant diseases. All tissue specimens were washed several times with Leibovitz (L15) medium, and extensively cut with a scalpel. The preparations were then enzymatically proteolysed for 2 hours at 37.degree. C. with gentle shaking in Leibovitz medium containing collagenase type I (200 u/ml), hyaluronidase (100 u/ml), penicillin (1000 units/ml), streptomycin (1 mg/ml), amphotericin B (2.5 .mu.g/ml) and gentamycin (80 .mu.g/ml). Tissue preparations were centrifuged 10 minutes at 200.times.g and the pellets were suspended in RPMI 1640 medium, supplemented with 10% fetal calf serum, penicillin (100 u/ml) and streptomycin (100 .mu.g/ml) and plated in 100 mm Petri dishes. Cells were grown for one to three weeks to a density of 8.times.10.sup.6 cells and histopathological diagnoses and cytotoxic assays were performed. Normal leukocytes from peripheral blood and bone marrow aspirates for cytotoxic assays were obtained by diluting whole blood in one volume of phosphate-buffered saline. The diluted sample was placed over an equal volume of Ficoll-Paque and centrifuged for 10 minutes at 200.times.g. The cells were resuspended and plated in RPMI 1640 medium containing 20% fetal calf serum, 4 mM 1-glutamine, 50 .mu.M .beta.-mercaptoethanol, non-essential amino acids, 1 mM sodium pyruvate, penicillin (100 units/ml) and streptomycin (100 .mu.g/ml).

[0065] The cytotoxic effect of the chimeric protein was variable (Table 2) with up to three-fold differences in ID.sub.50 observed in colon, breast and ovarian primary cultures originated from different patients (FIGS. 3A, 3C and 3D respectively).

2TABLE 2 Cytotoxic Effects of Met-GnRH-PE66 on Various Primary Cultures ID.sub.50 Origin (.mu.g total protein/well).sup.a Colon carcinoma 0.8-2.5.sup.b Renal cell carcinoma 1.2 Breast carcinoma 1-3.sup.c Ovarian carcinoma 1.6-3.sup.d Bladder Carcinoma no effect control cells: Colon no effect.sup.e Fibroblasts no effect.sup.e Bone marrow no effect.sup.e Leukocytes no effect.sup.e .sup.aThe ID.sub.50 values show the effect of the insoluble fraction enriched with the chimeric protein .sup.bn = 3 .sup.cn = 4 .sup.dn = 2 .sup.eIncreasing concentration of Met-GnRH-PE66 did not affect cell growth

[0066] In cases where metastasis biopsies could also be obtained, cultures of primary tumors alongside with the metastasis were examined for Met-GnPH-PE66 cytotoxicity. The metastatic cells responded in the same manner, and their ID.sub.50 were even lower than those of the primary tumors. This may be explained by the high homogeneity of the metastasis culture compared with that of the primary culture.

[0067] Met-GnRH-PE66 was also tested on cultures of benign colon peripheral blood bone marrow and skin fibroblasts from healthy donors. The addition of up to 15 .mu.g/ml of the chimeric protein did not result in any measurable dose dependent killing (FIG. 3F).

Experiment 6

Histopathological Diagnosis of Primary Cultures

[0068] One of the basic questions regarding the veracity of the primary culture assays is of the epithelial origin of the cells. The tendency of cells in primary culture to lose their epithelial morphology has been described in several carcinomas. To confirm the absence of any substantial amount of "contaminating" fibroblasts, differential staining was performed.

[0069] Cells were stained as follows: 10,000 cells were plated on a microscope slide using a cytospin, followed by several minutes incubation at room temperature. Dried slides were fixed by soaking in -20.degree. C. cold methanol for 15 minutes and in -20.degree. C. cold acetone for a few seconds. Slides were kept at -20.degree. C. until staining. Staining was carried out with anti-desmin and anti-keratin antibodies to distinguish fibroblast from epithelial cells, respectively. This staining indicated that the vast majority of the cells (80-100%) were indeed epithelial, even in cases where the cultures exhibited a fibroblast-like shape (FIGS. 4A and 4B).

[0070] Further confirmation was achieved by staining with specific antitumor marker antigens according to the type of cancer. Formalin fixed sections from the original tumors and the primary cultures cells displayed the same pattern and intensity of staining.

Experiment 7

Specific Binding by Met-GnRH-PE66

[0071] To support the findings that colon adenocarcinoma cell lines and primary cultures can be targeted and killed by Met-GnRH-PE66, the ability of plasma membrane fractions from a colon adenocarcinoma cell line to specifically bind GnRH, was examined. The addition of increasing concentrations of Met-GnRH-PE66 chimeric toxin resulted in dose-related displacement of the .sup.1251-GnRH bound to these membranes. A semiconfluent 100 mm dish of the SW-48 colon adenocarcinoma cell line was washed and the cells were scraped off the plate with a rubber policeman. The collected cells were homogenized in ice-cold assay buffer (10 mM Tris HCl, pH 7.6, 1 mM dithiothreitol, 0.15% bovine serum albumin, 1 mM EDTA) and centrifuged at 250.times.g for 15 minutes (4.degree. C.). The resulting pellet was discarded and the supernatant was centrifuged at 20,000.times.g for 30 minutes (4.degree. C.). The plasma membrane pellet was resuspended in cold assay buffer. Aliquots containing 70 .mu.g plasma membrane protein in a final volume of 100 .mu.l, were incubated for 2 hours on ice with 6.times.10.sup.-6 M (240,000 cpm) .sup.125I-GnRH either in the presence or absence of (10.sup.-410.sup.-10 M) unlabeled GnRH authentic peptide and analog (des-Gly, [d-Ala]-LHRH) or (2.5.times.10.sup.-5-10.sup.-9 M) Met-GnRH-PE66 chimeric toxin. Following incubation, samples were washed through Whatman GF/C filters with 10 ml of cold assay buffer and counted in a gamma counter.

[0072] The addition of increasing concentrations of Met-GnRH-PE66 chimeric toxin resulted in dose related displacement of the .sup.125I-GnRH bound to these membranes. Unlabeled authentic GnRH peptide and the analogue des-Gly10 [D-Ala6]-LHRH produced similar results. As can be seen in FIG. 5, binding of the labeled GnRH to SW-48 colon adenocarcinoma cell line was specific and displacement by the Met-GnRH-PE66 chimeric toxin was as efficient as that by the GnRH analogue peptide. There was 37% non-specific binding.

Experiment 8

Met-GnRH-PE40 and Met-GnRH-PE66 Purification

[0073] The pellet of the insoluble fraction was suspended and stirred on ice in denaturation buffer (6 M guanidium HCl, 0.1 M Tris HCl, pH 8.6, 1 mM EDTA, 0.05M NaCl and 10 mM DDT). After an additional centrifugation, the reduced and denatured protein was diluted 1:100 in refolding buffer (50 mM Tris HCl, pH 8, 1 mM EDTA, 0.25M NaCl, 0.25 M L-arginine and 5 mM DTT) and kept at 4.degree. C. for 48 hours. Refolded protein solutions were diluted to 8 mM in TE20 buffer (20 mM Tris, pH 8.0, 1 mM EDTA). DEAE Sepharose was added and stirred for half an hour at 4.degree. C. before being packed onto a column. Washing of the column was done with 80 mM NaCl, in TE20 buffer for Met-GnRH-PE66 and 50 mM NaCl in TE20 buffer for Met-GnRH-PE40. Elution was performed with the linear gradient of 2.times.200 ml of 0.08-0.35 M NaCl, in TE20 (20 mM Tris, pH 8.0, 1 mM EDTA) buffer. The peak fractions were pooled,.0.5M L-arginine was added and stirred cell was used for concentration. 3 ml of the pooled fractions from the ion exchange column were loaded onto a Sepharyl S-200 HR gel filtration column, in 0.5 M NaCl, 0.15 M K-phosphate buffer, pH 6.0. The peak fractions were pooled, dialyzed against phosphate saline buffer and kept in aliquots at -20.degree. C. Purification of Met-GnRH-PE66 and Met-GnRH-PE40 is demonstrated in FIGS. 6 and 7, respectively.

Experiment 9

Effect of Highly-Purified Met-GnRH-PE Chimeric Proteins on SW-48 Colon Adenocarcinoma Cell Line

[0074] The cytotoxic activity of the purified Met-GnRH-PE66 and Met-GnRH-PE40 on the SW-48 colon adenocarcinoma cell line was assessed by measuring the inhibition of protein synthesis. The chimeric proteins were found to kill cells in a dose dependent manner. The ID.sub.50 of the purified Met-GnRH-PE66 chimeric toxin was two to three times lower than the refolded insoluble fraction. The ID.sub.50 of the Met-GnRH-PE40 purified protein was three to four times lower than the purified Met-GnRH-PE66 (FIG. 8).

Sequence CWU 1

1

4 1 1152 DNA Escherichia coli 1 atggagcact ggtcctattg gctgcgccct ggagaagctt ttgttaacgc ccatatggcc 60 gaagagggcg gcagcctggc cgcgctgacc gcgcaccagg cttgccacct gccgctggag 120 actttcaccc gtcatcgcca gccgcgcggc tgggaacaac tggagcagtg cggctatccg 180 gtgcagcggc tggtcgccct ctacctggcg gcgcggctgt cgtggaacca ggtcgaccag 240 gtgatccgca acgccctggc cagccccggc agcggcggcg acctgggcga agcgatccgc 300 gagcagccgg agcaggcccg tctggccctg accctggccg ccgccgagag cgagcgcttc 360 gtccggcagg gcaccggcaa cgacgaggcc ggcgcggcca acgccgacgt ggtgagcctg 420 acctgcccgg tcgccgccgg tgaatgcgcg ggcccggcgg acagcggcga cgccctgctg 480 gagcgcaact atcccactgg cgcggagttc ctcggcgacg gcggcgacgt cagcttcagc 540 acccgcggca cgcagaactg gacggtggag cggctgctcc aggcgcaccg ccaactggag 600 gagcgcggct atgtgttcgt cggctaccac ggcaccttcc tcgaagcggc gcaaagcatc 660 gtcttcggcg gggtgcgcgc gcgcagccag gacctcgacg cgatctggcg cggtttctat 720 atcgccggcg atccggcgct ggcctacggc tacgcccagg accaggaacc cgacgcacgc 780 ggccggatcc gcaacggtgc cctgctgcgg gtctatgtgc cgcgctcgag cctgccgggc 840 ttctaccgca ccagcctgac cctggccgcg ccggaggcgg cgggcgaggt cgaacggctg 900 atcggccatc cgctgccgct gcgcctggac gccatcaccg gccccgagga ggaaggcggg 960 cgcctggaga ccattctcgg ctggccgctg gccgagcgca ccgtggtgat tccctcggcg 1020 atccccaccg acccgcgcaa cgtcggcggc gacctcgacc cgtccagcat ccccgacaag 1080 gaacaggcga tcagcgccct gccggactac gccagccagc ccggcaaacc gccgcgcgag 1140 gacctgaagt aa 1152 2 383 PRT Escherichia coli 2 Met Glu His Trp Ser Tyr Trp Leu Arg Pro Gly Glu Ala Phe Val Asn 1 5 10 15 Ala His Met Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His 20 25 30 Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro 35 40 45 Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu 50 55 60 Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln 65 70 75 80 Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly 85 90 95 Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu 100 105 110 Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp 115 120 125 Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val 130 135 140 Ala Ala Gly Ala Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu 145 150 155 160 Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp 165 170 175 Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu 180 185 190 Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly 195 200 205 Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly 210 215 220 Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr 225 230 235 240 Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu 245 250 255 Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr 260 265 270 Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu 275 280 285 Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro 290 295 300 Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly 305 310 315 320 Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val 325 330 335 Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu 340 345 350 Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro 355 360 365 Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys 370 375 380 3 1869 DNA Escherichia coli 3 atgcagcact ggtcctattg gctgcgccct ggagaagctt tcgacctctg gaacgaatgc 60 gccaaagcct gcgtgctcga cctcaaggac ggcgtgcgtt ccagccgcat gagcgtcgac 120 ccggccatcg ccgacaccaa cggccagggc gtgctgcact actccatggt cctggagggc 180 ggcaacgacg cgctcgagct ggccatcgac aacgccctca gcatcaccag cgacggcctg 240 accatccgcc tcgaaggcgg cgtcgagccg aacaagccgc tgcgctacag ctacacgcgc 300 caggcgcgcg gcaggtggtc gctgaactgg ctggtaccga tcggccacga gaagccctcg 360 aacatcaagg tgttcatcca cgaactgaac gccggcaacc agctcagcca catgtcgccg 420 atctacacca tcgagatggg cgacgagttg ctggcgaagc tggcgcgcga tgccaccttc 480 ttcgtcaggg cgcacgagag caacgagatg cagccgacgc tcgccatcag ccatgccggg 540 gtcagcgtgg tcatggccca gacccagccg cgccgggaaa agcgctggag cgaatgggcc 600 agcggcaagg tgttgtgcct gctcgacccg ctggacgggg tctacaacta cctcgcccag 660 caacgctgca acctcgacga tacctgggaa ggcaagatct accgggtgct cgccggcaac 720 ccggcgaagc atgacctgga catcaaaccc acggtcatca gtgaagagct ggagtttccc 780 gagggcggca gcctggccgc gctgaccgcg caccaggctt gccacctgcc gctggagact 840 ttcacccgtc atcgccagcc gcgcggctgg gaacaactgg agcagtgcgg ctatccggtg 900 cagcggctgg tcgccctcta cctggcggcg cggctgtcgt ggaaccaggt cgaccaggtg 960 atccgcaacg ccctggccag ccccggcagc ggcggcgacc tgggcgaagc gatccgcgag 1020 cagccggagc aggcccgtct ggccctgacc ctggccgccg ccgagagcga gcgcttcgtc 1080 cggcagggca ccggcaacga cgaggccggc gcggccaacg ccgacgtggt gagcctgacc 1140 tgcccggtcg ccgccggtga atgcgcgggc ccggcggaca gcggcgacgc cctgctggag 1200 gcgaactatc ccactggcgc ggagttcctc ggcgacggcg gcgacgtcag cttcagcacc 1260 cgcggcacgc agaactggac ggtggagcgg ctgctccagg cgcaccgcca actggaggag 1320 cgcggctatg tgttcgtcgg ctaccacggc accttcctcg aagcggcgca aagcatcgtc 1380 ttcggcgggg tgcgcgcgcg cagccaggac ctcgacgcga tctggcgcgg tttctatatc 1440 gccggcgatc cggcgctggc ctacggctac gcccaggacc aggaacccga cgcacgcggc 1500 cggatccgca acggtgccct gctgcgggtc tatgtgccgc gctcgagcct gccgggcttc 1560 taccgcacca gcctgaccct ggccgcgccg gaggcggcgg gcgaggtcga acggctgatc 1620 ggccatccgc tgccgctgcg cctggacgcc atcaccggcc ccgaggagga aggcgggcgc 1680 ctggagacca ttctcggctg gccgctggcc gagcgcaccg tggtgattcc ctcggcgatc 1740 cccaccgacc cgcgcaacgt cggcggcgac ctcgacccgt ccagcatccc cgacaaggaa 1800 caggcgatca gcgccctgcc ggactacgcc agccagcccg gcaaaccgcc gcgcgaggac 1860 ctgaagtaa 1869 4 622 PRT Escherichia coli 4 Met Gln His Trp Ser Tyr Trp Leu Arg Pro Gly Glu Ala Phe Asp Leu 1 5 10 15 Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val 20 25 30 Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly 35 40 45 Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala 50 55 60 Leu Glu Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu 65 70 75 80 Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Leu Arg Tyr 85 90 95 Ser Tyr Thr Arg Gln Ala Arg Gly Arg Trp Ser Leu Asn Trp Leu Val 100 105 110 Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu 115 120 125 Leu Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile 130 135 140 Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe 145 150 155 160 Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile 165 170 175 Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg 180 185 190 Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu 195 200 205 Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn 210 215 220 Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn 225 230 235 240 Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser Glu Glu 245 250 255 Leu Glu Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln 260 265 270 Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg 275 280 285 Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val 290 295 300 Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val 305 310 315 320 Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu 325 330 335 Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala 340 345 350 Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu 355 360 365 Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala 370 375 380 Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu 385 390 395 400 Ala Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val 405 410 415 Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu 420 425 430 Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr 435 440 445 His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val 450 455 460 Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile 465 470 475 480 Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro 485 490 495 Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val 500 505 510 Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala 515 520 525 Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu 530 535 540 Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg 545 550 555 560 Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile 565 570 575 Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp 580 585 590 Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp 595 600 605 Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys 610 615 620

Claims

What is claimed is:

1. A fused chimeric protein comprising a linear genetically engineered molecule consisting essentially of peptide bonds, produced by fusing, at the level of cDNA: A. DNA encoding at least one cell targeting moiety consisting essentially of Met-GnRH or a Met-GnRH analog that specifically binds to GnRH binding sites on Caco2 adenocarcinoma cells; and B. DNA encoding at least one cell killing moiety.

2. A fused chimeric protein according to claim 1, produced by fusing at the cDNA level an oligonucleotide encoding gonadotropin releasing hormone (GnRH) or a GnRH analog, preceded by a Met, to a mutated DNA fragment of the full length Pseudomonas exotoxin (PE), encoding the protein Met-GnRH-PE66.

3. A fused chimeric protein according to claim 1, produced by fusing at the cDNA level an oligonucleotide encoding gonadotropin releasing hormone (GnRH) or a GnRH analog, preceded by a Met, to a DNA fragment comprising domains II and III of the Pseudomonas exotoxin (PE), encoding the protein Met-GnRH-PE40.

4. A method for the production of a chimeric protein as defined in claim 3, comprising ligating an oligonucleotide encoding gonadotropin releasing hormone (GnRH) or a GnRH analog, preceded by a Met, upstream of a DNA fragment encoding a mutated form of PE, under conditions sufficient to produce a chimeric protein comprising Met-GnRH-PE66.

5. A method for the production of a chimeric protein as defined in claim 4 that targets adenocarcinoma cells, comprising ligating an oligonucleotide encoding gonadotropin releasing hormone (GnRH) or a GnRH analog, preceded by a Met, upstream to a DNA fragment encoding domains II and III of the PE, under conditions sufficient to produce a chimeric protein comprising Met-GnRH-PE40.

6. A composition useful for treatment in cancer therapy comprising as active ingredient, a chimeric protein as defined in claim 1.

7. A plasmid comprising a promoter operably linked to a DNA molecule encoding a fused chimeric protein as defined in claim 1.

8. A chimeric protein comprising a genetically engineered molecule comprising a fusion of at least one cell targeting moiety consisting essentially of gonadotropin releasing hormone (GnRH) preceded by a Met (Met-GnRH) or a Met-GnRH analog that specifically binds to GNRH binding sites on Caco2 adenocarcinoma; and at least one cell killing moiety.

9. A fusion protein as claimed in claim 8, wherein said cell killing moiety comprises Pseudomonas exotoxin A.

10. A fusion protein as claimed in claim 8, that is a single protein.

11. A fusion protein as claimed in claim 8, that has no linking moiety between said cell killing moiety and said cell targeting moiety.

12. A fusion protein as claimed in claim 8, wherein said chimeric protein recognizes and/or binds to GnRH-binding sites on adenocarcinoma and hepatocarcinoma cells.

13. A fusion protein as claimed in claim 8, wherein said cell targeting moiety is a Met-GnRH analog having the sequence of Met-GnRH but having a glycine residue as the sixth amino acid of GnRH.