WO2000061183A2 - Methodes de traitement de lymphomes par la verotoxine - Google Patents

Methodes de traitement de lymphomes par la verotoxine Download PDF

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WO2000061183A2
WO2000061183A2 PCT/CA2000/000371 CA0000371W WO0061183A2 WO 2000061183 A2 WO2000061183 A2 WO 2000061183A2 CA 0000371 W CA0000371 W CA 0000371W WO 0061183 A2 WO0061183 A2 WO 0061183A2
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verotoxin
cells
lymphoma
cell
vti
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PCT/CA2000/000371
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WO2000061183A3 (fr
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Clifford A. Lingwood
Gerald Arbus
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Hsc Research And Development Limited Partnership
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Publication of WO2000061183A3 publication Critical patent/WO2000061183A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to verotoxin pharmaceutical compositions and to methods of treating mammalian neoplasia, particularly, lymphomas, brain, ovarian and skin cancers, therewith.
  • Bacteriocins are bacterial proteins produced to prevent the growth of competing microorganisms in a particular biological niche.
  • a preparation of bacteriocin from a particular strain of E. coli (HSCi Q) has long been shown to have anti-neoplastic activity against a variety of human tumour cell lines in vitro (1,2). This preparation, previously referred to as PPB (partially purified bacteriocin (2)) or ACP (anti-cancer proteins (2)) was also effective in a murine tumour model of preventing metastases to the lung (2).
  • PPB partially purified bacteriocin (2)
  • ACP anti-cancer proteins (2)
  • Verotoxins also known as SHIGA-like toxins, comprise a family known as Verotoxin 1 , Verotoxin 2, Verotoxin 2c and Verotoxin 2e of subunit toxins elaborated by some strains of E. coli (3). These toxins are involved in the etiology of the hemolytic uremic syndrome (3,4) and haemorrhagic colitis (5). Cell cytotoxicity is mediated via the binding of the B subunit of the holotoxin to the receptor glycolipid, globotriaosylceramide, in sensitive cells (6).
  • the verotoxin family of E coli elaborated toxins bind to the globo series glycolipid globotriaosylceramide and require terminal gal ⁇ -1-4 gal residue for binding.
  • VT2e the pig edema disease toxin, recognizes globotetraosylceramide (Gb_ ⁇ ) containing an additional ⁇ 1-3 linked galNac residue.
  • Gb_ ⁇ globotetraosylceramide
  • These glycolipids are the functional receptors for these toxins since incorporation of the glycolipid into receptor negative cells renders the recipient cells sensitive to cytotoxicity.
  • the toxins inhibit protein synthesis via the A subunit.
  • the A subunit is an N-glycanase which removes a specific adenine base in the 28S RNA of the 60S RNA ribosomal subunit.
  • the specific cytotoxicity and specific activity is a function of the B subunit.
  • the verotoxin A subunit is the most potent inhibitor of protein synthesis yet described, being effective at a concentration of about 8 pM.
  • pathology and toxin targeting is restricted to tissues which contain the glycolipid receptor and these comprise endothelial cells of a subset of the blood vasculature.
  • Verotoxins have been strongly implicated as the etiological agents for hemolytic uremic syndrome and haemorrhagic colitis, microangiopathies of the glomerular or gastrointestinal capillaries respectively.
  • Human umbilical vein endothelial cells (HUVEC) are sensitive to verotoxin but this sensitivity is variable according to cell line.
  • Human adult renal endothelial cells are extraordinarly sensitive to verotoxin in vitro and express a correspondingly high level of Gb3-
  • HUS is primarily a disease of children under three and the elderly, following gastrointestinal VTEC infection. It has been shown that receptors for verotoxin are present in the glomeruli of infants under this age but are not expressed in the glomeruli of normal adults.
  • HUNEC can be sensitized to the effect of verotoxin by pretreatment by tumour necrosis factor which results in a specific elevation of Gb3 synthesis (7,8).
  • Human renal endothelial cells on the other hand, although they express high levels of Gb3 in culture, cannot be stimulated to increase Gb ⁇ synthesis (8). It has been suggested that the transition from renal tissue to primary endothelial cell culture in vitro results in the maximum stimulation of Gb3 synthesis from a zero background (9).
  • HUS in the elderly is the result of verotoxemia and a concomitant stimulation of renal endothelial cell Gb3 synthesis by some other factor, eg. LPS stimulation of serum ⁇ T ⁇ F.
  • Gb3 is the pk blood group antigen (17). Tissue surveys using anti-pk antisera have shown that human ovaries do not express this glycolipid (18, 19). Sensitivity to VTI cytotoxicity in vitro has been shown to be a function of cell growth, the stationary phase cells being refractile to cytotoxicity (20). The sequence homology between the receptor binding B subunit and the human ⁇ 2-interferon receptor and the B cell marker CD 19 suggests that expression of Gb3 is involved in the mechanism of ⁇ 2-interferon and CD 19 signal transduction (12). On surface ligation, Gb3 has been shown to undergo a retrograde intracellular transport via the rough endoplasmic reticulum to the nuclear membrane (21).
  • the astrocytoma is the most common primary human brain tumour.
  • the majority of astrocytomas are malignant neoplasms which infiltrate diffusely into regions of normal brain.
  • no such promising therapy has yet been found for the patient with a malignant astrocytoma.
  • the median survival for patients with glioblastoma multiforme, the most malignant form of astrocytoma, is approximately 12 months and accordingly, it is imperative that new therapeutic treatments for malignant astrocytomas be found.
  • VTs consist of a 30kDa enzymatic A subunit which is capable of inhibiting protein synthesis.
  • the A subunit is noncovalently associated with a pentameric 7kDa B subunit array which binds to Gb3.
  • VTI and the receptor binding B subunit alone, also induce morphological changes and DNA fragmentation characteristic of apoptosis in Gb3-positive cells (22, 23).
  • Verotoxin-producing Escherichia coli J. Infect. Dis. 151:775. 5. Riley, L.W., R.S. Remis, S.D. Helgerson, H.B. McGee, J.G. Wells, B.R. Davis,
  • CD 19 has a potential CD77 (globotriaosyl ceramide) binding site with sequence similarity to verotoxin B-subunits: Implications of molecular mimicry for B cell adhesion and enterohemorrhagic
  • verotoxin particularly Verotoxin 1
  • purified Verotoxin 1 has potent anti-neoplasia effect in vitro and in vivo.
  • the invention provides a pharmaceutical composition for the treatment of mammalian neoplasia comprising a non-lethal anti-neoplasia effective amount of a verotoxin, preferably, verotoxin 1, or the pentameric B subunit of verotoxin and a suitable pharmaceutically acceptable diluent, adjuvant or carrier therefor.
  • the invention preferably provides a pharmaceutical composition and method of treatment for mammalian lymphomas, skin cancers, brain cancers and ovarian cancers.
  • the invention provides a process for the manufacture of a pharmaceutical composition for the treatment of mammalian neoplasia, said process comprising admixing an agent, e.g., verotoxin, the pentameric B subunit of verotoxin 1, verotoxin 2, or verotoxin 2c, with a pharmaceutically acceptable carrier, adjuvant or diluent therefor.
  • an agent e.g., verotoxin, the pentameric B subunit of verotoxin 1, verotoxin 2, or verotoxin 2c
  • the present invention provides selective, specific cancer treatments wherein the agent, e.g., verotoxin, the pentameric B subunit of verotoxin 1, verotoxin 2, or verotoxin 2c, selectively binds with Gb3 in Gb3-containing cells.
  • the agent e.g., verotoxin, the pentameric B subunit of verotoxin 1, verotoxin 2, or verotoxin 2c
  • the agent e.g., verotoxin
  • the pentameric B subunit of verotoxin 1, verotoxin 2, or verotoxin 2c selectively binds with Gb3 in Gb3-containing cells.
  • the treatment is of value against cutaneous T-cell lymphomas, particularly, Mycosis Fungoides, sezary syndrome, related cutaneous disease lymphomatoid papilosis, and post transplant lymphoprohferative disorder (PTLD), e.g. PTLD following a transplant, e.g., a renal, heart, liver, or lung transplant.
  • PTLD post transplant lymphoprohferative disorder
  • Mycosis fungoides lesions in humans have been cleared without any observed adverse systemic effects by the application of VTI (5ng in 2 ml. solution) by interdermal injection in patients.
  • the invention provides a method of treating mammalian neoplasia comprising treating said mammal with a non-lethal anti-neoplasia effective amount of an agent, e.g. a verotoxin, e.g., preferably verotoxin 1, the pentameric B subunit of verotoxin 1, verotoxin 2, or verotoxin 2c.
  • an agent e.g. a verotoxin, e.g., preferably verotoxin 1, the pentameric B subunit of verotoxin 1, verotoxin 2, or verotoxin 2c.
  • the agent may be Pag adhesin linked to a toxin (e.g.. ricin) or an antibody to Gb3 linked to a toxin (e.g., ricin).
  • the agent e.g., verotoxin or its B subunit
  • sub-cutaneous application is preferred.
  • Verotoxin I has been injected intramuscularly into a patient with advanced ovarian carcinoma. No adverse affects were monitored on lymphocyte or renal function and a serum tumour marker was found to continue to rise when the patient was treated with relatively high doses of Verotoxin 1. This tumour was refractory to all conventional cancer therapies. No effect was found on hemoglobin levels.
  • the agent e.g., verotoxin or its B subunit
  • a suitable vehicle in which the active agent, e.g., verotoxin or B subunit, ingredient is either dissolved or suspended in a liquid, such as serum to permit the verotoxin to be delivered for example, in one aspect from the bloodstream or in an alternative aspect sub-cutaneously to the neoplastic cells.
  • solutions are, typically, alcohol solutions, dimethyl sulfoxide solutions, or aqueous solutions containing, for example, polyethylene glycol containing, for example, polyethylene glycol 400, Cremophor-EL or Cyclodextrin.
  • Such vehicles are well-known in the art, and useful for the purpose of delivering a pharmaceutical to the site of action.
  • multi-drug resistant cell lines were found to be hypersensitive to Verotoxin 1.
  • multidrug resistant ovarian cancer cell lines SKVLB and SKOVLC were more sensitive to VT cytotoxicity than corresponding non-multidrug resistant ovarian cancer cell line SKOV3.
  • Such an observation indicates the possible beneficial effect for patients bearing the SKVLB cell line cancer than those with the SKOV3 cell line under VT treatment.
  • our observed binding of VTI to the lumen of blood vessels which vascularize the tumour mass, in addition to the tumour cells per se may result in an anti-angiogenic effect to augment the direct anti-neoplastic effect of verotoxin.
  • a series of human Gb containing astrocytoma cell lines were tested for sensitivity to VT.
  • verotoxin and its B subunit may be significantly enhanced by a prior treatment of the neoplastic cells with a sensitizer, such as sodium butyrate.
  • Figure 1 shows the selective neutralization of ACP cytotoxicity by anti VTI and or anti VTI B subur ⁇ t but not by anti VT2 antibodies as determined by cell density measurement after 48 hours;
  • Figure 2 shows the viability of selected ovarian and breast tumour cell lines to verotoxin concentration
  • Figure 3 represents VTI contained within ACP preparation binding to Gb ⁇ (and
  • Figure 4 represents VT thin layer chromatography overlay of ovarian tumour and ovary glycolipids
  • Figure 5 represents VT thin layer chromatography overlay of selected cell line glycolipids
  • Figure 6 represents in three graphs ovarian cell line sensitivity to VTI, VT2 and VT2c;
  • Figure 7 represents glioblastoma multiforme cell line sensitivity to VTI, VT2 and VT2c;
  • Figure 8 represents the distribution of labelled VTI B subunit (VTB- 131 I) administered IP (inter-peridinually) in a Gb3 tumour bearing nude mouse;
  • Figure 9 represents the results of a three-day treatment of several human astrocytoma cell lines with VTI ;
  • Figures 10A - 10G represents a graph of the anti-proliferative effects of VTI on human astrocytoma cells
  • Figures 11A and 1 IB provide a comparison of SF-539 and XF-498 sensitivity to VTI holotoxin
  • FIGS. 12A and 12B represent the detection of the VT-Receptor glycolipid, Gb3 in human astrocytoma cell lines;
  • Figure 13 shows the sensitivity of two astrocytoma cell lines to VTI after sensitizing culture; and Figure 14 shows the sensitivity to the B subunit of verotoxin VTI of the two cell lines used in tests shown in Figure 13.
  • Figures 15 a and b depict the FITC- VTB staining of LPD liver, showing the membrane staining of single cells dispersed throughout the tissue.
  • Figures 15 c and d show the FITC- VTB staining of the LPD adenoid.
  • Figure 15 e depicts background staining using FITC VT B of a normal liver.
  • Figure 15 f depicts a normal liver using double labels, anti-CD20 and FITC VTB, it shows coincident expression of the lymphoid antigen and VT receptor.
  • Figure 16 depicts a fixed liver section processed for EBV nucleotide sequences using in situ hybridization. EBV positive cells are stained.
  • Figure 17 depicts a gel of glycolipid extracts of EBV positive B cell lines, MB and TH9. The left panel of the figure shows orcinol detection and the right panel shows the VTI overlay which was used to detect the presence of Gb3.
  • Figure 18 depicts a graph of VTI cytotoxicity of MB and TH9 EBV transformed B cells.
  • the present invention pertains to methods and agents capable of inducing the death of lymphoma cells.
  • Lymphoma is an art recognized term and refers to a potentially malignant neoplasm of the lymph and reticuloendoleitiial tissues.
  • the term "lymphoma cell” is art recognized and includes the cells of the neoplasm as well as cells which have been exposed to the agent which caused the neoplasm or which could potentially cause a neoplasm.
  • Epstein-Barr virus which is a double stranded DNA virus of the herpesvirus family.
  • the virus is transmitted by saliva, infects nasopharygeal epitheal cells and B lymphocytes, and is ubiquitous in human populations worldwide. It infects human B cells by binding specifically to the type 2 complement receptor (CR2) followed by receptor mediated endocytosis.
  • CR2 type 2 complement receptor
  • Two types of cellular infections can occur.
  • a lytic infection viral DNA, RNA and protein synthesis begin, followed by assembly of viral particles and lysis of the host cell.
  • a latent non-lytic infection can occur, in which the viral DNA is incorporated into the host genome indefinitely.
  • Epstein-Barr nuclear antigens include at least four different nuclear proteins that are expressed early in lytic infections and may also be expressed by some latently infected cells.
  • EBNAs are well-characterized EBV antigens which have been shown to be targets for specific cytolytic T lymphocytes (CTLs).
  • CTLs cytolytic T lymphocytes
  • Other viral structural protein antigens are expressed within infected cells and on released viral particles during lytic infections, including viral capsid antigens (VCAs). Antibodies specific for VCAs are present in acutely infected, recovering and remotely infected individuals.
  • Epstein-Barr virus has profound effects on B lymphocyte growth characteristics in vitro.
  • the virus is a potent, T-cell independent polyclonal activator of B cell proliferation.
  • EBV can immortalize normal human B cells so that they will proliferate in culture indefinitely.
  • the resulting long term B lymphoblastoid cell lines are latently infected with the virus and may express EBNA proteins, but they do not have a malignant phenotype (Abbas, A.K. et al, Cellular and Molecular Immunology, (W.B. Saunders Company: Philadelphia, 1991) p.343).
  • EBV positive cells may also express Gb3.
  • B cell lymphomas e.g. Burkitt's lymphoma and PTLD
  • T-cell immunodeficient individuals including individuals with congenital immunodef ⁇ ciences, AIDS patients, and kidney, renal or heart allograft recipients receiving immunosuppressive drugs. These individuals have deficiencies in normal T cell function.
  • EBV infection proceeds unchecked in these individuals and EBV-induced polyclonal proliferation of B cells increases the chances of errors made by recombinases or isotype switching enzymes, resulting in a relatively high frequency of genetic translocations to Ig loci, potentially resulting in deregulation of genes and subsequent abnormal expression.
  • B cell lymphomas e.g. Burkitt's lymphoma and PTLD, are thought to be sequelae of unchecked EBV infections.
  • the present invention provides a method for inducing cell death, inhibiting protein synthesis, inducing apoptosis, in lymphoma cells, e.g., lymphoma cells of B cell origin, e.g., lymphoma cells of B cell origin which are EBV positive, and which bear Gb 3 receptors.
  • lymphoma cells e.g., lymphoma cells of B cell origin, e.g., lymphoma cells of B cell origin which are EBV positive, and which bear Gb 3 receptors.
  • Cells which express Gb 3 receptors are defined herein as "Gb 3 receptor positive cells.”
  • Cells which are of B cell origin are defined herein as cells which are descendent from B cells.
  • Cells which are EBV positive are defined herein as cells expressing EBNA.
  • lymphomas include Burkitt's lymphoma and PTLD (post-transplant lymphoproliferative disease) e.g., PTLD involving renal and/or liver transplantation.
  • PTLD includes disorders afflicting subjects who have undergone organ transplant surgery, e.g., renal transplantation, liver transplantation, and may be characterized by infiltrating lymphoma cells.
  • Another aspect of the invention pertains to treating a subject, e.g., a human, having a disorder characterized by (or associated with) infiltrating lymphoma cells, e.g., lymphoma cells of B cell origin, e.g., lymphoma cells of B cell origin which are EBV positive.
  • the human is infected with HIV.
  • these methods include the steps of administering an effective amount of an agent, e.g., a toxin of this invention, which is capable of inducing the cell death of the infiltrating lymphoma cells, e.g., by inhibiting the infiltrating lymphoma cell's protein synthesis, or by inducing apoptosis in the infiltrating lymphoma cell, such that treatment occurs.
  • agents e.g., a toxin of this invention
  • Non-limiting examples of disorders or diseases characterized by infiltrating lymphoma cells include lymphoma, e.g., Burkitt's lymphoma or PTLD, e.g., PTLD associated with renal, liver, heart, or lung transplant.
  • disorder includes a condition of a living organism or one of its parts which impairs normal or regular functioning, e.g., a disease, e.g., PTLD or Burkitt's lymphoma.
  • treating or “treatment” includes a reduction or alleviation of at least one adverse effect or symptom of a disorder or disease, e.g., a disorder or disease characterized by or associated with angiogenesis, a disorder characterized by infiltrating lymphoma cells.
  • the methods of the current invention also include administering an effective amount of an agent which binds Gb3 and induces cell death of infiltrating lymphoma cells, such that treatment occurs.
  • the agent binds via Gb3 to cells which are involved in causing the disorder, e.g., infiltrating lymphoma cells, e.g., lymphoma cells of B cell origin, e.g., lymphoma cells of B cell origin which are EBV positive, and induces cell death.
  • the agent may induce cell death of the cells causing the disorder through a variety of mechanisms.
  • the agent may, for example, bind the cell via Gb3, and become incorporated into the cell thus inducing cell death (e.g., by inhibition of protein synthesis, by induction of apoptosis).
  • VTs also known as SHIGA-like toxins, comprise a family known as VTI, VT2, VT2c, and VT2e of subunit toxins elaborated by some strains of E. coli.
  • Cell toxicity is mediated via the binding of the B subunit of the holotoxin to Gb3- VTs are described in U.S. Patent Application Serial Number 08/563,394, entitled "Verotoxin Pharmaceutical Compositions and Medical Treatments Therewith", filed November 28, 1995. The isolation and purification of VTs have been earlier described.
  • VTI can be prepared genetically from the high expression recombinant E.
  • VT2 can be obtained from R82 (Infect. Immun. 56:1926-1933 (1988)) and purified by protein purification procedures (FEMS MicrobioL Lett. 48:379- 383 (1987)).
  • VT2c can be obtained from clinical strain E32511 and purified by protein purification procedures (FEMS MicrobioL Lett. 51:211-216 (1988)).
  • VTI B subunit can be prepared according to Ramatour, et al. Biochem. J. 272:805-811 (1990).
  • the VTs consist of a 30kDa enzymatic subunit which is capable of inhibiting protein synthesis.
  • the A subunit is noncovalently associated with a pentameric 7kDa B subunit array which binds to Gb ⁇ .
  • VTI protein synthesis
  • receptor binding subunit alone, also induce morphological changes and DNA fragmentation characteristic of apoptosis in Gb3-positive cells.
  • VTI B subunit Cell binding of the VTI B subunit alone can induce apoptosis in B cells and Gb3 containing B cells are prone to apoptosis during B-cell differentiation.
  • Sensitivity to VTI is a function of cell cycle and cells at Gl/S boundary are particularly sensitive while stationary phase cells are refractory.
  • Gb3-bound VTI can follow a unique pathway of intracellular retrograde transport to the Golgi/ER and nuclear membrane. Gb3 binding is involved in ⁇ -interferon receptor function, and in CD 19 signal transduction in germinal center B cells to mediate homotypic adhesion and apoptosis.
  • agents capable of inducing cell death in infiltrating lymphoma cells include, among others, PagG adhesin (Kihlberg, et al. J. Am. Chem. Soc. 111:6364-6368 (1989) and antibodies to Gb3 or CD77 which can be linked to a toxin capable of inhibiting angiogenesis.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • a variety of monoclonal antibodies to Gb3 or CD77 are discussed in Oosterwijk, et al. (199V Int. J.
  • anti- Gb3 is commercially available (AN 1003566, Biodesign International, Kennebunkport, ME, USA).
  • Toxins which can be linked to these antibodies include, among others, VTs, and other immunotoxins known in the art, e.g., ricin.
  • the agents capable of inducing cell death in infiltrating lymphoma cells may be administered to the subject by methods well-known in the art, namely, intravenously, intra-arterially, topically, subcutaneously, by ingestion, intra-muscular injection, inhalation, and the like, as is appropriately suitable to the disease.
  • subcutaneous application is preferred.
  • the VT or its B subunit is typically administered in a suitable vehicle in which the active VT or B subunit ingredient is either dissolved or suspended in a liquid, such as serum to permit the VT to be delivered, for example, in one aspect from the bloodstream or in the alternative aspect subcutaneously to the cells.
  • a suitable vehicle in which the active VT or B subunit ingredient is either dissolved or suspended in a liquid, such as serum to permit the VT to be delivered, for example, in one aspect from the bloodstream or in the alternative aspect subcutaneously to the cells.
  • solutions are typically alcohol solutions, dimethyl sulfoxide solutions, or aqueous solutions containing, for example, polyethylene glycol containing, for example, polyethylene glycol 400, Cremophor-EL, or Cyclodextrin.
  • Such vehicles are well- known in the art and useful for the purpose of delivering a pharmaceutical to the site of action.
  • the invention further provides a method for monitoring a previously diagnosed subject with a disorder characterized by abnormal cell proliferation, e.g., infiltrating lymphoma cells, e.g., lymphoma, e.g., Burkitt's lymphoma, PTLD, e.g., PTLD associated with liver transplantation, PTLD associated with renal transplantation.
  • a disorder characterized by abnormal cell proliferation e.g., infiltrating lymphoma cells, e.g., lymphoma, e.g., Burkitt's lymphoma
  • PTLD e.g., PTLD associated with liver transplantation, PTLD associated with renal transplantation.
  • the method involves contacting a biological sample, e.g., a tissue sample, from the subject with an agent capable of detecting Gb3, e.g., fluorescently labeled VTI, determining the amount of Gb3 expressed in the sample, comparing the amount of Gb3 expressed in the sample to a the amount of Gb3 expressed in a sample previously obtained from the same subject to determine the progression of the disease, e.g., measuring the increase or decrease in levels of Gb3 over time in a subject.
  • a biological sample e.g., a tissue sample
  • an agent capable of detecting Gb3, e.g., fluorescently labeled VTI e.g., fluorescently labeled VTI
  • Verotoxin 1 was prepared genetically from the high expression recombinant E. coli pJB28, J. Bacteriol 166:37S and 169:4313. The generally protein purification procedure described in FEMS MicrobioL Lett_. 41 :63, was followed.
  • 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 clinical strain E32511 and purified according to FEMS MicrobioL Lett. 51 :211-216 (1988).
  • VTI B subunit was prepared according to Ramotar (24). VTs were aliquoted in PBS and stored at 70°C. The appropriate dilution for the treatment of astrocytoma cell lines was prepared freshly in media and added to the cells.
  • Pellet Preparation may be conducted as follows:
  • the supernatants should be quite yellow and the bacterial pellet should become more fine and diffuse with each extraction step.
  • step 3 Add 50 ⁇ g/ml carbenicillin to each of the 5L jugs (from step 1). Seed each jug with 2 ml of seed (step 2) and incubate for 24 hours at 37°C with shaking of approximately 120 rpm. 4. Heat incubator to 45°C and incubate for 30 minutes.
  • Cibachron blue (CB) 11. Equilibrate cibachron blue (2 cm diameter, 82 ml volume, PIERCE) with 100 ml of lOmM sodium phosphate buffer (wash buffer).
  • Histidine buffer (0.025M) 2.0g/500 ml H O pH 6.2 with HC1
  • next few steps should preferably be done at 4°C: 8. Add crystalline ammonium sulphate very slowly, with stirring to pooled supernatants to 30%> saturation. 9. Let stir for 20 min and then remove precipitate by centrifugation (lOOOOg for 10 min).
  • VTI verotoxin 1 doses
  • VTI was purified from the E. coli strain as previously described which overexpresses the cloned toxin genes.
  • the purified toxin was free of endotoxin contamination.
  • the protein concentration of this batch of verotoxin was determined and the toxin aliquoted and stored at -70°C.
  • VTI was diluted into injection grade sterile saline containing 0.2%> v/v of the patient's own serum. 210 ⁇ l of sterile patient serum was added to 10 ml of sterile injection saline and 93.9 ml of purified VTI (6.7 g/ml) added to give a final toxin concentration of 62.5 ng/ml or 12.5 ng per 0.2 ml. dose.
  • the final toxin preparation was sterile-filtered using a 0.2 mm syringe filter and dispensed in 2 ml aliquots into 10 ml vials. One working vial may be stored at 4°C and the remaining vials frozen until needed.
  • FITC labeling of VTI FITC was added directly to VTI (in a 1 :1, w/w ratio) in 0.5M Na2CO3/NaHCO3 conjugated buffer pH 9.5 and the mixture gently rotated for 1.2 hours at room temperature. Free FITC was removed by a Centricon filter.
  • Fluorescent Staining of Cells 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 lh at room temperature. The cells were washed 5 times with PBS, mounted with DABCO and observed under a Polyvar fluorescent microscope.
  • SKOV3 drug sensitive human ovarian cell line
  • SKOVLC SKOV3, resistant to Vincristine
  • SKOVLB SKOV3, resistant to Vinblastine
  • SKOVLC & SKOVLB drug resistant cell lines
  • This material may be made by the following procedure. 1. Dissolve 20 mg of iodogen in 2.0 ml of chloroform (10 mg/ml). Make a 1:10 dilution by adding 0.25 ml of the 10 mg/ml solution to 2.25 ml chloroform (1 mg/ml). 2. Dispense 20 ⁇ l of this dilute solution into a clean, dry sterilized glass tub. Add
  • HPLC 11. Conduct sterility and pyrogen tests.
  • SF-126, SF-188, SF- 539, U 87-MG, U 251-MG, and XF-498) were selected for study.
  • SF-126, SF-188, and SF-539 were kindly provided by Dr. Mark Rosenblum, Henry Ford Hospital.
  • U 87-MG and U 251-MG were kindly provided by Dr. Jan Ponten, University of Uppsala, Sweden; and
  • XF-498 was a gift of Dolores Dougherty, University of California San Francisco.
  • Astrocytoma cells were cultured in alpha-MEM, nonessential amino acids, glutamine, gentamycin, and 10% heat-inactivated fetal bovine serum.
  • the cultures were incubated at 37°C and equilibrated in 5%> CO 2 and air.
  • Cells were harvested with 0.25%> trypsin (Gibco, Santa Clara, CA) in Ca ++ ⁇ and Mg ++ ⁇ free Hank's balanced salt solution and were subcultured weekly.
  • Human capillary endothelial cells were isolated after the method of Costello (25) and were derived from samples of normal human brain taken from patients undergoing neurosurgical procedures for epilepsy, trauma, and resection of arteriovenous malformations.
  • the capillary cells were grown as described above in media supplemented with 15 ⁇ g/ml endothelial growth factor (Sigma, St. Louis) (26).
  • the endothelial origin of the cells in culture was established by immunocytochemical analysis using anti-human factor- VHI-related antigen antisera (Dako, Santa Barbara, CA) as described previously (27).
  • VTI VTI
  • VT2c the B subunit of VTI, VT2, and VT2c was added alone to the astrocytoma cells at same concentrations listed above.
  • a single dose of VTI, VT2, and VT2c was added to confluent astrocytoma cells in microtiter wells.
  • Cell survival at 72 hours was monitored by staining with 0.1%) crystal violet, and measuring the optical density at 590 nm using a Dynatek microtiter plate reader.
  • TBS Tris Buffer Salin
  • SF-539 cells grown on the cover slips overnight were incubated at 37°C with VTI B-subunit (50 ⁇ g/ml) for 1.5 hrs or 10 hrs and fixed (with 1%> paraformaldehyde for 3 minutes), permeabilized with 0.1 %> Triton X in 100 mm PBS for 5 min, and stained with 5 ⁇ g/ml propidium iodide (Sigma). After extensive wash with 50 mm PBS, the fixed cells were mounted with DABCO (1 ,4-Diazabicyclo-Octane, sigma), and nuclear staining observed under incident uv illumination.
  • Apoptosis of astrocytoma cells incubated with 10 ng/ml of VTI for 24-36 hrs in the presence of 10%> bovine fetal serum was analyzed on an Epics Profile Analyzer (Coulter Electronics, Pathology, University of Toronto). After treatment, cells were trypsinized and the 200Xg centrifuged cell pellet was suspended in 1ml of hypotonic fluorochrome solution of 50 ⁇ g/ml propidium iodide (Sigma) and stained for 30 min at 4 C. To remove RNA prior to staining, cells were treated with 100 ⁇ l of 200 ⁇ g/ml DNase-free RNase A at 37 C for 30 min. Cell cycle distribution was determined using manual gating.
  • Flow cytometric quantitation of apoptotic cells within the propidium iodide-stained population was performed. Debris and dead cells were excluded on the basis of their forward and side light-scattering properties. Astrocytoma cells grown simultaneously in the absence of VTI served as controls.
  • Fig. 1 relates to the neutralization of ACP cytotoxicity by anti-VT.
  • KHT cell monolayers were incubated with 35 ng/ml ACP from E.coli HSCi Q, or 10 pg/ml VTI, VT2 or VT2c in the rresence of monoclonal anti-VTl(PHl), monoclonal anti VT2 or polyclonal rabbit anti VTI B subunit.
  • the cells were incubated for 72 hours at 37°C and viable adherent cells were detected by fixation and staining with crystal violet. Cytotoxity of VTI and ACP was completely neutralized in the presence of anti VTI or anti VT1B subunit (anti-VT2 serum had no effect).
  • VTI cytotoxic assay
  • vero cells cells from Africa green monkey kidney that are very sensitive to verotoxin
  • the ACP preparation contained 0.05%> VTI.
  • This concentration of purified VTI was as effective as ACP in inhibiting the growth of various tumour cell lines in vitro (Fig. 2).
  • VTI mimics the anti-neoplastic effect of ACP in vitro.
  • VTI was tested for the ability to inhibit the metastases of KHT fibrosarcoma cells in the mouse model as had been previously reported for ACP.
  • the equivalent dose of VTI was as effective as ACP, reducing the number of lung metastases to background levels, following a primary subcutaneous tumour inoculum (Table 1).
  • Table 1 Response of KHT cells, growing as lung modules, to treatment with VTI or ACP.
  • mice were treated with VT-1 or ACP(l-p) 1 day after cell injection (1000 KHT cells/mouse i-v).
  • mice were treated with VT- 1 or ACP(i-p) 1 day after cell injection (1000 KHT cells/mouse).
  • VT-1 B (lOug/mouse +/- Freund's Adjuvant (FA) given (i-p) 4 weeks and 2 weeks before cell injection.
  • FA adjuvant
  • ACP was tested for glycolipid binding by TLC overlay using monoclonal anti-
  • VT1 or anti-VT2c show extensive binding of a component within the ACP preparation to globotriaosylceramide and galabiosyl ceramide (Fig. 3). This binding specificity is identical to that reported for purified VT1(8). No binding component reactive with anti-VT2 was detected. In Fig. 3 anti VT antibodies were used to detect binding to the immobilized glycolipids. Arrows indicate position of standard (from the
  • VTI demonstrated in vitro activity against a variety of ovarian carcinoma cell lines.
  • a large number of primary human ovarian tumour biopsies were screened for the expression of Gb3 via TLC overlay using purified VTI . It was found that Gb3 was barely detectable in normal ovary tissue, whereas in all cases a significant increase in expression of Gb3 was observed in the ovarian carcinoma.
  • elevated levels of Gb3 were found in acites tumour and in tumours that had metastized to the omentum, (Fig. 4) which defines lane 1, ovarian omentum metastasis; lane 2: tumour biopsy; lane 3, tumour biopsy; lanes 3-6, normal ovary; lane 7, human kidney Gb3 standard.
  • Fig 2 shows human ovarian tumour cell lines sensitive to ACP tested for VT sensitivity.
  • Human ovarian and breast tumour derived cell lines were tested for VTI sensitivity wherein ovarian I, 2, 3, 4 and 5 are denoted ⁇ , +, x, N and o respectively, and breast- SKBR3 , 468 ⁇ , 453 >, 231 A .
  • the cell lines 1-ovarian, 453 and SKBR3, previously shown to be resistant to ACP, were also resistant to up to 20 ng/ml VTI.
  • the 1, 2, 3 and 4 cells were from ovarian cancer patients; the 453 cells were from a breast cancer patient; 231 and SKBR3 are breast adenocarcinoma cell lines, and 5, SKOV3 and SKOVLB are adenomacarcinous ovarian cancer cell lines.
  • the lines 1, 453 and SKBR3, resistant to ACP, were co-resistant to VTI.
  • Fig. 5 shows VT sensitive and resistant cell lines tested for the presence of Gb3 by VT binding in tic overlay. Glycolipid from an equal number of cells were extracted and separated by tic prior to toxin binding. In Fig.
  • SKBR3, 468, 231 and 453 are derived from breast tumours. Only 231 is sensitive to VTI.
  • SKOVLB is a multiple drug resistant ovarian tumour cell line derived from SKOV3.
  • Ovarian tumour cells were highly sensitive to VT (Fig. 3) and contained elevated levels of the VT receptor, Gb3 (Fig. 4). Breast cancer cells were for the most part, toxin resistant (Fig. 3) and receptor negative (Fig. 5). Low levels of Gb3 were detected in normal ovarian tissue but these were markedly elevated for the ovarian tumour tissue samples.
  • Fig. 6 human derived ovarian tumour cell lines were tested for VTI, VT2, and VT2c sensitivity. The cells were grown to confluence in 48- well plates, then incubated for 48 hrs. in the presence of increasing doses of VTs.
  • SKOVLB the multiple drug resistant variant of SKOV3 ovarian line, showed the most sensitivity to VT's with SKOVLC being the next most sensitive to the VT's.
  • Fig. 7 shows the effect after 48 hrs. of treatment of the brain tumour SF-539 cell line derived from a recurrent, right temporoparictal glioblastoma multiform with VTI, VT2, and VT2c. This cell line, as others, was highly sensitive to VT's.
  • Fig. 8 provides the results from imaging a nude mouse with 131I-VT1B (CPM distribution in different organs).
  • VTlB-l ⁇ l cpm distribution in nude mouse with implanted ovarian tumour showed that a considerable amount of radiolabled VTI B had been concentrated ir the ovarian tumour. Only a trace amount of VTI B was located in the brain where the potential VTI side effect was considered. Since the lung in human adult is not the site of concern for VTI toxicity this does not present a problem for treatment of human adult with ovarian tumour.
  • the CPM in kidney includes the excreted radiolabelled VTI B subunit. Accordingly, based on this test, imaging with labelled VTI B subunit can be a very useful method for screening the susceptible patient to VTI cytotoxiciry.
  • Fig. 9 shows the sensitivity of a variety of human astrocytoma cell lines to VTI. All these cells contain Gb3 but show variable sensitivity to VTI induced cytotoxicity. This suggests that certain astrocytomas will be more susceptible to verotoxin than other astrocytomas. This is important since astrocytomas are very refractory to treatment at the present time and cell sensitivity in vitro to concentrations as low as 5ng per/ml is rare.
  • Figs. 10A - 10G show the anti -pro liferative effects of VTI on human astrocytoma cells. All astrocytoma cell lines showed at least some inhibition of growth following VTI treatment. The most sensitive cell line was SF-539 (Fig. 10A), and the least sensitive was SF-126 (Fig. 10F). Human cerebral capillary endothelial cells were largely resistant to the growth-inhibitory effects of VTI except at high doses (100 ng/ml) (Figs. 10G). U-251 MG and U-87 MG were sensitive to VTI (Figs. 10B and 10C), whereas XF 498 and SF-188 were somewhat less sensitive to VTI (Figs.
  • FIGs. 11 A and 1 IB provide a comparison of SF-539 and XF-498 sensitivity to VTI holotoxin (upper panel) and B-subunit (lower panel). Forty-eight hrs following the treatment of SF-539 and XF-498 cells in monolayer culture, the percent cell survival was calculated. VTI was cytotoxic to SF-539 astrocytoma cells at doses as low as 0.01 ng/ml (upper panel). XF-498 cells were resistant to VTI holotoxin. When the VTI B- subunit was employed, only SF-539 was sensitive to this toxin (lower panel).
  • Figs. 12 A and 12B represent the detection of the VT-Receptor glycolipid, Gb3 in human astrocytoma cell lines.
  • Astrocytoma neutral glycolipids were prepared from 1 x 106 cells and separated by TLC.
  • Glycolipids were visualized by orcinol and bands representing Gb3 are seen in all astrocytoma cell lines.
  • B The same blot was assayed by VTI overlay. In this study, VTI binds to Gb3 extracted from astrocytoma cells as shown (arrow). SF-539 astrocytoma cells showed maximal binding of Gb3/YT1.
  • Fig. 13 compares the sensitivity of two astrocytoma cell lines SF539 (sensitive),
  • XF498 (less sensitive) and XF 498, following three days of culture of XF498 in sodium butyrate. It is seen that the sensitivity of XF498 is increased to that or even more than that of the most sensitive cell line SF539.
  • Fig. 14 shows the same effect for the B subunit of verotoxin 1.
  • Figs. 10A - 10G show that all astrocytoma cell lines studied were sensitive to VTI.
  • the most sensitive cell line in terms of growth inhibition was SF-539 (Fig. 10A) and the least sensitive was SF-188 (Fig. 10E).
  • SF-539 When treated with other members of the VT family including VT2, and VT2c, SF539 was growth inhibited.
  • VT-1 was the most potent species (Fig. 11).
  • human cerebral endothelial cells were largely resistant to the growth inhibitory and cytotoxic effects of VT-1 (Fig. 10G). Only when doses as high as 100 ng/ml were used were endothelial cells inhibited.
  • FIG. 11 A and Fig. 11 B A comparison between the sensitivity of SF 539 and XF498 for VTI and VTI B subunit is shown in Fig. 11 A and Fig. 11 B.
  • XF498 cells were considerably less sensitive to the B subunit than to the VT-1 holotoxin.
  • SF 539 astrocytoma cells were significantly more sensitive to the B subunit alone than were XF 498 astrocytoma cells, since 50%> cell death was observed in the presence of 50 ng/ml.
  • the glycolipid profile of the 6 human astrocytoma cell lines analyzed for Gb3 content as detected with orcinol is shown in Fig. 12 A. All of the astrocytoma cell lines
  • SF-539 cells expressed the highest levels of Gb3 with maximal binding to VTI.
  • astrocytoma cells were analysed by flow cytometry.
  • apoptotic cells show less propidium iodide fluorescence than viable cells and can be quantified as the "subdiploid" population or pre-Gl position in cell cycle (Fig. 13, arrow head). Presence of cells with fractional DNA content, typical of apoptosis was more marked in SF-539 than XF-498 cells.
  • a cell cycle analysis of the non- apoptotic cell population revealed marked differences in the proportion of cells in the respective phases of the cell cycle.
  • VTI- sensitive SF- 539 cells a pronounced loss of S phase cells from 33 to 15 and 10%
  • VTI sensitive XF-498 cells the loss of S phase cells observed was only 75 to 69 and 65%. Changes in the proportion of cells in G2-M phase were also seen (Fig. 13).
  • VTI treated cells displayed characteristic features of apoptosis, such as marked reduction in diameter, condensed chromatin. Nuclear segmentation and subnuclear bodies were prominent in cells treated with, VTI B-subunit for 1.5 or 10 hours.
  • VTl-treated astrocytoma cells By electron microscopy, VTl-treated astrocytoma cells (SF-539, XF-498 demonstrated characteristic features of apoptosis such as, blebbing of the cytoplasmic membrane, fragmentation of heterochromatin, condensation of the nucleolar membrane, loss of cell junctions and microvilli, nuclear disintegration, and apoptotic bodies.
  • the results herein show that VTI inhibits the growth of a series of human astrocytoma cell lines. All cell lines showed significant sensitivity to VTI, contained the Gb3 receptor for VT, and demonstrated ultrastructural features indicative of apoptosis following VT treatment.
  • VTs provide the basis of new agents active against human astrocytoma cells.
  • results show that the most toxin sensitive astrocytoma cell line, SF-539, is also highly sensitive to B subunit induced apoptosis.
  • apoptosis Definitive morphological evidence of apoptosis (nuclear shrinkage and choromatine condensation) were observed within 1.5 hrs of toxin or B subunit administration to astrocytoma cells. This is considerably more rapid than has previously been described for induced apoptosis by anticancer drugs. Accumulation of VTI -treated astrocytoma cells in pre-Gl position in cell cycle (Fig. 13) is strong evidence for apoptosis. Additional evidence in support of VTI causing apoptosis in sensitive astrocytoma cells include nuclear staining with propidium iodide and ultrastructural alterations indicative of apoptosis.
  • verotoxins are an effective agent for binding specifically to cells which upregulate Gb 3 .
  • Case 1 A previously healthy 9 year old boy presented acute fulminant hepatitis on November 1990. Although the etiology was believed to be viral, all serology screening tests were negative including HAV, BHV, CMV and EBV. The diagnosis given was non A non B hepatitis with histologic findings on liver biopsy of syncytial giant cell hepatitis. Despite full supportive conservative treatment, including a trial of prostaglandin E, the patients' condition deteriorated, from acute hepatic failure, with impaired gluconeogenesis and abnormal coagulation functions, urea cycle dysfunction, and encephalopathy. One month after his presentation the patient received a reduced size liver transplant. Induction immuno suppression included methylprednisolone, cyclosporine and azathioprine.
  • the patient was stable until day 10 post-transplant when he developed an acute, grade III rejection, which was treated with a ten day course of OKT3. Concomitantly, the patient developed Staphylococcus aureus sepsis.
  • Antibiotic treatment included vancomycin initially, followed by a combination of cloxacillin, ampicillin and gentamicin. Ganciclovir treatment was initiated because of an increase in the Anti- EBVCA titer.
  • liver function and the patient's general condition continued to deteriorate.
  • a liver biopsy revealed changes suggestive of cholangitis secondary to biliary obstruction.
  • a second 14 day course of OKT3 was administrated.
  • the patient's course was complicated by a colon laceration and hemothorax from a percutaneous needle liver biopsy, causing a serious bleeding event requiring a number of surgical interventions for hemostasis and hematoma evacuation.
  • a laceration of the left femoral artery following a dialysis line insertion was also surgically repaired.
  • the patient's course was complicated by acute renal failure presumed to have been caused by both acute tubular necrosis related to sepsis with hypotension and drug toxicity.
  • the patient became oligo-anuric leading to the institution of continuous arterio-venous hemofiltration followed by hemodialysis.
  • liver function continued to deteriorate necessitating a second liver transplant, there was no indication of a malignant process in all imaging studies and histopathologic investigations.
  • a second liver transplant was attempted three months after the first one, during which the patient died from uncontrollable bleeding.
  • a subsequent histopathologic examination of the first graft revealed a malignant lymphoma infiltrating the bile duct and the peripheral tissues with patchy parenchymal involvement.
  • Azathioprene treatment was stopped and treatment with gancyclovir and alpha-interferon was started for a period of six months. Prednisone and cyclosporine were continued according to the standard liver transplant protocol. No evidence of tumor spread was found on an extensive imaging work-up.
  • liver function remained stable apart from a few episodes of mild rejection which responded well to an increased dose of prednisone.
  • Case 3 This girl was born with right sided reflux nephropathy and a left non- functioning dysplastic kidney which was surgically removed in infancy. In addition the child had multiple congenital malformations including: anal stenosis, spinal bifida, cicornuate uterus, Wolff-Parkinson- White syndrome, mild sensorineural hearing loss, fused left radius and ulna, hypoplastic femoral condyles and mild scoliosis; mental development was normal. In infancy she had a partial bowel resection due to volvulus and throughout childhood was treated with bronchodilators for moderate asthma.
  • Induction immunosuppression for her transplant consisted of prednisone, azathioprine and a 7-day course of OKT3. Cyclosporine was started on day 3 post- transplant. Prior to transplant both recipient and donor were CMV positive, but only the donor was EBV positive. The post-transplant course was complicated by one episode of rejection which occurred 3 months after transplantation, which was successfully treated with an increased dose of methylprednisolone that was tapered down over a period of two weeks, maintenance azathioprine was replaced by mycophenolate mofetil following this rejection episode.
  • the patient was discharged after more than 1 month hospitalization but unfortunately presented a week after her discharge with similar symptoms of sinusitis and trachiitis deteriorating to airway obstruction requiring ventilation.
  • a biopsy from the nasal mucosa revealed infiltration of monoclonal B cells and was diffusely positive for EBV by in situ hybridization.
  • cyclosporine was stopped, prednisone continued to be gradually decreased to 10 mg/day and treatment with gancyclovir and CMV hyperimmune globulin was continued.
  • the patient developed diffuse swelling of her parotid glands which were biopsied and revealed EBV related large cell lymphoma. Aggressive chemotherapy was ineffective and the patient died.
  • FITC/VTI B Subunit staining of LPD liver section Serial 5 ⁇ M cryosections of samples were thawed, dried, blocked with BSA and stained with FITC- VTI B in PBS (0.5 ⁇ g/ml) containing 0/1 % BSA for 1 hr at room temperature. Sections were extensively washed with PBS, mounted without media with antifading agent DABCO and observed under incident UV illumination.
  • FITC- VTB staining of LPD livers from cases 1 and 2 ( Figure 15 a and b, respectively), and the adenoid from case 3 ( Figures 15 c and d) show the membrane staining of single cells dispersed throughout the tissue.
  • Background staining for FITC VT B was seen for a normal liver section (case 1, Figure 15 e).
  • Double labeling using anti-CD20 and FITC VTB showed coincident expression of the lymphoid antigen and VT receptor ( Figure 15 f).
  • Figure 16 shows a fixed liver section from case 1 which was processed for EBV nucleotide sequences using in situ hybridization techniques. The EBV positive cells are stained.
  • the EBV a known herpes virus, has been associated with several human cancers including nasopharyngeal carcinoma, BL, and a B cell lymphoma seen in immunodeficient hosts. All humans carry some EBV infected lymphocytes
  • lymphomas By giving immunosuppressive agents following organ transplantation, immunosurveillance is compromised and the EBV positive population is allowed to expand unchecked, increasing the likelihood of lymphoma development and PTLD.
  • the main histologic types of lymphomas seen include polyclonal B cell proliferation and a malignant monoclonal lymphoma.
  • Daudi Burkitt B cell lymphoma cell line and EBV induced B lymphoblasts express Gb3 receptors and are highly sensitive to VT cytotoxicity in vitro [Cohen, A. et al. J. Biol. Chem. (1987) 262:17088-17099].
  • Frozen tissue of the three reported cases above of PTLD were analyzed. At present, only single core biopsies are usually taken and the tissue is rarely frozen, therefore, access to frozen tissue is not readily available.. All three of the cases showed cells that are EBV induced in that they had markers for EBV. Additionally these same cells showed an up-regulation of the Gb3 receptor. None of the other tissue surrounding these cells whether from liver, adenoids or parotid gland showed Gb3 on their surface.
  • Immunodeficient mice in particular strains with severe combined immunodeficiency (SCID) accept human lymphoid xenografts and allow their endogenous expansion over periods of several months. If these grafts are derived from EBV carriers, then eventually PTLD will develop as surveillant T cells are slowly lost from the graft. This provides an excellent model for the natural course of PTLD.
  • SCID severe combined immunodeficiency
  • PTLD development requires several months in xenografted SCID mice and engraftment is successful in 705 of cases.
  • To examine the behavior of these lymphomas in an accelerated fashion requires engraftment of already (in vitro) EBV transformed B lineage cells. This is a valid model for PTLD, as only the long phase for escape from EBV immunosurveillance is bypassed. It has been proposed to employ this accelerated model for the study of VTI in vivo effectiveness.
  • the VT receptor species identified are, predominantly, the more slowly migrating Gb 3 isoforms which had been previously correlated in other cell lines with high sensitivity to VT in vitro and in vivo and with MDR expression.
  • both EBV positive B cell lines are highly susceptible to VTI (CD50 ⁇ 5pg/ml).

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Abstract

La présente invention concerne des méthodes de traitement du lymphome par l'administration d'un agent fixant le Gb3. Dans un mode de réalisation le lymphome peut être une affection lymphoproliférative après greffe.
PCT/CA2000/000371 1999-04-09 2000-04-07 Methodes de traitement de lymphomes par la verotoxine WO2000061183A2 (fr)

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WO1995022349A1 (fr) * 1994-02-22 1995-08-24 Geva, Ruth Compositions pharmaceutiques de verotoxines et traitements medicaux utilisant ces compositions
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LACASSE E C ET AL: "SHIGA-LIKE TOXIN PURGES HUMAN LYMPHOMA FROM BONE MARROW OF SEVERE COMBINED IMMUNODEFICIENT MICE" BLOOD,US,W.B. SAUNDERS, PHILADELPHIA, VA, vol. 88, no. 5, 1 September 1996 (1996-09-01), pages 1561-1567, XP000673518 ISSN: 0006-4971 *
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Cited By (5)

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Publication number Priority date Publication date Assignee Title
US7655412B2 (en) * 2001-11-30 2010-02-02 National Research Council Of Canada Self-assembly molecules
WO2004016148A3 (fr) * 2002-08-02 2004-10-28 Inst Curie Sous-unite b de toxine shiga, en tant que vecteur pour le diagnostic de tumeurs et l'administration de medicaments a des tumeurs d'expression gb3
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US7981400B2 (en) 2002-08-02 2011-07-19 Institut Curie Shiga toxin B-subunit as a vector for tumor diagnosis and drug delivery to GB3 expressing tumors
US8313731B2 (en) 2002-08-02 2012-11-20 Institut Curie Shiga toxin B-subunit as a vector for tumor diagnosis and drug delivery to GB3 expressing tumors

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