Note: Descriptions are shown in the official language in which they were submitted.
216371 6
VEROTOXIN PHARMACEUTICAL COMPOSITIONS AND
MEDICAL TREATMENTS THEREWITH
Field of the Invention
This invention relates to verotoxin pharmaceutical compositions and to
methods of treating mammalian neoplasia, particularly, brain, ovarian and skin
cancers,
therewith.
Backiuound to the Invention
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 o) 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).
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 a-1-4 gal residue
for binding. In
addition, VT2e, the pig edema disease toxin, recognizes globotetraosylceramide
(Gb4)
216371s'
2
containing an additional 13 1-3 linked galNac residue. 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 - an N- glycanase which removes a specific adenine base in the
28S RNA of the
60S RNA ribosomal subunit. However, the specific cytotoxicity and specific
activity is a
function of the B subunit. In an in vitro translation system, the verotoxin A
subunit is the most
potent inhibitor of protein synthesis yet described, being effective at a
concentration of about 8
pM. In the rabbit model of verocytotoxemia, 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 exquisitely sensitive to verotoxin in vitro and express
a correspondingly
high level of Gb3. However, 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. HUVEC 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 Gb3 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). We therefore suspect that 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 a TNF. Thus under these conditions,
the majority of
individuals (excepting the very young) would not be liable to VT induced renal
pathology
following systemic verotoxemia.
It has also been shown that the verotoxin targets a sub-population of human B
cells in vitro (10). These Gb3 containing B cells are found within the
germinal centres of lymph
nodes (11). It has been proposed that Gb3 may be involved in a germinal centre
homing by
3 2163716
CD19 positive B cells (12) and that Gb3 may be involved in the mechanisms of
antigen
presentation (13).
Elevated levels of Gb3 have been associated with several other human tumours
(14-16), but ovarian tumours have not been previously investigated. 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 VTl 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 a2-
interferon
receptor and the B cell marker CD19 suggests that expression of Gb3 is
involved in the
mechanism of a2-interferon and CD19 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. Despite the advent of promising adjuvant therapies and drugs
which have
impacted positively on patient survival in other tumor types in recent times,
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.
In addition to the cytotoxic effects of VTs on a wide range of cells by the A
subunit inhibition of protein synthesis, recent evidence suggests that VT1,
and the receptor
binding B subunit alone, also induce morphological changes and DNA
fragmentation
characteristic of apoptosis in Gb3-positive cells (22, 23).
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2163 716
4
Reference List
The present specification refers to the following publications.
1. Farkas-Himsley, H. and R. Cheung. Bacterial Proteinaceous Products
(bacteriocins as
cytotoxic agents of neoplasia). Cancer Res. 36:3561-3567, (1976).
2. Hill, R.P. 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, M.A. Infection by Verocytotoxin-producing Escherichia coli. Clin.
Microbiol.
Rev. 2:15-38 (1989).
4. Karmali, M.A., M. Petric, C. Lim, P.C. Fleming, G.S. 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, L.W., R.S. Remis, S.D. Helgerson, H.B. McGee, J.G. Wells, B.R.
Davis, R.J_
Hebert, E.S. Olcott, L.M. Johnson, N.T. Hargrett, P.A. Blake and M.C. Cohen.
Haemorrhagic colitis associated with a rare Escherichia coli serotype. N.
Engl. J. Med.
308:681 (1983).
6. Lingwood, C.A., Advances in Lipid Research. R. Bell, Y.A. Hannun and A.M.
Jr.
Academic Press. 25:189-211 (1993).
7. van de Kar, N.C.A.J., L.A.H. Monnens, M. Karmali and V.W.M. van Hinsbergh.
Tumour necrosis factor and interleukin-1 induce 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, C.A. Verotoxin-binding in human renal sections. NeQhron. 66:21-28
(1994).
10. Cohen, A., V. Madrid-Marina, Z. Estrov, M. Freedman, C.A. Lingwood and
H_M.
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.
2163716
Irmnunol. 2:1-8 (1990).
11. Gregory, C.D., T. Turz, C.F. Edwards, C. Tetaud, M. Talbot, B. Caillou,
A.B.
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, M.D. and C.A. Lingwood, CD19 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 E. coli
pathogenesis. J.
Exp. Med. 180: 191-201, (1994).
13. Maloney, M. and C. Lingwood. Interaction of verotoxins with
glycosphingolipids.
TIGG. 5:23-31 (1993).
14. Li, S.C., S.K. Kundu, R. Degasperi and Y.T. Li. Accumulation of
globotriaosylceramide
in a case of leiomyosarcoma. Biochem. J. 240:925-927 (1986).
15. 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
S.I. Hakomori. Changes in glycolipid expression in human testicular tumours.
Int. J.
Cancer. 45:1040-1044, (1990).
17. Naiki, M. and D.M. Marcus. Human erythrocyte P and Pk 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 defmed 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
S.I.
Hakomori. Tissue distribution of the Pk antigen as determined by a monoclonal
antibody. J. Immunogenet. 12:213 (1985).
20. Pudymaitis, A. and C.A. Lingwood. Susceptibility to verotoxin as a
function of the cell
2163716
cycle. J. Cell Physiol. 150:632-639 (1992).
21. 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. Mangeney, M., Lingwood, C.A., Caillou, B., Taga, S., Tursz, T. and Wiels,
J. Apoptosis
induced in Burkitt's lymphoma cells via Gb3/CD77, a glycolipid antigen. Cancer
Res.
53: 5314-5319, 1993.
23. Sandvig, K. and van Deurs, B. Toxin-Induced Cell Lysis: Protection by 3-
Methyladenine and Cycloheximide. Exp Cell Res. 200: 253-262, 1992.
24. Ramotar, K., Boyd, B., Tyrrell, G., Gariepy, J., Lingwood, C.A. and
Brunton, J.
characterization of Shiga-like toxin I B subunit purified from overproducing
clones of
the SLT-1 B cistron. Biochem. J. 272: 805-811, 1990.
25. Costello, R. and Delmaestro, R., Human cerebral endothelium; Isolation and
characterization of cell drived from microvessels of non-neoplastic and
malignant glial
tissue. J. Neuro-oncol. 8:231-243, 1990.
26. Pintus, C., Ransom, J. and Evans, C. Endothelial cell growth supplement: a
cell cloning
factor that promotes the growth of monoclonal antibody producing hybridoma
cells. J.
Immunological Methods. 61:195-200, 1983.
27. Rutka J.T., Kleppe-Hoifodt H., Emma D.A., Giblin J.R., Dougherty D.V.,
McCulloch
J.R., DeArmond S.J. and Rosenblum M.L., Characterization of normal human brain
cultures: Evidence for the outgrowth of leptomeningeal cells. Laboratory
Investi atgion
55: 71-85, 1986.
Although anti-neoplastic effects of bacterial preparations have been known for
over twenty years, the neoplastic effect of verotoxin per se has, to-date,
remained unknown. As
a result of extensive investigations, we have discovered that verotoxin,
particularly Verotoxin 1,
is an active component within the ACP and that purified Verotoxin 1 has potent
anti-neoplasia
effect in vitro and in vivo. Most surprisingly, we have found effective in
vivo anti-cancer
treatments of human beings commensurate with non-toxic administered dosages.
2163716
~
Summary of the Invention
It is an object of the present invention to provide a pharmaceutical
composition
for the treatment of mammalian neoplasia and, particularly, brain, skin cancer
and ovarian
cancer.
It is a further object of the present invention to provide a method of
treating
mammalian neoplasia, particularly, skin, brain and ovarian cancers.
Accordingly, in one aspect 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 skin cancers, brain cancers and ovarian cancer.
In a further aspect the invention provides a process for the manufacture of a
pharmaceutical composition for the treatment of mammalian neoplasia, said
process comprising
admixing verotoxin or the pentameric B subunit of verotoxin with a
pharmaceutically
acceptable carrier, adjuvant or diluent therefor.
The present invention provides selective, specific cancer treatments wherein
verotoxin or the pentameric B subunit of verotoxin selectively binds with Gb3
in Gb3-
containing cells. This is in contrast to the use of broad spectrum anti-
neoplastic agents such as
most chemotherapeutic agents, in that non-Gb3 containing cells are not
affected by verotoxin.
The present invention thus provides a most beneficial, cell-selective,
therapeutic treatment.
The treatment is of value against cutaneous T-cell lymphomas, particularly,
Mycosis Fungoides, sezary syndrome and related cutaneous disease lymphomatoid
papilosis.
For example, Mycosis fungoides lesions in humans have been cleared without any
observed
adverse systemic effects by the application of VTl (5ng in 2 ml. solution) by
interdermal
injection in patients.
In a further aspect, the invention provides a method of treating mammalian
neoplasia comprising treating said mammal with a non-lethal anti-neoplasia
effective amount of
2163716
8
a verotoxin, preferably Verotoxin 1 or the pentameric B subunit of verotoxin.
The verotoxin or its B subunit may be administered to the patient 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. For treatment of a skin cancer, sub-cutaneous application is
preferred.
In the practice of the present invention, Verotoxin 1 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 verotoxin or its B subunit is, typically, administered in a suitable
vehicle in
which the active 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 altemative aspect sub-cutaneously to the neoplastic
cells. Alternative, for
example, 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.
Several multi-drug resistant cell lines were found to be hypersensitive to
Verotoxin 1. For example, 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. Further, our observed binding of VT1 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 Gb3 containing astrocytoma cell lines were tested for
sensitivity to VT. Although all cells were sensitive, the sensitivity varied
over a 5000-fold
range despite approximately equivalent Gb3 levels. We have found that
treatment of the least
sensitive cell line with sodium butyrate initiated a 5000-fold increase in VT
sensitivity
2163716
9
concomitant with an alteration in intracellular VT targeting.
Thus, we have also found that the efficacy of verotoxin and its B subunit may
be
significantly enhanced by a prior treatment of the neoplastic cells with a
sensitizer, such as
sodium butyrate.
Brief Description of the Drawings
In order that the invention may be better understood preferred embodiments
will
now be described, by way of example only, with reference to the accompanying
drawings
wherein:
Fig. 1 shows the selective neutralization of ACP cytotoxicity by anti VT1 and
or
anti VT1 B subunit but not by anti VT2 antibodies as determined by cell
density measurement
after 48 hours;
Fig. 2 shows the viability of selected ovarian and breast tumour cell lines to
verotoxin concentration;
Fig. 3 represents VT1 contained within ACP preparation binding to Gb3 (and
Gb2).
Fig. 4 represents VT thin layer chromatography overlay of ovarian tumour and
ovary glycolipids;
Fig. 5 represents VT thin layer chromatography overlay of selected cell line
glycolipids;
Fig. 6 represents in three graphs ovarian cell line sensitivity to VT1, VT2
and
VT2c;
Fig. 7 represents glioblastoma multiforme cell line sensitivity to VT1, VT2
and
VT2c;
Fig. 8 represents the distribution of labelled VTl B subunit (VTB-131I)
administered IP (inter-peridinually) in a Gb3 tumour bearing nude mouse;
Fig. 9 represents the results of a three-day treatment of several human
astrocytoma cell lines with VTI;
Figs. l0A - lOG represents a graph of the anti-proliferative effects of VTl on
human astrocytoma cells;
2163716
Figs. 11A and 11B provide a comparison of SF-539 and XF-498 sensitivity to
VT1 holotoxin;
Figs. 12A and 12B represent the detection of the VT-Receptor glycolipid, Gb3
in
human astrocytoma cell lines;
5 Fig. 13 shows the sensitivity of two astrocytoma cell lines to VTl after
sensitizing culture; and
Fig. 14 shows the sensitivity to the B subunit of verotoxin VT1 of the two
cell
lines used in tests shown in Fig. 13.
10 Detailed Description of the Invention
Experimental
The isolation and purification of verotoxins VTI, VT2 and VT2c have been
earlier described.
Verotoxin 1 was prepared genetically from the high expression recombinant E.
coli pJB28, J. Bacteriol 166:375 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).
VT1 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.
Purification of VT1 from JB28
Pellet Preparation may be conducted as follows:
1. Prepare 6 x 1L LB broth in 3 x 5L jugs (media) and autoclave.
Add carbenicillin to give a 100 g/ml fmal conc. when cool.
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11
2. Seed at least 6 ml of penassay (tubes in cold room) + 100 g/rnl
carbenicillin with JB28
and incubate O/N @ 37 C.
3. Seed jugs (1 ml seed/litre broth) next moming and incubate for 24 hours at
37 C at 200
rpm (vigorous shaking).
4. Spin down bugs at 9K for 15 min. at 4 C and scrape pellet into a freezer
bag for future
use. Freeze at -70 C.
Preparation of Crude Toxin Extract:
1. Retrieve pellet and dump into beaker. Resuspend in 400 ml of PBS containing
0.1
mg/ml polymyxin B, 50 mg PMSF using a blender. Blend thoroughly then sonicate
on
ice for - 1 minute to disperse further.
2. Incubate in shaking incubator, 200 rpm, or with vigorous stirring @37 C for
1 hour.
3. Spin down cells @ 9K for 15 minutes.
4. Pour off supematant and keep. Resuspend pellet in 400 ml PBS with 0.1 mg/ml
polymyxin B and PMSF. Blend and sonicate as before.
5. Incubate with vigorous shaking/stirring at 37 C for 1 hour.
6. Spin at 10K for 15 minutes and save supematant.
7. The supernatants should be quite yellow and the bacterial pellet should
become more
fine and diffuse with each extraction step.
8. Filter the combined supematants through Whatmari filter paper than through
a glass
fibre filter to clarify. This step is optional, but will greatly speed the
concentration step.
~
9. Amicon the combined supematants at 70 psi (max.) using a YMIO membrane
(takes
about 200 hours) to concentrate to < 50 ml.
Chromatography:
Hy,droxylapatite
l. Equilibrate hydroxylapatite column with 10mM K or Na phosphate (several
column
3 G volumes).
* Trade-mark
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12
2. Load sample and wash with equilibration buffer until absorbance of effluent
is
negligible.
3. Add 2 column volumes (150 ml) of 100mM K phosphate (until yellow-coloured
fractions emerge) and collect 3 ml fractions.
4. Wash column with 500mM K phosphate and re-equilibrate with 10mM K
phosphate.
Add 0.05% sodium azide.
Chromatofocussing
5. Measure fractions (A280) and Pool peak fractions from HA.
6. Dialyse against 2L 0.025M imidazole-HCI pH 7.4 O/N. Also equilibrate the
chromatofocussing column O/N with the same (300 ml).
7. Load sample and follow with 400 ml polybuffer-HCI pH 5.0 (50m1 polybuffer
74 +
350m1 dH2O, a 1:7 dilution, -pH to 5.0 with HCI). NOTE: make sure the sample
is
equilibrated to the temperature that the column will be run at (usually room
temperature) prior to loading. If the column is to be run at 40 then buffers
must be pH'd
at 4 C and the column equilibrated at this temperature.
8. Collect 1 ml fractions and test them for A280 and pH.
9. Plot the A280 and pool peak fractions at about pH 6.8 for VT-1 (pool side
peaks
separately).
10. Clean column with 100 ml 1M NaCI. if really dirty follow with 100 ml 1M
HCI, but
quickly equilibrate column with imidazole. Store colum.n in 20% ethanol in
25mM
imidazole.
Cibachron blue
11. Equilibrate cibachron blue with 10mM Na phosphate buffer, pH 7.2 (100ml).
12. Load sample directly from CF and follow with 60m1 of same buffer.
13. Elute with 0.5M NaCl in above buffer and collect fractions.
14. Test fractions for A280 and cytotoxicity and pool appropriate ones.
15. Clean column with 25m1 each of 8M Urea in wash buffer and 1M NaCI in wash
buffer.
16. Reequilibrate column with 10mM phosphate containing 0.1 % azide.
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13
17. Dialyse peak fractions against wash buffer with one change.
18. Lyophilize and resuspend in I ml dH2O.
19. Do protein assay and run SDS-PAGE to check purity.
Solutions:
HA column
potassium phosphate buffer (0.5M stock)
17.42g K2HPO4 up to 300 ml with dH2O
6.8g KH2PO4 pH 7.2 with KOH
CF column
imidazole buffer
0.851 g/500 ml H20
pH 7.4 with HCI
CB column
sodium phosphate buffer (Wash buffer-WB)
0.71g/500m1 NazHPO4
pH 7.2 with HAc
degas
Elution buffer Cleaning Buffers
2.922g NaCI/100 ml WB 12.012g Urea/25 ml WB
1.461 g NaCI/25m1 WB
Purification of VT2 from R82
Pellet Preparation:
~
1. Prepare 3 x 2L penassay broth (Antibiotic Meida 3, DIFCO; pH 7.0) in 3 x 5L
jugs and
autoclave at 121 C for 20 minutes. Allow broth to cool to room temperature
before use.
2. Seed minimum 3 x 2ml of penassay broth containing 75 g/ml carbenicillin
(Disodium
salt, SIGMA) with R82 and incubate ovemight at 37 C, with shaking.
* Trade-mark
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14
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.
5. Reduce temperature to 37 C and incubate for another 3 hrs.
6. Spin down culture solution at 9,000xg for 15-20 min at 4 C. Discard
supematant and
store pellets at -20 C.
Preparation of Crude Toxin Extract:
1. Resuspend pellets in 100 ml of PBS (phosphate buffered saline, OXOID; pH
7.3).
2. Add 0.3 mg/ml PMSF (phenylmethyl-sulfonyl fluoride, SIGMA) dissolved in 0.5
ml
acetone to pellet solution. Let acetone evaporate. Sonicate on ice at highest
output
possible for 5 min or until an homogeneous solution is obtained.
3. Spin down cell at 9,000xg at 4 C for 20 min. Discard pellets.
4. Concentrate supematants using ultrafiltration (Model 8400 standard
infiltration cell,
AMICON) with N2 no higher than 70 psi and using a 10,000 MW cutoff membrane
filter (YM10 membrane, AMICON).
5. Using 12-14,000 MW cutoff tubing (SPECTRAPOR) (now and in all dialysis
steps),
dialyse toxin solution against 4L of 10mM potassium phosphate overnight, with
stirring
at 4 C.
Chromatography:
Hydroxylapatite (HA
1. Equilibrate hydroxylapatite column (BSA binding capacity: 32 mg/g,
approximately 113
ml volume; CALBIOCHEM (BEHRIIVG DIAGNOSTICS)) with 2 column volumes of
10mM potassium phosphate.
2. Load sample and follow with 1 column volume 10mM potassium phosphate.
3. Add 2 column volumes of 200mM potassium phosphate and collect 2 ml
fractions. The
fractions containing the toxin should be coloured differently from the other
fractions.
* Trade-mark
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4. Wash column with 1 column volume of 500mM potassium phosphate and
reequilibrate
with I column volume of 10mM potassium phosphate. Add azide to the top of the
column for storage.
5 Chromatofocussing (CF)
5. Pool peak fractions from HA column either by colour or by cytotoxicity test
on Vero
cells (10-fold dilutions).
6. Dialyse pooled fractions against 4L 0.025M Histidine-HCl pH 6.2 (SIGMA)
overnight.
Also equilibrate the chromatofocussing column (PBE (polybuffer exchanger) 94,
1.5
10 cm diameter, 57 ml volume; PHARMACIA) ovemight with the same buffer (300
ml).
7. Loan sample and follow with 400 ml polybuffer-HCI pH 4.0 (50 ml polybuffer
74
(PHARMACIA) + 350 ml dHzO - pH to 4.0 with HC1),
8. Collect 2 rnl fractions and test the pH of each fraction. Once the pH has
dropped to
3.95, stop collecting fractions. Test the fractions using absorbance of 280 nm
or by
15 cytotoxicity on Vero cells (10-fold dilutions).
9. Pool peak fractions, and return pH to 7.0 using 1N NaOH.
10. Clean column with 200 ml 1M NaC1. If dirty follow with 100m1 1M HCI, but
quickly
equilibrate column with 0.025M imidazole, otherwise equilibrate with 24% EtOH-
H20.
Cibachron blue (CB)
11. Equilibrate cibachron blue (2 cm diameter, 82 ml volume, PIERCE) with 100
ml of
10mM sodium phosphate buffer (wash buffer).
12. Load sample and follow with 60 ml of wash buffer.
13. Elute with 0.5M NaC1 in wash buffer and collect 2 ml fractions.
14_ Test fractions for absorbance at 280 nm using the elution buffer as a
blank and
cytotoxicity on Vero cells and pool appropriate fractions.
15. Clean column with 25 ml each of 8M Urea in wash buffer and IM NaCI in wash
buffer.
16. Reequilibrate column with 100 ml of wash buffer and add azide to the top
of the column
for storage.
17. Dialyse peak fractions against 4L 0.01M Tris-CL (pH 7.0, SIGMA).
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18. Lyophilize sample and resuspend in 1-2 ml dHZO (OPTIONAL).
1E
19. Do protein assay (BCA Protein assay reagent, PIERCE) and rune SDS-PAGE gel
(Schagger, 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 (0.5M stock)
17.42g K2HPO4 up to 300 ml with dH2O
6.8g KH2PO4 pH 7.2 with KOH
CF column
Histidine buffer (0.025M)
2.0g/500 ml H20
pH 6.2 with HC1
CB column
Sodium phosphate buffer (Wash buffer-WB)
0.71 g/500rn1 Na2HPO4
pH7.2withHAc
degas
Elution buffer (0.5M) Cleaning Buffers
2,922g NaCI/100ml WB 12.O1g Urea/25 ml WB
1.46 NaCI/25 ml WB
0.01 M Tris
ak
4.84 g Trizma Base
4 L ddH2O
pH to 7.2 with HCI
Purification of VT2c from E32511
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Pellet Preparation:
1. Prepare 3 x 2L penassay broth (Antibiotic Media 3, DIFCO; pH 7.0) in 3 x 5L
jugs and
autoclave at 121 C for 20 minutes. Allow broth to cool to room temperature
before use.
2. Seed minimum 3 x 2 ml of penassay broth with E32511 and incubate overnight
at 37 C.
3. Add 0.2 g/ml Mitomycin C (1 ml of 0.4 mg/ml) (add 5 ml of ddHzO to the
vial) to
each of the 5L jugs (from step 1). Seed each jug with 2 ml of seed (step 2)
and incubate
for 6 hrs at 37 C with shaking of approximately 120 rpm. It is very important
to stagger
the incubation by about 45 min/flask because the toxin begins to deteriorate
after 6 hour
exposure to Mitomycin C.
4. Spin down culture solution at 9,000xg for 15-20 min at 4 C. Discard
supematant and
store pellets at -20 C.
Preparation of Crude Toxin Extract:
1. Resuspend pellets in 150 ml of PBS (Phosphate buffered saline, OXOID; pH
7.3).
2. Add 0.3 mg/ml PMSF (phenylmethyl-sulfonyl fluoride, SIGMA) dissolved in 0.5
ml
acetone to pellet solution. Let acetone evaporate. Sonicate on ice at highest
output
possible for 3 min or until an homogeneous solution is obtained.
3. Add 0.1 mg/ml polymyxin B sulphate (Aerosporin, BURROUGHS WEL_LCOME INC.;
500,000 units) to solution and incubate with gentle shaking at 37 C for 1 hr.
4. Spin down cells at 9,000xg at 4 C for 20 min (to remove all cells and cell
debris from
solution).
5. Decant supernatant and store at 4 C. Resuspend pellet in 75 ml PBS and add
0.1 mg/ml
polymyxin B.
6. Incubate with gentle shaking at 37 C for 1 hr.
7. Spin down cell at 9,000xg at 4 C for 20 min and pool supematants (from step
5).
Discard pellets.
The next few steps should preferably be done at 4 C:
8. Add crystalline ammonium sulphate very slowly, with stirring to pooled
supernatants to
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18
30% saturation.
9. Let stir for 20 min and then remove precipitate by centrifugation (10000g
for 10 min).
10. Add crystalline ammonium sulphate very slowly, with stirring to pooled
supernatants to
70% saturation.
11. Let stir for 20 min and then centrifuge at 10000g for 10 min.
12. Resuspend pellet from step 11 in 15 ml of 0.01 M Potassium phosphate
buffer.
13. Using 12-14,000 MW cutoff tubing (SPECTRAPOR) (now and in all dialysis
steps),
dialyse toxin solution against 4L of 10mM potassium phosphate overnight, with
stirring
at 4 C.
Chromatography:
Hydroxylapatite (HA)
l. Equilibrate hydroxylapatite column (BSA binding capacity: 32 mg/g,
approximately 113
ml volume; CALBIOCHEM (BEHRING DIAGNOSTICS)) with 2 column volumes of
10mM potassium phosphate.
2. Load sample and follow with 1 column volume 10mM potassium phosphate.
3. Add 2 column volumes of 100mM-200mM potassium phosphate and collect 2 ml
fractions. The fractions containing the toxin should be coloured differently
from the
other fractions.
4. Wash column with 1 column volume of 500mM potassium phosphate and
reequilibrate
with 1 column volume of 10mM K phosphate. Add azide to the top of the column
for
storage.
Chromatofocussing(CF)
5. Pool peak fractions from HA column either by colour or by cytotoxicity test
on Vero
cells (10-fold dilutions).
6. Dialyse pooled fractions against 4L 0.025M imidazole-HCI pH 7.4 (SIGMA)
overnight.
Also equilibrate the chromatofocussing column (PBE (polybuffer exchanger) 94,
1.5
cm diameter, 57 ml volume; PHARMACIA) overnight with the same buffer (300 ml).
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19
7. Load sample and follow with 200 ml polybuffer-HCl pH 5.0 (25 ml polybuffer
74
(PHARMACIA) + 175 ml dH2O - pH to 5.0 with HCl).
8. Collect 2 ml fractions and test the pH of each fraction. Once the pH has
dropped to
5.95, stop collecting fractions. Test the fractions for cytotoxicity on Vero
cells (10-fold
dilutions).
9. Pool peak fractions.
10. Clean column with 200 ml iM NaCI. If really dirty follow with 100 ml 1M
HCI, but
quickly equilibrate column with 0.025M imidazole.
Cibachron blue (CB)
11. Equilibrate cibachron blue (2 cm diameter, 82 ml volume, PIERCE) with 100
ml of
10mM sodium phosphate buffer (wash buffer).
12. Load sample and follow with 60 ml of wash buffer.
13. Elute with 0.5M NaCI in wash buffer and collect 2 ml fractions.
14. Test fractions for absorbance at 280 nm using the elution buffer as a
blank and
cytotoxicity on Vero cells and pool appropriate fractions.
15. Clean column with 25 ml each of 8M Urea in wash buffer and 1M NaCI in wash
buffer.
16. Reequilibrate column with 100 ml of wash buffer and add azide to the top
of the column
for storage.
17. Dialyse peak fractions against 4L 0.01 M Tris-CL (pH 7.0, SIGMA).
18. Lyophilize sample and resuspend in 1-2 ml dH2O (OPTIONAL).
19. Do protein assay (BCA Protein assay reagent, PIERCE) and run SDS-PAGE gel
(Schagger, 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 (0.5M stock)
17.42g K2HPO4 up to 300 ml with dH2O
6.8g KH2PO4 pH 7.2 with KOH
2163716
CF column
imidazole buffer (0.025M)
0.851 g/500 ml H20
pH 7.4 with HCl
5 CB column
sodium phosphate buffer (Wash buffer-WB)
0.71g/500 ml Na2HPO4
pH 7.2 with HAc
degas
10 Elution buffer Cleaning buffers
2.922 g NaCI/100m1 WB 12.012g Urea/25m1 WB
1.461 g NaCI/25m1 WB
0.01 M Tris
4.84 g Trizma Base
15 4 L ddHZO
pH to 7.2 with HCl
Affinit,ypurification of verotoxins
500 g globotriaosyl ceramide in 1 ml chloroform was mixed and dried with 1 g
20 of dried celite. The chloroform was evaporated and the celite suspended in
PBS and poured in
a column. Crude polymyxin extract 20 ml (25 mg protein) the toxin producing E.
coli was
applied to the column and incubated at room temp for 15 mins. The column was
washed with
PBS and purified verotoxin eluted with 10 ml 1M Tris pH 9.6. The eluate was
neutralized and
dialysed. This method is applicable for purification of all verotoxins.
(Boulanger, J., Huesca,
M., Arab, S and Lingwood, C.A. "Universal method for the facile production of
glycolipid/lipid
matrices for the affmity purification of binding ligands" Anal Biochem 217: 1-
6 [1994])
Preparation of verotoxin 1 doses
VT1 was purified from the E. coli strain as previously described which
overexpresses the cloned toxin genes. The purified toxin was free of endotoxin
contamination.
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The protein concentration of this batch of verotoxin was determined and the
toxin aliquoted
and stored at -70 C.
To prepare VT1 doses for patients, VT1 was diluted into injection grade
sterile
saline containing 0.2% v/v of the patient's own serum. 210 ul of sterile
patient serum was
added to 10 nil of sterile injection saline and 93.9 ml of purified VTl (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 fmal
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 labelling of VT1: FITC was added directly to VT1 (in a 1:1, w/w ratio) in
0.5M
NazCO3/NaHCO3 conjugated buffer pH 9.5 and the mixture gently rotated for 1.2
hours at room
temperature. Free FITC was removed by centricon.
Fluorescent Staining of Sections: Samples of surgically removed ovarian
tumours were
embedded in OCT compound, flash frozen in liquid nitrogen, and stored at -70 C
until use.
Five m sections of frozen sample were thawed, allowed to dry and stained with
FITC-labelled
VTI in PBS (0.5 mg.ml) containing 0.1% BSA for 1 h at room temperature.
Sections were
extensively washed with PBS and mounted with mounting medium containing DABCO.
Sections were observed under a Polyvar fluorescent microscope.
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-VTl for lh at room temperature. The cells were washed 5 times with PBS,
mounted with
DABCO and observed under a Polyvar fluorescent microscope.
Quantification of VT1 antitumour activity: SKOV3 (drug sensitive human ovarian
cell line),
SKOVLC (SKOV3, resistant to Vincristine, and SKOVLB (SKOV3, resistant to
Vinblastine)
were each grown in a- MEM supplemented with 10% fetal calf-serum and tested
for their
sensitivity to VTs. Equal numbers of cells (approximately 1000 per/ml of
media) were added to
the wells of Linbro 98 well plate. 10-fold dilution of VTs were tested in
triplicate and
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22 incubated for 48h at 37 C in a humidified atmosphere containing 5% CO2.
Cells were then
fixed with 2% Formalin, stained with Crystal Violet, and read with ELISA plate
reader.
To quantify the anticancer activity of VT1, SKOV3, SKOVLC, and SKOVLB
(human ovarian cell line) were incubated with 10-fold dilution of VT1 for 48h.
SKOVLC &
SKOVLB (drug resistant cell lines) are more sensitive to VT1 antitumour
activity than SKOV3.
Preparation of131I-VT1B
This material may be made by the following procedure.
1. Dissolve 20 mg of iodogen in 2.0 ml of chloroform (10 mg/mI). Make a 1:10
dilution
by adding 0.25 ml of the 10 mg/mi solution to 2.25 ml chloroform (1 mg/ml).
2. Dispense 20 ul of this dilute solution into a clean, dry sterilized glass
tub. Add 500 ul of
chloroform and evaporate to dryness under N2.
3. Add 1.5 mg. in 0.66 ml of VT1B subunit to the test tube.
4. Add 5 MCi of13'I sodium iodide in 100 ul. Allow labelling to proceed for 10
mins.
5. Wash a PD- 10 column with 25 ml of Sodium Chloride Injection USP.
6. Dilute13'I-VT1B to 2.5 ml total volume with 1% HSA in Sodium Chloride
Injection
USP. Load onto PD-10 column. Elute colarnn with 3.5 ml 1% HSA in saline.
7. Measure 1311 activity of eluant and column to determine LE. Draw up pooled
fractions
into a syringe with spinal needle attached. Detach spinal needle and attach
MillexGV
filter.
8. Filter into a sterile 10 ml multidose vial. Note volume filtered and assay
vial for 131 I in
dose calibrator. Calculate concentration.
9. Draw up 0.1 ml of 13'I-VTIB and dispense 0.05 ml into each of two 5 ml
sterile
multidose vials (one for sterility test and one for pyrogen test). Vials
already contain 2
ml saline (=1:50 dilution).
10. Determine RCP by PC (Whatman No. 1) in 85% MeOH and by size exclusion
HPLC.
11. Conduct sterility and pyrogen tests.
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AstroWoma Cell Lines, Endothelial Cells and Culture Conditions:
Six permanent human malignant astrocytoma cell lines (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%
COZ 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 normaI 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-VIII-
related antigen
antisera (Dako Santa Barbara, CA) as described previously (27).
Approximately 1-5 x 104 cells were added to 24-well plates and incubated in a-
MEM in 5% COz at 37 C. After 24 hours, the growth medium was replaced with
medium
containing various concentrations of the holotoxin VTI (0, 0.1, 5, 50, and 100
ng/ml). The
treated astrocytoma cell lines and endothelial cells were trypsinized and
counted at intervals
throughout the growth curve. Cell viability was assessed by trypan blue dye
exclusion. Cell
counts were plotted again time for the various concentrations of VTl and B
subunit. For each
time point analyzed, the wells were set-up in triplicate.
For selected cell lines, the B subunit of VTI, VT2, and VT2c was added alone
to the astrocytoma cells at same concentrations listed above. In these
experiments, a single dose
of VT1, VT2, and VT2c was added to confluent astrocytoma cells in microtiter
wells. Cell
survival at 72 hours was monitored by*aining with 0.1% crystal violet, and
measuring the
opticai density at 590 nm using a Dynatek microtiter plate reader.
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VT Receptor Analysis of Human Astrocytoma Cells:
Cultured human astrocytoma cells were homogenized in a minimum volume of
PBS and extracted with 20 volumes of a 2:1 by volume chloroform:methanol
solution. The
extract was partitioned against water and the lower phase partitioned again
against theoretical
upper phase. The lower phase was dried completely and dissolved in a known
volume of 2:1
chlorofornl:methanol. The presence of Gb3 was detected by TLC overlay binding
with VTI.
Astrocytoma lower phase and standard Gb3 from human kidney each were separated
by TLC
[(chloroform:methanol:water = 65:25:4 (v/v/v)]. The TLC plates were dried and
blocked with
1% gelatin in water at 37 C ovemight. Then they were washed three times with
50 mm TBS
(Tris Buffer Salin) for 5 min and incubated with 0.1 g/ml VTI for 1 hour.
After further.
washing with TBS, the plates were incubated with a mouse monoclonal PHI and
anti-VTl
antibody (2 g/ml), followed, after washing, by peroxidase-conjugated goat
anti-mouse
antibody or peroxidase conjugated goat anti-rabbit antibody as appropriate.
Finally, the plates
were washed with TBS, and VTI binding was visualized with 4-chloro-1 naphthol
peroxidase
substrate. A similar plate was prepared and stained with orcinol carbohydrate
spray for
comparison.
Nuclear staining with propidium iodide:
SF-539 cells grown on the cover slips overnight were incubated at 37 C with
VT 1 B-subunit (50 g/ml) for 1.5 hrs or 10 hrs and fixed (with 1%
paraformaldehyde for 3
lE
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.
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Flow Cytometry:
Apoptosis of astrocytoma cells, incubated with 10 ng/ml of VTl for 24-36 hrs
in
the presence of 10% bovine fetal serum was analyzed on an EpictProfile
Analyzer (Coulter'~'
5 Electronics, Pathology, University of Toronto). After treatment, cells were
trypsinized and the
200Xg centrifuged cell pellet was suspended in 1 ml 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 ul of 200 ug/ml DNase-free RNase A at 37
C for 30 min.
Cell cycle distribution was determined using manual gating. Flow cytometric
quantitation of
10 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 VT1 served as
controls.
Ultrastructural Analysis of VT-treated Astrocytoma Cells:
15 Cells were cultivated on a transferable 9 mm cyclopore membrane (0.45 um
pore size, Falcon) to form a confluent monolayer and were incubated at 37 C
with VTI (10
ng/ml). Cells were fixed at room temperature by addition of 1.6%
glutaraldehyde to the well
and then incubated in 0.066 M Sorensen buffer (pH 7.4) containing 1.5%
glutaraldehyde for 1 h
at 4 C. After 2 h of washing with 0_ 1 M phosphate buffer, cells were post-
fixed in 2% osmium
20 tetroxide in the same buffer. After dehydration in graded ethanols and
propylene oxide, Epon
embedding and uranyl-lead staining were performed. Thin sections were examined
in a Philips
EM 400 electron microscope and ultrastructural features of apoptosis was
analysed.
Fig. I relates to the neutralization of ACP cytotoxicity by anti-VT. KHT cell
monolayers were incubated with 35 ng/ml ACP from E.coli HSCjo, or lOpg/ml VT1,
VT2 or
25 VT2c in the presence of monoclonal anti-VTl(PH1), monoclonal anti VT2 or
polyclonal rabbit
antiVTl 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 VTl or anti VT1B subunit (anti-
VT2 serum had
no effect).
From measurement of the cytotoxic assay of ACP on vero cells (cells from
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Africa green monkey kidney that are very sensitive to verotoxin), relative to
a pure VTI
standard, it was estimated that the ACP preparation contained 0.05% VT1. This
concentration
of purified VTI was as effective as ACP in inhibiting the growth of various
tumour cell lines in
vitro (Fig. 2). Thus, 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 VT-
1 or ACP.
GP TREATMENT # OF # OF LUNG MEA WT LOSS
MICE NODULES/MOUSE N /GAIN*
EXPT 1
1 Control 9 34,24,39,47,28,32,26 32.6 +5%
,29,34
2 ACP-0.25 ug/mouse 4 12,31,25,15 20.8 0
3 ACP-1.0 ug/mouse 6 1,2,2,5,1 2.2 0
(1 death)
4 ACP-4 ug/mouse 5 0,0,0,0,0 0 -13%
5 VT-1 0.009 ug/mouse 5 29,41,34,29,21 30.8 +5%
6 VT-1 0.036 ug/mouse 5 7,16,29,16,6 14.8 +5%
7 VT-1 0.144 ug/mouse 5 1,4,2,3,1 2.2 +5%
EXPT2
1 Control 4 15,12,8,12 11.75 <5%
2 ACP-2 ug/mouse 5 0,1,0,0,0 0.2 <5%
3 VT-1 0.1 ug/mouse 4 0,0 0 <5%***
(2 deaths)
4 VT-1B-0.2 ug/mouse 5 13,14,9,7,19 12.4 <5%
5 VT-lB-lO ug/mouse 5 8,3,9,11 6.8 <5%
Mice were treated with VT-1 or ACP(1-p) I day after cell injection (1000 KHT
cells/mouse i-v).
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27
Lung nodules counted @ 20 days after cell injection.
* Mean change in gp wi-max during 10 days (Expl 1) or 4 days (Expt 2) after VT-
1 or ACP
injection. Max wt loss @ days 7-8.
** Death occurred @ days 2-3 after ACP injection
*** Deaths occurred @ days 7-8
Purified VTl was found to mimic the anti-metastatic effect of ACP on the
growth of this tumour from a primary subcutaneous site. Lung metastasis was
completely
inhibited. Moreover, prior immunization of mice with the purified B-subunit of
verotoxin
completely prevented any protective effect of ACP when the animals were
subsequently treated
with the tumour and ACP (Table 2).
Table 2. Response of KHT lung nodules, growing to immunized mice, to treatment
with VT1
or ACP.
GP IIVIlVIUNI- TREATMENT # OF # OF LUNG MEA WT LOSS/
ZATION* MICE NODULES/ N GAIN*
MOUSE
1 None None 6 34,47,53, 48.5 <5%
62,43,52
2 None VT-1 -0,2 5 5 deaths
ug/mouse (dy 6-8)**
3 None ACP-2.0 5 0,1,2,0,0 0.6 -8%
ug/mouse
4 VT-1B+FA None 5 43,40,47, 39.2 -6%
43,23
5 VT-1B+FA VT-1 -0,2 6 26,44,49, 36.7 <5%
ug/mouse 21,43,37
6 VT-1B+FA ACP-2.0 6 50,38,33, 43.3 <5%
ug/mouse 41,48,50
7 FA only None 5 44,60,19, 37.6 <5%
25,40
8 FA only VT-1 -0,2 5 5 deaths
ug/mouse (dy 6-8)***
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28
GP IMMUNI- TREATMENT # OF # OF LUNG MEA WT LOSS/
ZATION* MICE NODULES/ N GAIN*
MOUSE
9 FA only ACP -2.0 5 1,1,2,1,0 1 -6%
ug/mouse
Mice were treated with VT-1 or ACP(i-p) 1 day after cell injection (1000 KHT
cells/mouse).
Lung nodules counted @ 20 days after cell injection (i-v).
*Immunization was 2 injections of VT-1B (IOug/mouse +/- Freund's Adjuvant (FA)
given (i-p)
4 weeks and 2 weeks before cell injection.
** Mean change in gp wt - max during 13 days. Maximum weight loss @ day 7-8.
ACP was tested for glycolipid binding by TLC overlay using monoclonal anti-
VTI or anti-VT2c. Anti-VTI shows 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 top) galabiosyl
ceramide,
globotriaosyl ceramide and globotetraosyl ceramide. Panel 1-detection using
anti VT1, panel 2-
detection using anti VT2c.
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. Similarly, 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. Surprisingly, we have found that
multi-drug resistant
variants of ovarian tumour cell lines were considerably more sensitive to VTI
cytotoxicity than
the drug sensitive parental cell line (Figs 2, 5 and 6). Similar effects had
been observed for
ACP. Fig. 2 shows human ovarian tumour cell lines sensitive to ACP tested for
VT sensitivity.
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29
Human ovarian and breast tumour derived cell lines were tested for VT1
sensitivity wherein
ovarian 1, 2, 3, 4 and 5 are denoted 0, +, x, ^ and o respectively, and breast-
SKBR3 , 468*,
4530, 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 VT1.
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 VT1. Fig. 5 shows VT sensitive
and resistant
cell lines tested for the presence of Gb3 by VT binding in tlc overlay.
Glycolipid from an equal
number of cells were extracted and separated by tlc prior to toxin binding. In
Fig. 5, lane
1:SKBR3, lane 2:468, lane 3:231, lane 4:453, lane 5 Gb3 standard, lane
6:SKOV3, lane
7:SKOVLB. Cell lines SKBR3, 468, 231 and 453 are derived from breast tumours.
Only 231 is
sensitive to VT1. 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.
The specific elevation of Gb3 in ovarian tumours as opposed to normal ovary
tissue provides the feasibility of using the toxin in the management of this
malignancy. Ovarian
tumours are often refractory to chemotherapy and prognosis is poor. Indeed,
preliminary phase
1 clinical trials using a ACP injected directly into skin malignancies
(Mycosis fungoides) have
proven successful without adverse systemic effects.
With reference now to 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.
We have found that both drug resistant cells are approximately 500 to 1000
times more sensitive to verotoxin cytotoxicity than the parental SKOV3 cell
line.
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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 VT1, 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 13'I-VTIB (CPM
5 distribution in different organs). VT1B-1311 cpm distribution in nude mouse
with implanted
ovarian tumour showed that a considerable amount of radiolabled VTIB had been
concentrated
in the ovarian tumour. Only a trace amount of VT1B 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 VTl toxicity this does not present a problem for treatment of
human adult with
10 ovarian tumour. In addition the CPM in kidney includes the excreted
radiolabelled VT1 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 VT1 cytotoxicity.
Fig. 9 shows the sensitivity of a variety of human astrocytoma cell lines to
VT1.
All these cells contain Gb3 but show variable sensitivity to VT1 induced
cytotoxicity. This
15 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 - lOG show the anti-proliferative effects of VT1 on human
astrocytoma cells. All astrocytoma cell lines showed at least some inhibition
of growth
20 following VT1 treatment. The most sensitive cell line was SF-539 (Fig. I
OA), and the least
sensitive was SF-126 (Fig. lOF). Human cerebral capillary endothelial cells
were largely
resistant to the growth-inhibitory effects of VTl except at high doses (100
ng/ml) (Figs. lOG).
U-251 MG and U-87 MG were sensitive to VT1 (Figs. 10B and 10C), whereas XF 498
and SF-
188 were somewhat less sensitive to VT1 (Figs. lOD, 10E and lE) than were U-
251 MG and U-
2 5 87 MG.
Figs. 1lA and I1B 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
stu-vival was
calculated. VTl was cytotoxic to SF-539 astrocytoma cells at doses as low as
0.01 ng/ml
30 (upper panel). XF-498 cells were resistant to VTl holotoxin. When the VTl B-
subunit was
2163 71J
31
employed, only SF-539 was sensitive to this toxin (lower panel).
Figs. 12A and 12B represent the detection of the VT-Receptor glycolipid, Gb3
in
human astrocytoma cell lines. Astrocytoma neutral glycolipids were prepared
from I x 106
cells and separated by TLC. (A) 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, VT1 binds to Gb3 extracted from astrocytoma cells as shown
(arrow). SF-539
astrocytoma cells showed maximal binding of Gb3/VT1. Lane 1, U87 MG; lane 2,
U251 MG;
lane 3, SF-126; lane 4, SF-188; lane 5, XF-498; lane 6, SF-539, lane 7,
standard Gb3 (0.3
ug/mi).
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
Anti-Proliferative Effects of Verotoxin on Human Astrocytoma Cells:
Figs. l0A - lOG show that all astrocytoma cell lines studied were sensitive to
VT1. The most sensitive cell line in terms of growth inhibition was SF-539
(Fig. IOA) and the
least sensitive was SF-188 (Fig. IOE). 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). Interestingly, human cerebral endothelial cells were largely
resistant to the growth
inhibitory and cytotoxic effects of VT-1 (Fig. IOG). Only when doses as high
as 100 ng/ml
were used were endothelial cells inhibited.
A comparison between the sensitivity of SF 539 and XF498 for VTI and VT1 B
subunit is shown in Fig. 11A and Fig. I 1B. XF498 cells were considerably less
sensitive to the
B subunit than to the VT-1 holotoxin. By comparison, 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.
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32
VT-Receptor Analysis of Human AstrocYtoma Cells:
The glycolipid profile of the 6 human astrocytoma cell lines analyzed for Gb3
content as detected with orcinol is shown in Fig. 12A. All of the astrocytoina
cell lines
expressed significant levels of Gb3 and showed binding with VT1 in the overlay
assay used
(Fig. 12B). SF-539 cells expressed the highest levels of Gb3 with maximal
binding to VTl.
Flow Cytometric Analysis:
To determine the extent of astrocytoma cells death by apoptosis, cells were
analysed by flow cytometry. SF-539 and XF-498 astrocytoma cells exposed to VT1
(10 ng/ml)
revealed the characteristic features of apoptosis. As a result of chromatin
condensation and
DNA cleavage, apoptotic cells show less propidium iodide fluorescence than
viable cells and
can be quantified as the "subdiploid" population or pre-G1 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. In VT1-
sensitive SF-539 cells, a pronounced loss of S phase cells from 33 to 15 and
10% was seen,
whereas with the less VT1 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).
Propidium iodide stains:
For detection of apoptotic morphology in cells treated with VTI or VTI B-
subunit, permeabilized SF-539 and XF-498 cells were stained with the DNA-
intercalating agent
propidium iodide and were analyzed by fluorescence microscopy. 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
VT1 B-subunit for 1.5 or 10 hours.
33 2163716
Ultrastructural Analysis of VT-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 VT1 inhibits the growth of a series of human
astrocytoma cell lines. All cell lines showed significant sensitivity to VT1,
contained the Gb3
receptor for VT, and demonstrated ultrastructural features indicative of
apoptosis following VT
treatment. These results show that VTs provide the basis of new agents active
against human
astrocytoma cells. The results show that the most toxin sensitive astrocytoma
cell line, SF-539,
is also highly sensitive to B subunit induced apoptosis.
Defmitive 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 VT1-treated astrocytoma
cells in pre-
G1 position in cell cycle (Fig. 13) is strong evidence for apoptosis.
Additional evidence in
support of VT1 causing apoptosis in sensitive astrocytoma cells include
nuclear staining with
propidium iodide and ultrastructural alterations indicative of apoptosis.
We have found that there was relative insensitivity of human cerebral
capillary
endothelial cells to VT.
While the invention has been described in detail and with reference to
specific
embodiments thereof, it will be apparent to one skilled in the art that
various changes and
modifications can be made therein without departing from the spirit and scope
of the invention.
