Verotoxin pharmaceutical compositions and medical treatments therewith

IS 2 149 347 T3

DESCRIPTION

Pharmaceutical compositions of verotoxins and medical treatments with them.

Invention field

This invention relates to pharmaceutical compositions of verotoxins, and to methods for treating mammalian neoplasms, particularly ovarian and skin cancers.

Background of the invention

Bacteriocins are bacterial proteins produced to prevent the growth of competing microorganisms in a particular biological niche. A bacteriocin preparation from a particular strain of E. coli (HSC10) has long been shown to have anticancer activity in vitro against a variety of human tumor cell lines (1,2). This preparation, which was previously referred to as PPB (from partially purified bacteriocin = partially purified bacteriocin (2)) or ACP (anti-cancer proteins = anti-cancer proteinas (2)) was also effective in a murine tumor model, to prevent metastasis in the lung (2).

Verotoxins (VT), also known as SHIGA-like toxins, comprise a family of toxin subunits, known as Verotoxin 1, Verotoxin 2, Verotoxin 2c, and Verotoxin 2e, made by some strains of E. coli. (3). These toxins are implicated in the etiology of hemolytic uremic syndrome (HUS) (3,4) and hemorrhagic colitis (5). Cytotoxicity for cells is mediated by the binding of the B subunit of holotoxin to the receptor glycolepid, balloon-triaosyl-ceramide, in sensitive cells (6).

The verotoxin family, of toxins made by E. coli, target the globe-triaosyl-ceramide, globe-triaosyl-ceramide series, and require a terminal gal-a-1-4-gal residue for attachment. Furthermore, VT2e, the porcine edematous disease toxin, recognizes globotetetraosylceramide (Gb4) which contains an additional β-1-3 linked galNac residue. These glycolepids are the functional receptors for these toxins, since the incorporation of the glycolepid into receptor-negative cells renders the cells of the recipient organism sensitive to cytotoxicity. Toxins inhibit protein synthesis through the A subunit

- an N-glycanase that removes a specific adenine base in 28S RNA from the 60S RNA ribosomal subunit. However, specific cytotoxicity and specific activity are a function of subunit B. In an in vitro translation system, subunit A verotoxin is the most potent inhibitor of protein synthesis ever described, being effective at a concentration of approximately 8 pM. In the rabbit model of verocytotoxemia, the pathology and localization of the toxins are restricted to tissues that contain the glycolepid receptor, and these comprise endothelial cells = endothelial cells (EC) of a subgroup of the blood vasculature. Verotoxins have been widely implicated as the etiological agents for hemolytic uremic syndrome and hemorrhagic colitis, microangiopathies of the glomerular or gastrointestinal capillaries, respectively. Human umbilical vein endothelial cells (HUVEC) are sensitive to a verotoxin, but this sensitivity is variable depending on the cell line. Human adult kidney endothelial cells are exquisitely sensitive to a verotoxin in vitro and express a correspondingly high level of Gb3. However, HUS is primarily a disease of children under three years of age and of adults, as a continuation of a gastrointestinal infection by VTEC (verotoxin endothelial cells). Receptors for verotoxins have been shown to be present in the glomeruli of children below this age, but are not expressed in the glomeruli of human adults. HUVECs can be sensitized to the effect of a verotoxin by prior treatment with tumor necrosis factor, which results in a specific elevation of Gb3 synthesis (7,8). On the other hand, human renal endothelial cells, although they express high levels of Gb3 in culture, cannot be stimulated to increase Gb3 synthesis (8). Transition from kidney tissue to primary endothelial cell culture in vitro has been suggested to result in maximal stimulation of Gb3 synthesis from zero base (9). The authors therefore suspect that HUS in adults is the result of verotoxemia and a concomitant stimulation of Gb3 synthesis in renal endothelial cells by some other factor, eg. ex. stimulated by LPS (lipopolysaccharides) of a serum TNF α. Therefore, under these conditions, the majority of individuals (except the very young) will not be susceptible to VT-induced renal disease as they followed from systemic verotoxemia.

Verotoxin has also been shown to localize to a subpopulation of human B cells in

ES 2 149 347 T3 vitro (10). These B cells that contain Gb₃ they are found within the germinal centers of the lymph nodes (11). It has been proposed that the Gb₃ may be implicated in a host of germinal centers by CD19-positive B cells (12) and that Gb₃ it may be involved in antigen presentation mechanisms (13).

Elevated levels of Gb3 have been associated with several other human tumors (14-16), but ovarian tumors have not been previously investigated. Gb3 is the antigen of the blood group p^k (17). Tissue examinations using anti-p antisera^k have shown that human ovaries do not express this glycolipid (18,19).

The sensitivity to cytotoxicity of VT1 in vitro has been shown to be a function of cell growth, these cells in stationary phase being refractory to cytotoxicity (20). The sequence homology between the receptor-binding B subunit and the human interferon α2 receptor and the B cell marker CD19 suggests that the expression of Gb3 is involved in the transduction mechanism of interferon α2 and CD19 signals (12 ). Upon surface ligation, Gb3 has been shown to undergo retrograde intracellular transport through the rough endoplaosmic retoculum to the cell membrane (21). The use of a verotoxin to induce apoptosis in Burkitt lymphoma cells through the glycolopid antigen Gb3 / CD77 has also been described (in 22).

List of references

This specification refers to the following publications:

1. Fakas-Himsley, H. and R. Cheung. Bacterial Proteinaceous Products (bacteriocins as cytotoxic agents of neoplasia). Cancer Res. 36: 3561-3567, (1976).

two. Hill, RP and H. Farkas-Himsley. Further studies of the action of a partially purified bacteriocin against a murine fibrosarcoma. Cancer Res. 51: 1359-1365 (1991).

3. Karmali, MA Infection by Verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2: 1538 (1989).

Four. Karmali, MA, M. Petric, C. Lim, PC Fleming, GS Arbus and H. Lior, 1985. The association between hemolytic uremic syndrome and infection by Verotoxin-producing Escherichia coli, J. Infect. Dis. 151: 775.

5. Riley, LW, RS Remis, SD Helgerson, HB McGee, JG Wells, BRDavis, RJ Hebert, ES Olcott, LM Johnson, NT Hargrett, PA Blake, and MC Cohen. Haemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308: 681 (1983).

6. Lingwood, CA, Advances in Lipid Research. R. Bell, YA Hannun and AM Jr. Academic Press. 25: 189-211 (1993).

7. van de Kar, NCAJ, LAH Monnens, M. Karmali and VWM van Hinsbergh. Tumor necrosis factor and interleukin-1 induces expression of the verotoxin receptor globotriaosyl ceramide on human endothelial cells. Implications for the pathogenesis of the Hemolytic Uremic Syndrome. Blood. 80: 2755, (1992).

8. Obrig T., C. Louise, C. Lingwood, B. Boyd, L. Barley-Maloney, and T. Daniel. Endothelial heterogeneity in Shiga toxin receptors and responses. J. Biol. Chem. 268: 15484-15488 (1993).

9. Lingwood, CA Verotoxin-binding in human renal sections, Nephron. 66: 21-28 (1994).

10. Cohen, A., V. Madrid-Marina, Z. Estrov, M. Freedman, CA Lingwood and HM Dosch. Expression of glycolipid receptors to Shiga-like toxin on human B lymphocytes: a mechanism for the failure of long-lived antibody response to dysenteric disease. Int. Immunol. 2: 1-8 (1980).

eleven. Gregory, CD, T. Turz, CF Edwards, C. Tetaud, M. Talbot, B. Caillou, AB Rickenson, and M. Lipinski. 1987. Identification of a subset of normal B cells with a Burkitt's lymphoma (BL) -like phenotype. J. Immunol. 139: 313-318 (1987).

12. Maloney, MD and CA Lingwood, CD19 has a potential CD77 (globotriaosyl cermide) binding site with sequence similarity to verotoxin B-subunits: Implications of molecular mimicry for B cell adhesion and enterohemorrhagic E. coli pathogenesis. J. Exp. Med. 180: 191-201, (1994).

IS 2 149 347 T3

13. Maloney, M. and C. Lingwood. Interaction of verotoxins with glycosphingolipids. TIGG. 5: 23-31 (1993).

14. Li, SC, SK Kundu, R. Degasperi, and YT Li. Accumulation of globotriaosyl ceramide in a case of leiomyosarcoma. Biochem. J. 140: 925-927 (1986).

fifteen. Mannori G., O. Cecconi, G. Mugnai and S. Ruggieri. Role of glycolipids in the metastatic process: Characteristics neutral glycolipids in clones with different metastatic potentials isolated from a murine fibrosarcoma cell line. Int. J. Cancer. 45: 984-988 (1990).

16. Ohyama, C., Y. Fukushi, M. Satoh, S. Saitoh, S. Orikasa, E. Nudelman, M. Straud, and SI Hakomori. Changes in glycolipid expression in human testicular tumors. Int. J. Cancer. 45: 1040-1044, (1990).

17. Naiki, M. and DM Marcus. Human erythrocyte P and p^k blood group antigens: Identification as glycosphingolipids. Biochem. Biophys. Res. Comm. 60: 1105-1111, (1974).

18. Pallesen, G. and J. Zeuthen. Distribution of the Burkitt's-lymphoma-associated antigen (BLA) in normal human tissue and malignant lymphoma as defined by immunohistological staining with monoclonal antibody 38:13. J. Cancer Res. Clin. Oncol. 113: 78-86 (1987).

19. Kasai, K., J. Galton, P. Terasaki, A. Wakisaka, M. Kawahara, T. Root, and SI Hakomori. Tissue distribution of the Pk antigen as determined by a monoclonal antibody. J. Immunogenet. 12: 213 (1985).

twenty. Pudymaitis, A. and CA Lingwood. Susceptibility to verotoxin as a function of the cell cycle. J. Cell Physiol. 150: 632-639 (1992).

twenty-one. Sandvig, K., O. Garred, K. Prydz, J. Kozlov, S. Hansen, and B. van Deurs. Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum. Nature. 358: 510-512 (1992).

22. Mageney, M et al. Apoptosis induced in Burkitt's lymphoma cells via Gb3 / CD77, a glycolipid antigen. Cancer Research. 53: 5314-5319 (1993).

Although anti-neoplastic effects of bacterial preparations have been known for more than 20 years, the anti-neoplastic effect of a verotoxin per se has remained unknown to date. As a result of intensive investigations, we have discovered that a verotoxin, particularly Verotoxin 1, is an active component within ACPs and that purified verotoxin 1 has a potent anti-neoplastic effect in vitro and in vivo. Most surprisingly, we have found effective in vivo anticancer treatments in humans commensurate with administered non-toxic dosages.

Summary of the invention

It is an object of the present invention to create a pharmaceutical composition for the treatment of neoplasms in mammalians, particularly cancers of the skin, brain and ovary.

Correspondingly, in one aspect, the invention provides the use of a verotoxin in the production of a medicament for the treatment of mammary neoplasms, comprising a non-lethal, anti-neoplasm effective amount of a verotoxin, preferably Verotoxin 1. The invention provides preferably a pharmaceutical composition for the treatment of skin cancers, brain cancers and ovarian cancers, of mammary glands.

The present invention provides specific and selective treatments for cancers, in which a verotoxin selectively binds with Gb3 in cells containing Gb3. This is in contrast to the use of a wide range of antineoplastic agents such as most chemotherapeutic agents, in which cells that do not contain Gb3 are not affected by a verotoxin. The present invention therefore provides a highly beneficial selective cell therapy treatment.

Treatment is valuable against cutaneous T-cell lymphomas, particularly mycosis fungoides, Saezary syndrome, and the related skin disease lymphomatoid papillosis. For example, mycosis fungoides lesions have been cured in humans without observed unfavorable systemic effects of any kind, by applying VT1 (5 ng in 2 ml of solution) by intradermal injection to patients.

IS 2 149 347 T3

Verotoxin can be administered to the patient by methods well known in the art, namely, intravenously, intraarterially, topically, subcutaneously, by ingestion, intramuscular injection, inhalation, and the like, as appropriate for the disease. . For the treatment of skin cancer, a subcutaneous application is preferred.

In the practice of the present invention, Verotoxin 1 has been injected intramuscularly into a patient with advanced ovarian carcinoma. No unfavorable effects on lymphocytes or renal function were observed and it was found that a serum tumor marker continued to grow when the patient was treated with relatively high doses of Verotoxin 1. This tumor was refractory to all conventional cancer therapies. No effect on hemoglobin levels was found.

The verotoxin is administered, topically, in an appropriate vehicle, wherein the active ingredient verotoxin is dissolved or suspended in a liquid, such as a serum, to allow the verotoxin to be delivered, for example, in one aspect, from the blood stream. or, in an alternative aspect, via subcutaoneal to neoplastic cells. Alternative solutions are, for example, topically, solutions in alcohols, solutions in dimethyl sulfoxide or aqueous solutions containing, for example, a polyethylene glycol, for example polyethylene glycol 400, Cremophor-EL or cyclodextrin. Such vehicles are well known in the art and are useful for the purpose of delivering a pharmaceutical agent to the site of action.

Several multi-drug resistant cell lines were found to be hypersensitive to Verotoxin 1. For example, the multi-drug resistant SKOVLB and SKOVLC ovarian cancer cell lines were more sensitive to VT cytotoxicity than the corresponding cell cancer cell lines. SKOV3 ovarian non-resistant to multiple drugs. This observation indicates the possible beneficial effect for patients who are carriers of the SKOVLB cell line cancer, greater than for the SKOV3 cell line carriers treated with a VT. Furthermore, the fixation of VT1 to the lumen of blood vessels that vascularize the tumor mass, which we have observed by the inventors, in addition to tumor cells per se, can result in an anti-angiogeonic effect to increase the anti-angiogeonic effect. direct neoplastic of a verotoxin.

Brief description of the drawings

In order that the invention may be better understood, preferred embodiments are now described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows the selective neutralization of ACP cytotoxicity by anti-VT1 and / or anti-VT1 subunit B antibodies but not by anti-VT2 antibodies, as determined by measuring cell density after 48 hours;

Figure 2 shows the viability of selected breast and ovarian tumor cell lines at a verotoxin concentration.

Figure 3 represents the VT1 contained in an ACP preparation that binds to Gb3 (and Gb2).

Figure 4 depicts a thin layer chromatography (TLC) overlay of an ovarian tumor and ovarian glycolipids;

Figure 5 depicts a VT thin-layer chromatographic overlay of glycolopids from selected cell lines;

Figures 6A, 6B and 6C represent, respectively, in three graphs the sensitivity of ovarian cell lines to VT1, VT2 and VT2c;

Figure 7 depicts the sensitivity to VT1, VT2 and VT2c of glioblastoma multiform cell lines;

Figure 8 represents the distribution of the labeled VT1 subunit B (VTB¹²¹I) administered by IP route (intra-peritoneal) in a mouse devoid of immunity, bearing a tumor with Gb3; and Figure 9 represents the results of a treatment with VT1 during three days of various cell lines of human astrocytomas.

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Detailed description of the invention

Experimental part

The isolation and purification of the verotoxins VT1, VT2 and VT2c have already been described previously.

Verotoxin 1 was genetically prepared from the highly expressing recombinant E. coli strain pJB28, J. Bacteriol 166: 375 and 169: 4313. The protein purification process that has been described in FEMS Microbiol was followed in general terms. Lett 41:63.

Verotoxin 2 was obtained from R82, Infect. Immun. 56: 1926-1933 (1988); and purified according to FEMS Microbiol Lett. 48: 379-383 (1987).

Verotoxin 2c was obtained from a clonal strain E32511 and purified according to FEMS Microbiol. Lett 51: 211-216 (1988).

Purification of VT1 from JB28

Sediment preparation can be done as follows:

1. Prepare 6 batches of 1L LB Broth (6 x 1L) in 3 5L jugs (media) and autoclave.

Carbenicillin is added to give a final concentration of 100 µg / ml when cold.

two. At least 6 ml of penassay (tubes in a river enclosure) + 100 µg / l of carbenicillin are seeded with JB28 and incubated overnight at 37 ° C.

3. The jars are seeded (1 ml of seed material / liter of broth) the following morning and incubated for 24 hours at 37^°C at 200 rpm (with vigorous shaking).

Four. The jars are centrifuged at 9K for 15 min at 4^°C and the sediment is scraped off into a freezer bag for future use. Freezes at -70^°C.

Preparation of the crude extract of toxins:

1. The sediment is recovered and poured into a beaker. Resuspend in 400 ml of PBS containing 0.1 mg / ml of polymyxin B and 50 mg of PMSF using a mixer. Mix thoroughly and then sonicate on ice for 1 minute to further disperse.

two. Incubate in a shaking incubator at 200 rpm or vigorously at 37^°C for 1 hour.

3. The cells are centrifuged at 9K for 15 minutes.

Four. The supernatant is racked and stored. The pellet is resuspended in 400 ml of PBS with 0.1 mg / ml of polymyxin B and PMSF. It is mixed and treated with ultrasound as before.

5. Incubate with shaking / vigorous shaking at 37^°C for 1 hour.

6. Centrifuge at 10K for 15 minutes and the supernatant is saved.

7. The supernatants should be quite yellow in color and the bacterial sediment should become more fine and diffuse with each extraction operation.

8. The combined supernatants are filtered through Whatman filter paper and then through a glass fiber filter to clarify. This operation is optional, but it will greatly speed up the concentration operation.⁹

9. The combined supernatants are treated in Amicon® at 482,000 N / m² (70 psi) (maximum) using a YM10 membrane (which takes about 200 hours) to concentrate to less than 50 ml.

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Chromatography:

Hydroxylapatite

1. A hydroxylapatite (HA) column is equilibrated with 10 mM sodium or potassium phosphate (various column volumes).

two. The sample is loaded and washed with equilibration buffer until the absorbance of the effluent is negligible.

3. 2 columnar volumes (150 ml) of 100 mM potassium phosphate are added (until yellow colored fractions emerge) and 3 ml fractions are collected.

Four. The column is washed with 500 mM potassium phosphate and re-equilibrated with 10 mM potassium phosphate. 0.05% sodium azide is added.

Chromatographic focus (CF)

5. The fractions (A₂₈₀) and pooled peak fractions from HA.

6. Dialyze against 2 L of 0.025 M imidazole-HCl pH 7.4 overnight. Also equilibrate the chromatographic focus column overnight with this (300 ml).

7. The sample is loaded and followed with 400 ml of polybuffer-HCl pH 5.0 (50 ml of polybuffer 74 + 350 ml of dH₂Or [distilled water] (a 1: 7 dilution, - adjust the pH to 5.0 with HCl). NOTE: Make sure the sample is equilibrated to the temperature at which the column is run (usually room temperature) before loading. If the column is to operate at 4 ° C, then the buffers must be adjusted to pH at 4 ° C and the column must be equilibrated at this temperature.

8. The 1 ml fractions are collected and assayed for A280 and alpH.

9. A280 and peak fractions pooled at approximately pH of

6.8 for VT1 (the secondary peaks of the grouping separately).

10. The column is cleaned with 100 ml of 1M NaCl. If it is really dirty, continue with 100 ml of 1M HCl but this column is quickly equilibrated with imidazole. The column is stored with 20% ethanol in 25 mM imidazole.

Cibachron Blue (RTM)

eleven. Cibachron blue (RTM) [CB] is equilibrated with 10 mM sodium phosphate buffer, pH 7.2 (100 ml).

12. The sample is loaded directly from the CF (chromatographic approach) and is followed with 60 ml of the same buffer.

13. Elute with 0.5M NaCl in the above buffer and collect the fractions.

14. Fractions are assayed for A280 and cytotoxicity and appropriate pooled.

fifteen. The column is cleaned with 25 ml each of 8 M urea in the wash buffer and 1 M NaCl in the wash buffer.

16. The column is re-equilibrated with 10 mM sodium phosphate containing 0.1% azide.

17. Peak fractions are dialyzed against a wash buffer with one change.

18. It is lyophilized and resuspended in 1 ml of dH2O.

19. Protein is analyzed and SDS-PAGE (sodium dodecyl sulfonate-polyacrylamide gel electrophoresis) is performed to check purity.

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Solutions:

HA column

Potassium phosphate buffer (0.5 M reserve)

17.42 g of K2HPO4 up to 300 ml with dH2O 6.8g of KH2PO4 is adjusted to pH 7.2 with KOH

CF column

Imidazole buffer

0.851 g / 500 ml of H2O is adjusted to pH 7.4 with HCl

CB column

Sodium phosphate buffer (wash buffer = WB) 0.71 g / 500 ml of Na2PHO4 is adjusted to pH 7.2 with HAc (acetic acid) is degassed

Elution buffer 2,922 g NaCl / 100 ml WB

Purification of VT2 from R82

Cleaning buffers

12.012 g of urea / 25 ml of WB 1.461 g of NaCl / 25 ml of WB

Sediment preparation:

1. Prepare 3 batches of 2 l of Penassay broth (Antibiotic Maida 3, DIFCO; pH 7.0) in 3 jars of 5 l capacity and are autoclaved at 121^°C for 20 minutes. The broth is allowed to cool to room temperature before use.

two. At least 3 2 ml batches of Penassay broth containing 75 μg / ml carbenicillin (disodium salt, SIGMA) are seeded with R82 and incubated overnight at 37^°C, with shaking.

3. 50 µg / ml carbenicillin is added to each of the 5 L jugs (from run 1). Each jar is seeded with 2 ml of seed material (from run 2) and incubated for 24 hours at 37^°C shaking at approximately 120 rpm.

Four. The incubator is heated to 45^°C and incubate for 30 minutes.

5. The temperature is reduced to 37^°C and incubate for another 3 hours.

6. The culture solution is centrifuged at 9,000 xg for 15-20 min at 4^°C. Discard the supernatant and store the pellets at -20.^°C.

Preparation of the crude extract of toxins:

1. The pellets are resuspended in 100 ml of PBS (phosphate buffered saline = phosphate buffered saline, OXOID; pH 7.3).

two. 0.3 mg / ml of PMSF (from phenylmethyl-sulfonyl fluoride = phenylmethyl-sulfonyl fluoride, SIGMA) dissolved in 0.5 ml of acetone are added to sediment the addition. The acetone is allowed to evaporate. It is treated with ultrasound on ice with the maximum possible performance for 5 min or until a homogeneous solution is obtained.

3. Coellules are centrifuged at 9,000 xg at 4^°C for 20 min. Sediments are discarded.

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Four. The supernatants are concentrated using an ultrafiltration (pattern 8400, AMICON (RTM) infiltration cell) with N2 at no more than 70 psi and using a membrane filter cut off at MW [molecular weight] 10,000 (YM10 membrane, AMICON ( RTM)).

5. Using a 12-14,000 MW cut-off pipe (SPECTRAPOR (RTM)) (now and in all other dialysis operations), the toxin solution is dialyzed against 4 L of 10 mM potassium phosphate overnight, with shaking. to 4 °.

Chromatography:

Hydroxylapatite (HA)

1. The hydroxylapatite column is equilibrated (BSA binding capacity (from bovine serum albumin = bovine serum albumin): 32 mg / kg, volume approximately 113 ml; CALBIOCHEM (BEHRING DIAGNOSTICS)) with 2 columnar volumes of potassium phosphate 10 mM .

two. The sample is loaded and followed with 1 columnar volume of 10 mM potassium phosphate.

3. 2 column volumes of 200 mM potassium phosphate are added and 2 ml fractions are collected. The fractions containing the toxin should be colored differently than the other fractions.

Four. The column is washed with 1 column volume of 500 mM potassium phosphate and re-equilibrated with 1 column volume of 10 mM potassium phosphate. Azide is added to the top of the column for storage.

Chromatographic focus (CF)

5. Peak fractions from the HA column are collected either by color or by Vero cell cytotoxicity assay (10-fold dilutions).

6. The pooled fractions are dialyzed against 4 L of 0.025 M histidine-HCl pH 6.2 (SIGMA) overnight. The chromatographic focusing column (PBE (from polybuffer exchanger = poly-buffer exchanger) 94 is also equilibrated with a diameter of 1.5 cm and a volume of 57 ml; of PHARMACIA overnight with the same buffer (300 ml)

7. The sample is loaded and followed with 400 ml of polybuffer-HCl of pH 4.0 (50 ml of polybuffer 74 (PHARMACIA) + 350 ml of dH2O, the pH is adjusted to 4.0 with HCl).

8. 2 ml fractions are collected and the pH of each fraction is assayed. Once the pH has dropped to 3.95, fraction collection is stopped. Fractions are assayed to an absorbance of 280 nm (A280) or for cytotoxicity with Vero cells (10-fold dilutions).

9. The peak fractions are pooled, and the pH is returned to 7.0 using 1N nAoh.

10. The column is cleaned with 200 ml of 1M NaCl. If it is dirty, it is followed with 100 ml of 1M HCl, but the column is rapidly equilibrated with 0.025 M imidazole, otherwise it is equilibrated with 24% EtOH-H2O

Cibachron Blue (RTM) (CB)

eleven. Cibachron blue (RTM) (2 cm diameter, 82 ml volume, from PIERCE, is equilibrated with 100 ml of 100 mM sodium phosphate buffer (wash buffer).

12. The sample is loaded and followed with 60 ml of wash buffer.

13. Elute with 0.9 M NaCl in wash buffer and collect 2 ml fractions.

14. Fractions are assayed for absorbance at 280 nm (A280) using the elution buffer as a vacuum sample and for cytotoxicity with Vero cells and proximate fractions are pooled.

fifteen. The column is cleaned with 25 ml in each case of 8 M urea in wash buffer and 1 M NaCl in wash buffer.

16. The column is re-equilibrated with 100 ml of a wash buffer and azide is added to the top of the column for storage.

17. Peak fractions are dialyzed against 4 L of 0.01 M Tris HCl (pH 7.0, SIGMA).

IS 2 149 347 T3

18. The sample is lyophilized and resuspended in 1-2 ml of dH2O (OPTIONAL).

19. Proteon is analyzed (BCA Protein Analysis Reagent, PIERCE) and a gel SDSPAGE is performed (Schnagger, H. and von Jagow, G., Analytical Biochem. 166, 368-379 (1987): 10% T Table 2; first line Table 3) to check purity.

Solutions:

HA column

Potassium phosphate buffer (reserve 0.5 M)

17.42 g of K2HPO4, up to 300 ml with dH2O

6.8 g of KH2PO4 is adjusted to pH 7.2 with KOH

CF column

Histidine buffer (0.025 M) 2.0 g / 500 ml H2O is adjusted to pH 6.2 with HCl

CB column

Sodium Phosphate Buffer (Wash Buffer - WB)

0.71 g / 500 ml of Na2HPO4 is adjusted to pH 7.2 with Hac degassed

Elution Buffer (0.5 M) 2.922 g NaCl /

100 ml of WB

Tris 0.01 M

4.84 g of Trizma base (RTM) l of dddH2O (tri-distilled water) adjust the pH to 7.2 with HCl

Purification of VT2c from E32511

Cleaning buffers

12.01 g of urea / 25 ml of WB 1.46 g of NaCl / 25 ml of WB

Sediment preparation:

1. Prepare 3 batches of 2 l of Penassay broth (Antibiotic Media 3, DIFCO, pH 7.0) in 3 5 l jars and autoclave at 121 ° C for 20 minutes. The broth is allowed to cool to room temperature before use.

two. At least 3 2 ml batches of Penassay broth are seeded with E32511 and incubated overnight at 37^°C.

3. 0.2 pg / ml of mitomycin C (1 ml of 0.4 mg / ml) are added (5 ml of ddH are added₂Or (double distilled water) to the vial) to each of the 5-liter jugs (from operation 1). Each jar is seeded with 2 ml of seed material (from operation 2) and incubated for 6 h at 37^°C with agitation at approximately 120 rpm. It is very important to stagger the incubation for approximately 45 min per flask, since the toxin begins to deteriorate after exposure to mitomycin C for 6 hours.

Four. The culture solution is centrifuged at 9,000 xg for 15-20 min at 4^°C. Discard the supernatant and store the pellets at -20^°C.

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Preparation of the crude extract of toxins:

1. The pellets are resuspended in 150 ml of PBS (phosphate buffered saline,

OXOID; pH 7.3).

two. 0.3 mg / ml of PMSF (phenylmethylsulfonyl fluoride, SIGMA) dissolved in 0.5 ml of acetone are added to settle the solution. The acetone is allowed to evaporate. It is treated with ultrasound on ice with the highest possible yield for 3 min or until a homogeneous solution is obtained.

3. 0.1 mg / ml of polymyxin B sulfate (Aerosporin, BURROUGHS WELLCOME INC .; 500,000 units) are added to the solution and incubated with gentle shaking at 37 ° C for 1 h.

Four. Cells are centrifuged at 9,000 xg at 4 ° C for 20 min (to remove all cells and cellular debris from the solution).

5. Decant the supernatant and store at 4^°C. The pellet is resuspended in 75 ml of PBS, and 0.1 mg / ml of polymyxin B is added.

6. Incubate with gentle shaking at 37^°C for 1 hr.

7. Cells are centrifuged at 9,000 xg at 4^°C for 20 min and collect the supernatants (from run 5). Sediments are discarded.

The following few operations should preferably be performed at 4^°C:

8. Crystalline ammonium sulfate is added very slowly, with stirring, to the pooled supernatants to a saturation of 30%.

9. It is left stirring for 20 min and then the precipitate is removed by centrifugation (at 10,000 xg for 10 min).

10. Crystalline ammonium sulfate is added very slowly, with stirring, to the pooled supernatants to 70% saturation.

eleven. Shake for 20 min and then centrifuge at 10,000 xg for 10 min.

12. The pellet from run 11 is resuspended in 15 ml of 0.01M potassium phosphate buffer.

13. Using a 12,000-14,000 MW cut-off tubing (SPECTRAPOR) (RTM) (now and in all dialysis operations), the toxin solution is dialyzed against 4 L of 10 mM potassium phosphate overnight, with shaking at 4^°C.

Chromatography:

Hydroxylapatite (HA)

1. The hydroxylapatite column (BSA binding capacity: 32 mg / g, volume approximately 113 ml; CALBIOCHEM (BEHRING DIAGNOSTICS) is equilibrated with 2 columnar volumes of 10 mM potassium phosphate.

two. The sample is loaded and followed with 1 columnar volume of 10 mM potassium phosphate.

3. 2 columnar volumes of 100 mM - 200 mM potassium phosphate are added and 2 ml fractions are collected. The fractions containing the toxin should be colored differently than the other fractions.

Four. The column is washed with 1 column volume of 500 mM potassium phosphate and re-equilibrated with 1 column volume of 10 mM potassium phosphate. Azide is added to the top of the column for storage.

Chromatographic focus (CF)

5. Peak fractions from the HA column are pooled either by color or by Vero cell cytotoxicity assay (10-fold dilutions).

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6. The pooled fractions are dialyzed against 4 L of 0.025 M imidazole-HCl pH 7.4 (SIGMA) overnight. The chromatographic focusing column (PBE = polybuffer-exchanger) 94, diameter 1.5 cm, volume 57 ml, is also equilibrated; PHARMACIA) overnight with the same buffer (300 ml).

7. The sample is loaded and followed with 200 ml of polybuffer-HCl of pH 5.0 (25 ml of polybuffer 74 (PHARMACIA) - 175 ml of dH2O - and the pH is adjusted to 5.0 with HCl).

8. 2 ml fractions are collected and the pH of each fraction is assayed. Once the pH has dropped to 5.95, the collection of the fractions is stopped. Fractions are assayed for cytotoxicity with Vero cells (10-fold dilutions).

9. The peak fractions are pooled.

10. The column is cleaned with 200 ml of 1 M NaCl. If it is really dirty, it is followed with 100 ml of 1 M HCl, but the column is quickly equilibrated with 0.025 M imidazole.

Cibachron Blue (CB) (from RTM)

eleven. Cibachron blue (RTM) (diameter 2 cm, volume 82 ml, PIERCE) is equilibrated with 100 ml of a 10 mM sodium phosphate buffer (wash buffer).

12. The sample is loaded and followed with 60 ml of the wash buffer.

13. Elute with 0.05 M NaCl in wash buffer and collect 2 ml fractions.

14. Fractions are assayed for absorbance at 280 nm using the elution buffer as a vacuum sample and for Vero cell cytotoxicity and the appropriate fractions are pooled.

fifteen. The column is cleaned with 35 ml in each case of 8 M urea in wash buffer and 1 M NaCl in wash buffer.

16. The column is re-equilibrated with 100 ml of wash buffer and azide is added to the top of the column for storage.

17. Peak fractions are dialyzed against 4 L of 0.01 M Tris-HCl (pH 7.0, SIGMA).

18. The sample is lyophilized and resuspended in 1-2 ml of dH2O (OPTIONAL).

19. Proteins are analyzed (BCA protein analysis reagent, PIERCE, and a gel SDSPAGE is performed (Schnagger, H. and von Jagow, G., Analytical Biochem 166, 368-379 (1987); 10% T Table 2; first line of Table 3) to check purity.

Solutions:

HA column

Potassium phosphate buffer (original 0.5 M)

17.42 g of K2HPO4, up to 300 ml with dH2O

6.8 g of KH2PO4 is adjusted to pH 7.2 with KOH

CF column

Imidazole buffer (0.025 M) 0.851 g / 500 ml H2O, adjust to pH 7.4 with HCl

CB column

Sodium phosphate buffer (wash buffer-WB) 0.71 g / 500 ml of Na2HPO4 is adjusted to pH 7.2 with Hac degassed

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Elution buffer 2,922 g NaCl / 100 ml WB

Cleaning buffers

12.012 g of urea / 25 ml of WB 1.461 g of NaCl / 25 ml of WB

Tris 0.01 M

4.84 g of Trizma base (RTM) l of ddH2O adjust the pH to 7.2 with HCl Purificacioán by affinity of the verotoxins

500 µg of balloon-triaosyl-ceramide was mixed in 1 ml of chloroform and dried with 1 g of dry Celite. The chloroform was evaporated and the Celite was suspended in PBS and then poured onto a column. The crude extract with polymyxin, 20 ml (25 mg protein) of E. coli producing the toxin were applied to the column and incubated at room temperature for 15 min. The column was washed with PBS and the purified verotoxin was eluted with 10 ml of 1 M Tris pH 9.6. The eluted material was neutralized and dialyzed. This method is applicable to the purification of all verotoxins. (Boulanger, J., Huesca, M., Arab, S. and Lingwood, CA “universal method for the easy production of glycolipid / lipid matrices for the affinity purification of binding ligands” Anal. Biochem 217: 1-6 [1994] ).

Verotoxin 1 Dose Preparation

VT1 was purified from the E. coli strain previously reported to overexpress cloned toxin genes. The purified toxin was free of endotoxin contamination. The protein concentration of this batch of verotoxin was determined and the toxin was aliquoted and stored at -70 ° C.

To prepare patient doses of VT1, VT1 was diluted in sterile, injectable grade saline containing 0.2% v / v (volume / volume) of the patients' own serum. 210 µ! of sterile serum from the patients to 10 ml of a sterile saline solution for injections and then 93.9 ml of purified VT1 (6.7 g / ml) were added to give a final toxin concentration of 62.5 ng / ml or 12.5 ng for each 0.2 ml dose. The final toxin preparation was filtered under sterile conditions using a 0.2 mm syringe filter and distributed in 2 ml aliquots into 10 ml capacity vials. One working vial can be stored at 4 ° C and the remaining vials can be frozen until needed.

FITC labeling of VT1: FITC was added directly to VT1 (in a 1: 1 ratio, w / w = weight / weight) in a conjugated buffer of Na2CO3 / NaHCO3 0.5 M, pH 9.5 and the mixture was gently swirled for 1.2 hours at room temperature. Free FITC was removed using a Centricon.

Fluorescent section stain: Surgically removed ovarian tumor samples were embedded in an OCT composition, snap frozen in liquid nitrogen, and stored at -70 ° C until use. 5 µm sections of frozen sample were thawed, allowed to dry, and stained with FITC-labeled VT1 in PBS (0.5 mg / ml) containing 0.1% BSA for 1 h at room temperature. Sections were washed extensively with PBS and mounted with mounting medium containing DABCO. The sections were viewed under a Polyvar fluorescent microscope.

Cells fluorescent stain: Cells growing on coverslips were washed once with PBS, fixed for 2 min at room temperature with 2% formalin, rinsed with PBS twice and incubated with FITC-VT1 for 1 h at room temperature. Cells were washed 5 times with PBS, mounted with DABCO, and viewed under a Polyvar fluorescent microscope.

Quantification of VT1 antitumor activity: SKOV3 (drug sensitive human ovary cell line), SKOVLC (SKOV3, vincristine resistant) and SKOVLB (SKOV3, vinblastine resistant) cell lines were grown in medium supplemented with α- MEM with 10% fetal calf serum and were tested for their sensitivity to VT's. Equal numbers of cells (approximately 1,000 per ml of medium) were added to the wells of a Linbro 98-well plate. Ten-fold dilutions of the VT's were tested in triplicate and incubated for 48 h at 37 ° C with a humidified atmosphere containing 5% CO.₂. Then the cells were fixed with 2% formalin, stained with

IS 2 149 347 T3

Crystal Violet and were read with an ELISA plate reader.

To quantify the anticancer activity of VT1, SKOV3, SKOVLC and SKOVLB (human ovary cell line) were incubated with a 10-fold dilution of VT1 for 48 h. SKOVLC and SKOVLB (drug resistant cell lines) are more sensitive to VT1 antitumor activity than SKOV3.

Preparation of ¹³¹I-VT1B

This material can be prepared by the following procedure.

1. 20 mg of iodoigen are dissolved in 2.0 ml of chloroform (10 mg / ml). A 1:10 dilution is made by adding 0.25 ml of the 10 mg / ml solution to 2.25 ml of chloroform (1 mg / ml).

two. 20 μl of this diluted solution is dispensed into a clean, dry sterilized glass tube. 500 μl of chloroform are added and evaporated to dryness under N₂.

3. 1.5 mg in 0.66 ml of the VT1 subunit B are added to the test tube.

Four. 5 MCi of iodide are added (¹³¹I) sodium in 100 μ !. The label is allowed to develop for 10 min.

5. A PD-10 column is washed with 25 ml of Sodium Chloride for Injections USP = Sodium Chloride Injection USP.

6. Diluted ¹³¹I-VT1B to a total volume of 2.5 ml with 1% HSA in Sodium Chloride for Injections USP. It is loaded onto a PD-10 column. The column is eluted with 3.5 ml of HAS in saline solution.

7. The activity of ¹³¹I from the eluent and from the column to determine the LE drawn pooled fractions into a syringe with an attached spinal needle. The spinal needle is detached and attached to the Millex (RTM) GV filter.

8. It is filtered and filled into a sterile 10 ml multi-dose vial. The filtered volume is noted and the vial is analyzed for¹³¹I in a dose calibrator. The concentration is calculated.

9. 0.1 ml of ¹³¹I-VT1B and 0.05 ml is dispensed into each of two sterile 5 ml multi-dose vials (one for the sterility test and the other for the pyrogens test). The vials already contain 2 ml of saline (= 1:50 dilution).

10. ACPs are determined by PC (Whatman n^° 1) in 85% MeOH and by size exclusion HPLC.

eleven. Sterility and pyrogenic tests are performed.

Figure 1 refers to the neutralization of ACP cytotoxicity by anti-VT. KHT cell monolayers were incubated with 35 mg / ml ACP from E. coli HSC10 or 10pg / ml from VT1, VT2 or VT2c in the presence of monoclonal anti-VT1 (PH1), monoclonal anti-VT2 or rabbit polyclonal anti-VT1 subunit B . Cells were incubated for 72 hours at 37^°C and viable adherent cells were detected by fixation and staining with Crystal Violet. The cytotoxicity of VT1 and ACP was completely neutralized in the presence of anti-VT1 or anti-VT1 subunit B (anti-VT2 serum has no effect).

From the measurement of the cytotoxicity analysis of ACP with Vero cells (cells from the kidney of an African green monkey that are very sensitive to verotoxin) in relation to a pure VT1 standard, it was estimated that the ACP preparation contained 0, 05% of VT1. This concentration of purified VT1 was as effective as ACPs in inhibiting the in vitro growth of tumor cell lines (Figure 2). Therefore, VT1 was tested for the ability to inhibit KHT fibrosarcoma cell metastasis in the mouse model, as previously reported for ACPs.

The equivalent dose of VT1 was as effective as ACP in reducing the number of lung methotasis to background levels, following an inoculum of a primary subcutaneous tumor (Table 1).

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TABLE 1

Response of KHT cells, which grow as lung nodules, to treatment with VT1 or ACP.

<td>GP</td><td>treatment © g / MOUSE)</td><td>No. of mice</td><td>N ° of lung nodules / mouse</td><td>Half</td><td>Weight loss / gain *</td>

<td colspan="6">Experiment 1</td>

<td> 1</td><td>Witness</td><td> 9</td><td> 34, 24, 39, 47, 28,</td><td> 32,6</td><td> +5%</td>

<td></td><td></td><td></td><td> 32, 26, 29, 34</td><td></td><td></td>

<td> 2</td><td>ACP (0.25)</td><td> 4</td><td> 12, 31, 25, 15</td><td> 20,8</td><td> 0</td>

<td> 3</td><td>ACP (1.0)</td><td> 6</td><td> 1, 2, 2, 5, 1</td><td> 2,2</td><td> 0**</td>

<td> 4</td><td>ACP (4)</td><td> 5</td><td> 0, 0, 0, 0, 0</td><td> 0</td><td> - 13%</td>

<td> 5</td><td>VT1 (0.009)</td><td> 5</td><td> 29, 41, 34, 29, 21</td><td> 30,8</td><td> +5%</td>

<td> 6</td><td>VT1 (0.036)</td><td> 5</td><td> 7, 16, 29, 16, 6</td><td> 14,8</td><td> +5%</td>

<td> 7</td><td>VT1 (0.144)</td><td> 5</td><td> 1, 4, 2, 3, 1</td><td> 2,2</td><td> +5%</td>

<td colspan="6">Experiment 2</td>

<td> 1</td><td>Witness</td><td> 4</td><td> 15, 12, 8, 12</td><td> 11,75</td><td> &lt;5%</td>

<td> 2</td><td>ACP-2</td><td> 5</td><td> 0, 1, 0, 0. 0</td><td> 0,2</td><td> &lt;5%</td>

<td> 3</td><td>VT1 (0.1)</td><td> 4</td><td> 0, 0</td><td> 0</td><td> &lt;5%***</td>

<td> 4</td><td>VT1B (0.2)</td><td> 5</td><td> 13, 14, 9, 7, 19</td><td> 12,4</td><td> &lt;5%</td>

<td> 5</td><td>VT1B (10)</td><td> 5</td><td> 8, 3, 9, 11</td><td> 6,8</td><td> &lt;5%</td>

Mice were treated with VT1 or ACP (ip) 1 day after cell injection (1,000 KHT cells / mouse iv).

Lung nodules were counted at 20 days after the injection of cells.

* Mean change in maximum group weight during 10 days (experiment 1) or 4 days (experiment 2) after injection of VT1 or ACP. Maximum weight loss at 7-8 days.

** One death occurred 2-3 days after ACP injection.

^*** There were two deaths on days 7-8.

Purified VT1 was found to mimic the antimetastatic effect of ACPs on the growth of this tumor from a primary subcutaneous site. Pulmoin metastasis was completely inhibited. Furthermore, prior immunization of mice with the purified verotoxin B subunit completely prevented any protective effect of ACPs when the animals were subsequently treated with tumor and ACP (Table 2).

TABLE 2

Response of lung nodules with KHT, growing in immunized mice, to treatment with VT1 or ACP.

<td>GP</td><td>Immunization*</td><td>Treatment</td><td>No. of mice</td><td>N ° of lung nodules / mouse</td><td>Half</td><td>Weight loss / gain *</td>

<td> 1</td><td>none</td><td>none</td><td> 6</td><td> 34, 47, 53, 62, 43, 52</td><td> 48,5</td><td> &lt;5%</td>

<td> 2</td><td>none</td><td>VT1 - 0.2 μg / mouse</td><td> 5</td><td></td><td></td><td>5 deaths (days 6-8) **</td>

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TABLE 2 (Continued)

<td>GP</td><td>Immunization*</td><td>Treatment</td><td>No. of mice</td><td>N ° of lung nodules / mouse</td><td>Half</td><td>Weight loss / gain *</td>

<td> 3</td><td>none</td><td>ACP - 2.0 μg / mouse</td><td> 5</td><td> 0, 1, 2, 0, 0</td><td> 0,6</td><td> - 8%</td>

<td> 4</td><td>VT1B + FA</td><td>none</td><td> 5</td><td> 43, 40, 47, 43, 23</td><td> 39,2</td><td> - 6%</td>

<td> 5</td><td>VT1B + FA</td><td>VT1 -</td><td> 6</td><td> 26, 44, 49, 21, 43, 37</td><td> 36,7</td><td> &lt;5%</td>

<td> 6</td><td>VT1B + FA</td><td>ACP - 2.0 μg / mouse</td><td> 6</td><td> 50, 38, 33, 41, 48, 50</td><td> 43,3</td><td> &lt;5%</td>

<td> 7</td><td>FA only</td><td>none</td><td> 5</td><td> 44, 60, 19, 25, 40</td><td> 37,6</td><td> &lt;5%</td>

<td> 8</td><td>FA only</td><td>VT1 - 0.2 μg / mouse</td><td> 5</td><td></td><td></td><td>5 deaths (days 6-8)</td>

<td> 9</td><td>FA only</td><td>ACP - 2.0 μg / mouse</td><td> 5</td><td> 1, 1, 2, 1,0</td><td> 1</td><td> - 6%</td>

The mice were treated with VT1 or ACP (ip) 1 day after the injection of the cells (1,000 KHT cells / mouse).

The lung nodules were counted at 20 days after the injection of the cells (iv) * The immunization consisted of 2 injections of VT1 B (10 μg / mouse +/- Freund's adjuvant (FA) administered (ip) at 4 weeks and 2 weeks before the injection of the cells.

^** Average change in maximum group weight during 13 days. Maximum weight loss at days 7-8.

ACP was tested for glycolopid binding by overlay on thin layer chromatography using monoclonal anti-VT1 or anti-VT2c. Anti-VT1 shows extensive binding of a component within the ACP preparation to either balloon-triaosyl-ceramide or gala-biosyl-ceramide (Figure 3). This binding specificity is identical to that reported for purified VT1 (8). No binding component that was reactive with anti-VT2 was detected. In Figure 3 anti-VT antibodies were used to detect immobilized glycolopid binding. The arrows indicate the position of the pattern (from the top) gala-biosyl-ceramide, balloon-triaosyl-ceramide, and balloon-tetraosyl-ceramide. Detection in panel 1 was done using anti-VT1, detection in panel 2 was done using anti-VT2c.

VT1 demonstrated in vitro activity against a variety of ovarian carcinoma cell lines. A large number of primary ovarian tumor biopsies were screened for Gb3 expression by TLC overlay using purified VT1. Gb3 was found to be poorly detectable in normal ovarian tissue, while in all cases a significant increase in Gb3 expression was observed in ovarian carcinomas. Similarly, elevated levels of Gb3 were found in an ascites tumor and in tumors that had metastasized to the omentum, (Figure 4) which defines lane 1, ovarian omentum metastasis; lane 2: tumor biopsy; lane 3 tumor biopsy; lanes 3-6, normal ovary; track 7 Gb pattern₃ of human kidney. Surprisingly, we have found that multiple drug resistant variants of ovarian tumor cell lines were considerably more sensitive to VT1 cytotoxicity than drug sensitive maternal cell lines (Figures 2, 5 and 6A-6C). . Similar effects have been observed for ACPs. Figure 2 shows ACP-sensitive human ovarian tumor cell lines tested for VT sensitivity. Cell lines from human breast and ovarian tumors were tested for sensitivity to VT1, with ovaries 1, 2, 3, 4, and 5 being designated x, and ◦ respectively, and breast SKBR3, 468 f, 435 · and 231 a. Cellular lones 1-ovary, 453 and SKBR3, which previously

ES 2 149 347 T3 were shown to be resistant to ACPs, they were also resistant to up to 20 ng / ml VT1.

Cells 1, 2, 3 and 4 came from patients with ovarian cancer; cells 453 came from a patient with breast cancer, 231 and SKBR3 are cell lines of adenocarcinoma of the breast, and 5 cells, SKOV3 and SKOVLB, are cell lines of adenomacarcinous ovarian cancer. Lines 1, 453 and SKBR3, resistant to ACP, were also resistant to VT1. Figure 5 shows VT-resistant and sensitive cell lines tested for the presence of Gb3 by fixation of VTs in the TLC overlap. Glycolopids from an equal number of cells were extracted and separated by TLC prior to toxin fixation. In Figure 5, lane 1: SKBR3, lane 2: 468, lane 3: 231, lane 4: 453, lane 5: Gb3 pattern, lane 6: SKOV3, lane 7: SKOVLB. SKBR3, 468, 231, and 453 cell lines are derived from breast tumors. Only the 231 is sensitive to VT1. SKOVLB is a multiple drug resistant ovarian tumor cell line derived from SKOV3.

Ovarian tumor cells were highly sensitive to VTs (Figure 3) and contained high levels of the VT receptor, Gb3 (Figure 4). Breast cancer cells were, for the most part, resistant to toxins (Figure 3) and receptor negative (Figure 5). Low levels of Gb3 were detected in normal ovarian tissue but these levels had been markedly elevated for tissue samples from ovarian tumors.

The specific elevation of Gb3 in ovarian tumors, in contrast to normal ovarian tissue, demonstrates the possibility of using the toxin in the treatment of this malignancy. Ovarian tumors are frequently refractory to chemotherapy and the prognosis is poor. Indeed, preliminary phase 1 clone testing using a PCA injected directly into skin malignancies (mycosis fungoides) has been shown to be successful, with no adverse systemic effects.

Referring now to Figures 6A, 6B and 6C, cell lines of ovarian tumors obtained from humans were tested for sensitivity to VT1, VT2 and VT2c. The cells were grown to confluence in 48-well plates and then incubated for 48 hours in the presence of increasing doses of VT's. SKOVLB, the variant of the SKOV3 ovary lone resistant to multiple drugs, which had the highest sensitivity to VT's, with SKOVLC being the next most sensitive to VT's.

Both drug resistant cells have been found to be approximately 500 to 1,000 times more sensitive to verotoxin cytotoxicity than the maternal SKOV3 cell line.

Figure 7 shows the effect after 48 hours of treatment with VT1, VT2 and VT2c of the SF-539 cell line of brain tumor originating from a recurrent right temporoparietal glioblastoma multiforme. This cell line, like others, was very sensitive to VT's.

Figure 8 presents the results of reproducing in images a mouse devoid of immunity with VT1B-¹³¹I (distribution of cpm in different organs). The cpm distribution of VT1B-¹³¹I, in a mouse devoid of immunity with implanted ovarian tumor, showed that a considerable amount of radiologically labeled VT1B had been concentrated in the ovarian tumor. Only a minute amount of VT1B was found in the brain when the potential side effect of VT1 was considered. Since the lung of an adult huma is not the site of concern for VT1 toxicity this poses no problem for the treatment of an adult human with an ovarian tumor. In addition, cpm in the kidney include radiolabelled and excreted VT1 subunit B. Correspondingly, based on this assay, imaging with labeled VT1 subunit B may be a very useful method to screen the susceptible patient with VT1 cytotoxicity.

Figure 9 shows the sensitivity of a variety of aVT1 human astrocytoma cell lines. All of these cells contain Gb3 but show variable sensitivity to VT1-induced cytotoxicity. This suggests that certain astrocytomas will be susceptible to verotoxins while others will not. This is important, since astrocytomas are currently highly refractory to treatment and in vitro cellular sensitivity at concentrations as low as 5 ng per ml is rare.