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INTRODUCTION |
The plant toxins have been employed in targeted therapies for the
treatment of cancer for many years (1). The main reason for their use
is their extreme potency; for example, it has been shown that a single
plant toxin molecule injected into the cytoplasm can kill a cell (2).
Therefore a selectively targeted plant toxin has the potential to be a
powerful anti-cancer therapy. Gelonin is a member of the type I
ribosome-inactivating plant toxin family (3). Members of this family
possess the catalytic A-chain necessary for protein synthesis
inhibition but lack the B-chain that is characteristic of the type II
toxins (e.g. ricin (1)). The B-chain is required for cell
binding and endosomal translocation of the type II toxins into the
cytoplasm of cells where protein synthesis is then inhibited (4). Since
gelonin has no active mechanism of cell entry it is relatively
non-toxic to intact cells, relying instead on nonspecific endocytosis
and fluid phase uptake for cellular entry (3). However, in cell-free translational systems gelonin is extremely efficient at inhibiting protein synthesis (IC50 10
9 M),
acting by preventing the association of elongation factors 1 and 2 with
the 60 S ribosomal subunit (1). As a result gelonin is a potential
candidate for use as a cytoplasmic targeted toxin, and previous studies
have shown it to be useful for targeting using transferrin (5),
gonadotrophin (6), and antibodies (7).
The use of such a potent toxin as a targeted therapy is limited unless
sufficient selectivity can be incorporated into the drug, thus reducing
the effect of nonspecific cell death. Therefore, targeting moieties
that exhibit high affinity for cell surface receptors selectively
up-regulated on tumor cells are required for this approach. One such
targeting ligand is folate, which exhibits high affinity for the folate
receptor (FR)1
(1010 M). The FR is overexpressed on a range
of cancers (8), in particular epithelial ovarian cancer where 90% of
cases exhibit up-regulation (9). In its unconjugated state folate is
non-immunogenic, it is also small in size (441.4 Da) and retains its
affinity for the FR upon conjugation to various proteins (8).
Additionally, it has been shown to be a useful targeting moiety for the
selective delivery of proteins into the cytoplasm of tumor cells
in vitro (10, 11). Therefore, folate is a potential
candidate ligand for the targeted delivery of gelonin into the
cytoplasm of tumor cells.
However, one difficulty with the synthesis of targeted toxins is the
effect of ligand conjugation upon toxin activity. For example, it has
been shown that modification of gelonin amino groups by a
heterobifunctional cross-linking reagent (12-14) and conjugation of
targeting agents to the toxin (3, 13) can result in up to 99% reduced
toxin activity. Therefore the method of ligand conjugation is critical
for retaining toxin activity while attaining sufficient selectivity for
target cells. In this study, we have compared two strategies for the
synthesis of folate-gelonin conjugates. The first exploits conjugation
of SH-folate to gelonin carbohydrate residues (folate-S-gelonin) using
3-(2-pyridyldithio)propionyl hydrazide (PDPH), a carbohydrate-selective
cross-linker. The second uses N-hydroxysuccinimide-folate to
conjugate the ligand directly to amino groups present in gelonin
(folate-CO-gelonin). We demonstrate by competitive inhibition of
[3H]folic acid binding to HeLa cells that both conjugates
bind to the FR with the same affinity as free folic acid. However,
folate-CO-gelonin is 225-fold less active at inhibiting translation
than both folate-S-gelonin and gelonin in a cell-free rabbit
reticulocyte lysate assay. Folate-S-gelonin also exhibits prolonged
inhibition of protein synthesis in HeLa and Skov3 cell lines in
vitro compared with gelonin.
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EXPERIMENTAL PROCEDURES |
Solid Phase Synthesis of SH-folate
A glass column with a join connection and glass frit (porosity
number 2) on the upper and lower end, respectively, was used as a
reaction vessel. This allowed stirring of the resin on a rotary
evaporator and washing after each reaction step. After each step, the
resin was stirred (20 min) and rinsed successively with
CH2Cl2 (5 × 10 ml) and CH3OH
(5 × 10 ml).
Cysteamine Grafting to the Resin via Thioether Bond
Formation--
4-Methoxytrityl chloride poly(styrene) (1% divinyl
benzene) resin (200 mg, 0.346 mmol of chloride group; Novabiochem,
Meudon, France) suspended in
CH2Cl2/CH3OH (2 ml; 3/1) and
pyridine (24 µl; 0.346 mmol) was poured into the glass column. Two
molar excess of cysteamine hydrochloride (78.6 mg, 0.692 mmol; Fluka,
St. Quentin Fallavier, France) was then added to the resin. The column
was connected to a rotary evaporator and stirred overnight at room temperature. The solvent was filtered off and the resin washed as
described above, followed by two washes with
CH2Cl2/pyridine (100/5) to avoid the resin
sticking to the glass wall. At this stage the resin turned yellow. A
positive Kaiser test (15) showed the presence of amino groups on the
resin, and Ellmans' reagent (16) showed no presence of free thiol groups.
Reaction of Cysteamine Bound onto the Resin with Folic
Acid--
Folic acid (763 mg, 1.73 mmol; Fluka) was slowly added in
Me2SO (2 ml) heated to 50 °C, and diisopropylethylamine
(303 µl, 1.73 mmol) was added to the dry resin.
Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
was then added (PyBOPTM; 900 mg, 1.73 mmol; Novabiochem)
and the mixture was stirred overnight at 30 °C. The mixture was
filtered and the resin washed with Me2SO (5 × 10 ml),
N,N-dimethylformamide (5 × 10 ml),
CH2Cl2 (5 × 10 ml), and CH3OH
(5 × 10 ml) resulting in an orange resin.
Cleavage of the Folic Acid-Cysteamine Conjugate from the
Resin--
A mixture of CH2Cl2/trifluoroacetic
acid (10 ml; 97/3) was added to the resin and stirred for 30 min at
room temperature. The mixture was filtered and the resin washed with
CH2Cl2 (10 ml) followed by CH3OH
(10 ml). After 3 cycles of cleavage/wash the combined organic fractions
were concentrated under vaccum in the presence of toluene. The crude
product was precipitated by addition of 40 ml of acetonitrile,
centrifuged, and washed twice with diethyl ether before drying under
vaccum. An orange powder was obtained (183 mg) which was identified by
1H NMR and mass spectroscopy. 1H NMR (300 MHz,
DMSO-d6) 
8.75 (s, 1H, C7-H1),
7.64 (d, 2H, J = 8, C13-H1/C15-H1), 6.62 (d, 2H, J = 8, C12-H1/C16-H1), 4.52 (s,
2H, C9-H2), 4.28 (dd, 1H, C19-H1), 3.18 (m, 2H,
C25-H2), 2.8-2.4 (m, 4H,
C21-H2/C22-H2), 2.50 (m, 2H,
C26-H2), 1.25 (s, 1H, S27-H1). Mass
spectroscopy (fast atom bombardment):
C21H24N8O5S,
m/z [M-H] 499.54, found
499.2.
Synthesis of Folate-S-Gelonin
Gelonin (500 µl of 1 mg/ml in water; Sigma, Dorset, United
Kingdom) was mixed with sodium periodate (500 µl of 1 mg/ml in phosphate-buffered saline, pH 7.4, Sigma) and incubated at room temperature for 1 h. Free sodium periodate was removed from the solution by size exclusion chromatography (PD-10 column; Amersham Pharmacia Biotech, Little Chalfont, UK) and fractions containing gelonin were pooled. The solution was treated with PDPH (40 µl of 0.1 M PDPH in ethanol to 1 ml of gelonin solution;
Perbio, Tattenhall, UK) and incubated stirring at room temperature for 5 h. Free PDPH was removed from the solution by size exclusion chromatography (PD-10 column) and fractions containing gelonin were
pooled. The gelonin solution was then mixed, under reduced conditions,
with SH-folate (10-fold molar excess of SH-folate:gelonin) and HEPES
buffer (pH 8.3) to give an overall HEPES molarity of 50 mM.
The solution was incubated stirring overnight at 4 °C. Free folate
was removed from the solution by size exclusion chromatography (PD-10
column) and fractions containing gelonin were pooled. The protein
content was determined using the BCA protein assay (Sigma) and the
number of folate moieties per gelonin was calculated using spectroscopic analysis (
365 nm = 9120.1 M1 × cm1). The molar
incorporation of folate:gelonin was calculated to be ~1:1.
Synthesis of N-Hydroxysuccinimide-folate (NHS-folate)
NHS-folate was synthesized according to the method of Lee and
Low (17). Folic acid (5 g, 11.3 mmol; Sigma) was dissolved in
Me2SO (100 ml) and triethylamine (2.5 ml) and reacted with N-hydroxysuccinimide (2.6 g, 22.6 mmol) and
dicyclohexylcarbodiimide (4.7 g, 22.7 mmol) overnight at room
temperature. The solution was filtered, concentrated under reduced
pressure at 37 °C, and NHS-folate precipitated in diethyl ether
(yellow-orange precipitate). The NHS-folate was washed three times in
anhydrous ether, dried under vacuum, and stored as a powder at
20 °C. 1H NMR analysis confirmed the presence of
N-hydroxysuccinimide on the 
-carboxyl (67%) and
-carboxyl (33%) groups of folic acid (data not shown).
Synthesis of Folate-CO-Gelonin
Folate-CO-gelonin was prepared by incubating gelonin (1 mg/ml in
50 mM HEPES buffer, pH 8.5) with NHS-folate (8:1 molar
ratio of NHS-folate:gelonin) stirring at 4 °C overnight. The
solution was spun to remove precipitates and free folate removed by
size exclusion chromatography (PD-10 column). The protein content was determined using the BCA protein assay and the number of folate moieties per gelonin was calculated using spectroscopic analysis (365 nm = 9120.1 M1 × cm1). The molar incorporation ratio of folate:gelonin was
calculated to be ~1:1.
Competitive Inhibition of [3H]Folic Acid Binding to
Cells by Folate-Gelonin
A sterile tube containing PBS (0.5 ml, pH 7.4), HeLa cells
(100,000 total), [3H]folic acid (approximately
109 M; Amersham Pharmacia Biotech), and
either folate-conjugated or unconjugated compounds (concentration
ranging from 0 to 20 µg/ml) was incubated at 4 °C for 30 min.
Cells were pelleted by centrifugation (2200 × g,
30 s), washed twice in PBS, and dissolved in urea buffer (9 M urea, 50 mM Tris-HCl, 0.15 M
-mercaptoethanol, pH 7.5). The samples were diluted in Ultima Flo AF
scintillation fluid (4 ml) and assayed for radioactivity in a Packard
1900TR liquid scintillation analyser (Packard, Berkshire, UK).
Cell-free Translational Reticulocyte Lysate Assay
Gelonin, folate-S-gelonin, or folate-CO-gelonin (concentrations
ranging from 1011 to 107 M) was
added to a sterile vial containing rabbit reticulocyte lysate reagent
(17 µl) and SP6 luciferase mRNA-luc (1 µg; Promega, Southampton, UK). The samples were incubated at 37 °C for 90 min. An
aliquot of each sample (1 µl) was assayed for luciferase activity by
the following method. Luciferin (250 µl of a stock solution consisting of: 10 mg of beetle luciferin; 0.47 ml of 1 M glycylglycine, pH 8.0; 15 ml water) was added to
luciferase assay reagent (5 ml: consisting of 1 M
glycylglycine, pH 8.0 (2.0 ml), 100 mM MgCl2 (1 ml), 500 mM EDTA (20 µl), dithiothreitol (50.8 mg), ATP
(27.8 mg), coenzyme A (21.3 mg), water (99.0 ml); final pH 8.0). This luciferin/luciferase reagent (100 µl) was added to the sample (1 µl) and the luminescence intergrated over 10 s in a luminometer (Lumat LB 9507; Berthold, Pforzheim, Germany).
Protein Synthesis Inhibition in HeLa and Skov3 Cells in Vitro
HeLa or Skov3 cells were grown in a 96-well plate (10,000 cells/well) in folate-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.). Gelonin or folate-S-gelonin (107
M) was added to each well and the cells incubated at
37 °C, 5% CO2 for various lengths of time. Media was
removed, the cells washed in PBS (pH 7.4, 2 washes) and methionine and
cysteine-free media (Sigma, Dorset, UK) was added to the cells for 30 min at 37 °C, 5% CO2.
[35S]Methionine/cysteine mixture (5 µCi/well; Amersham
Pharmacia Biotech) was added to the media and incubated at 37 °C,
5% CO2 for 1 h. Cells were then washed in ice-cold
PBS. Lysis buffer was added to each well (50 µl; Promega) and
incubated on ice for 30 min. Bovine serum albumin (50 µl of 10 mg/ml
in water; Sigma) was added to the lysis buffer followed by ice-cold
trichloroacetic acid (100% w/v; Sigma) and incubated on ice for 30 min. The precipitate was centrifuged (1,000 × g; 1 min) and washed twice in trichloroacetic acid (10% w/v). The
precipitate was resuspended in water and assayed for radioactivity in a
Packard 1900TR liquid scintillation counter (Packard, Berkshire, UK).
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RESULTS |
Synthesis of Thiol-derivatized Folate (SH-folate)--
We have
developed solid phase chemistry for the preparation of thiolated folic
acid. Folic acid was functionalized with cysteamine to introduce a
thiol group and the hydrochloride cysteamine was bound to the resin via
the formation of a thioether linkage (Fig. 1, 1). Under these conditions
only the thiol can react with the chlorotrityl moieties, as the amino
group is protonated. After cysteamine grafting onto the resin a Kaiser
test showed the presence of amino groups. No thiol groups were detected
with Ellman's reagent, proving the formation of a thioether bond.
Folic acid was reacted with PyBOPTM to generate an active
ester in situ and grafted onto the resin via formation of an
amide bond (Fig. 1, 2). The final step consisted of the
cleavage of the functionalized folic acid from the resin under acidic
conditions (Fig. 1, 3). The use of a methoxy chlorotrityl resin allows the cleavage of thiol derivatized folic acid from the
resin with a mildly acid solvent (3% trifluoroacetic acid in
CH2Cl2). Strong acidic conditions were avoided
as this lead to the breakage of the folic acid amide bond (data not
shown). The global yield for the conversion of folate acid to the
folate-thiol (Fig. 1, 3) was 63% (i.e. 86% per
step) as determined by thiol titration with Ellman's reagent (16).
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Fig. 1.
Solid phase synthesis of folate-SH.
Reagents: (a)
CH2Cl2:CH3OH, 3:1, pyridine 1 eq.,
room temperature, overnight; (b) Me2SO,
folic acid 5 eq, DIEA 5 eq., PyBOPTM 5 eq., 30 °C, o/n;
(c) CH2Cl2:trifluoroacetic acid,
97:3, room temperature, 3 × 30 min.
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Synthesis of Folate-S-Gelonin--
Folate-S-gelonin was prepared
with initial treatment of gelonin with sodium periodate to oxidize the
carbohydrate residues. The recovered protein yield after treatment with
NaIO4 was 95%. PDPH (Fig. 2,
5) is a heterobifunctional cross-linker containing an
oxidized carbohydrate-specific hydrazide and a pyridylthio reactive
group. Addition of PDPH to NaIO4-treated gelonin enabled conjugation of PDPH to oxidized carbohydrate residues and incorporation of a sulfhydryl-reactive group into the conjugate (Fig. 2,
6). The recovered protein yield from this step was 94%.
Folate was conjugated to gelonin by reacting SH-folate with the
pyridyldithio group of PDPH (Fig. 2, 7). The recovered
protein yield was 87% and the folate:gelonin ratio was ~1:1.
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Fig. 2.
Synthesis of folate-gelonin conjugates.
Formation of folate-gelonin conjugates via amino group (4, folate-CO-gelonin) or carbohydrate (5-7, folate-S-gelonin)
modification. Reagents for folate-CO-gelonin: (a)
Me2SO, NHS 2 eq., DCC 2 eq., Et3N 2 eq., room
temperature, o/n; (b) 50 mM HEPES, pH 8.5, 1 mg/ml gelonin (gelonin:folate-NHS, 1:8), 4 °C, o/n. Reagents for
folate-S-gelonin: (c) PBS, pH 7.4, 1 mg/ml gelonin, 1 mg/ml
NaIO4, room temperature, 1 h; (d) room
temperature, 5 h; (e) HEPES 50 mM,
N2, folate-SH 10 eq., 4 °C, o/n.
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Inhibition of [3H]Folic Acid Binding to HeLa Cells by
Folate-Gelonin Conjugates--
The effect of two different conjugation
chemistries on the ability of folate-gelonin to bind cellular folate
receptors were investigated using a [3H]folic acid
binding assay. The addition of folate conjugates to samples containing
[3H]folic acid has been previously shown to inhibit the
binding of the radioisotope to folate receptor positive cell lines (18, 19). Both folate-CO-gelonin and folate-S-gelonin inhibited
[3H]folic acid binding to HeLa cells (Fig.
3), with the latter showing slightly
higher inhibition levels at lower concentrations of the conjugate (0.4 µg/ml). Higher levels of folate conjugates (20 µg/ml) almost
totally inhibit radioisotope binding to the cells. In contrast,
unmodified gelonin at all concentrations was unable to inhibit
[3H]folic acid binding to HeLa cells, showing that the
folate-gelonin conjugates were binding to folate receptors present on
the HeLa cells. The folate-gelonin conjugates also inhibited the
binding of [3H]folic acid to the same level as a
folate-albumin-FITC conjugate (Table I).
This conjugate has an affinity for the FR of 1010
M, as measured by Scatchard analysis (data not shown).
Therefore the ability of folate to bind to its receptor is not impeded
by either of the conjugation procedures employed.
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Fig. 3.
Competitive inhibition of
[3H]folic acid binding to HeLa cells by
folate-gelonin. HeLa cells grown in folate-free Dulbecco's
modified Eagle's medium were incubated with [3H]folic
acid and gelonin ( ), folate-S-gelonin ( ), or folate-CO-gelonin
( ) at 4 °C for 30 min. Cells were pelleted, washed twice in PBS,
and dissolved in urea buffer. Radioactivity was assayed in a liquid
scintillation analyzer. Error bars show the ± S.D. of
three independent experiments.
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Table I
Inhibitory concentration (IC50) of folate-protein conjugates in
a [3H]folic acid cell binding assay
Folate-albumin-FITC is known to exhibit an affinity for the folate
receptor of 1010 M. The method described in the
legend to Fig. 3 was used except HeLa cells were incubated with
folate-albumin-FITC, folate-S-gelonin, or folate-CO-gelonin
(1010 to 106 M protein conjugate).
Errors are ± S.D. of three independent experiments. Values are
for the molarity of the protein.
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Inhibition of Protein Synthesis by Folate-Gelonin Conjugates in a
Cell-free Translational Assay--
The effects of different
conjugation chemistries on the ribosomal-inactivating properties of
folate-gelonin were investigated using a rabbit reticulocyte lysate
assay (Fig. 4). In this assay mRNA
encoding for luciferase is translated into protein which can be
detected using luminometry. Ribosomal inhibition by gelonin leads to
decreased mRNA translation, and thus lowered luciferase levels,
which can be quantified in the linear scale of the luminometer. Folate-S-gelonin exhibited a similar translational inactivating profile
compared with unmodified gelonin, with ribosomal inactivation apparent
at 1010 M and optimal at 108
M for both samples. There was no significant difference
between the inhibitory concentration (IC50) of these
samples (Table II), showing that
conjugation of folate via gelonin carbohydrate residues has no
detrimental effect on the ribosomal inactivating properties of the
toxin. In contrast, folate-CO-gelonin displayed a significantly altered
translational inactivating profile compared with both gelonin and
folate-S-gelonin, with ribosomal inactivation apparent at
107 M. The IC50 of
folate-CO-gelonin was over 225-fold higher than folate-S-gelonin (Table
II), showing that the amino conjugation procedure drastically reduces
the ribosomal inactivating property of the toxin.
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Fig. 4.
Effect of folate-gelonin conjugates on the
inhibition of protein synthesis in a cell-free translational
assay. Gelonin (), folate-S-gelonin (), folate-CO-gelonin
(), or water ( ) was added to a rabbit reticulocyte lysate assay
containing SP6 luciferase mRNA-luc and incubated at
37 °C for 90 min. An aliquot of each sample was assayed for
luciferase activity in a luminometer. Graph shows the
results from a single experiment.
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Table II
Inhibitory concentration (IC50) of gelonin conjugates in a
cell-free translational assay
The method as described in the legend of Fig. 4 was used. Errors are
the ± S.D. of three independent experiments.
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Inhibition of Protein Synthesis by Folate-S-Gelonin in HeLa and
Skov3 Cell Lines in Vitro--
The effect of folate-S-gelonin on
protein synthesis within intact cells was quantified by incorporation
of radiolabeled amino acids into cellular protein. HeLa cells treated
with 107 M folate-S-gelonin (Fig.
5a) exhibited over 50%
inhibition of protein synthesis after 1 h compared to untreated
cells. The protein synthesis inhibition remained at this level up to
2 h and subsequently increased to ~75% after 5 h. Protein
synthesis levels were restored to the same level as untreated cells
after 8 h. In contrast, cells treated with gelonin showed no
change in protein synthesis after incubation with the toxin for 2 h. However, after 5 h protein synthesis levels were increased and
reached almost 300% compared with untreated cells after 8 h.
Skov3 cells treated with 107 M of
folate-S-gelonin (Fig. 5b) exhibited a substantial decrease in translational levels after 1 h. After 8 h the level
remained below 40% of protein translation compared with untreated
cells. Skov3 cells treated with gelonin also exhibited decreased
protein translation after 1 h. However, by 2 h there was
evidence of a recovery of protein synthesis, and by 8 h the level
was almost 400% compared with untreated cells. Therefore the
folate-S-gelonin is causing a decrease in protein synthesis in both
cell lines compared with the gelonin control, probably due to increased
cellular uptake and/or cytoplasmic delivery of the toxin. We found that 0.01 µM of either folate-S-gelonin or gelonin had no
significant effect on HeLa or Skov3 protein synthesis over 8 h
(data not shown). Therefore the minimum concentration of
folate-S-gelonin that inhibits protein synthesis in these cell lines is
between 0.01 and 0.1 µM. Higher concentrations (1 µM) of either folate-S-gelonin or gelonin resulted in
significant inhibition of protein synthesis in both cell lines over
8 h (data not shown).
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Fig. 5.
Effect of folate-S-gelonin on protein
synthesis levels in HeLa and Skov3 cell lines in
vitro. HeLa (a) or Skov3 (b)
cells lines in a 96-well plate (10,000 cells/well) were exposed to
either gelonin () or folate-S-gelonin () (107
M) for various lengths of time at 37 °C, 5%
CO2. Cells were washed in PBS and methionine/cysteine-free
media was added for 30 min at 37 °C, 5% CO2.
[35S]Methionine/cysteine mixture was then added to the
cells for 1 h at 37 °C, 5% CO2. Cells were washed
in ice-cold PBS, lysed, and total protein precipitated in
trichloroacetic acid. Radioactivity was assayed in a liquid
scintillation analyzer. Error bars show the ±S.D. of three
independent experiments.
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DISCUSSION |
The formation of targeted toxins presents several problems of a
chemical and biochemical nature. First, the binding of the ligand to
the toxin must enable relatively simple and reproducible conjugation.
Second, the conjugation must occur at a site that will not impede
receptor-ligand interactions nor affect the activity of the toxin. Most
targeted toxins cause a decrease in at least one of these areas (3, 13,
14, 20, 21), therefore there is a pressing need for the design of
alternative conjugation chemistries. In the results presented here we
show that attachment of folate via gelonin carbohydrate residues
retains both toxin activity and ligand binding affinity. This work has
implications for the targeting of other glycoproteins via the FR and
also for other ligands where similar chemistries can be performed.
One advantage of using folate as a targeting ligand is the selective
up-regulation of the FR on many epithelial tumors (8). Indeed, Toffoli
and others (9) have shown that the folate receptor is up-regulated on
90% of ovarian carcinomas, making the FR a potential target for this
high mortality cancer. It has recently been described that tumor cells
isolated from the ascitic fluid of ovarian cancer patients internalize
folate-conjugated albumin via the FR (19). Therefore folate-targeted
toxins have the potential to be useful within a clinical setting. The
targeting of gelonin to the cytoplasm of cells using other targeting
ligands, such as transferrin (5) and antibodies (23), has been shown to lead to target cell death. However, the receptors for these ligands are
expressed on a range of neoplastic and non-neoplastic tissues. Therefore the use of these ligands may give rise to toxin uptake in
non-target cells, a phenomenon not observed with folate-targeted conjugates (19, 24, 25).
It has been reported that folate linked to a variety of proteins via
amino conjugation has no effect on the binding of the conjugates to the
FR (26). We show that this is also true of folate-CO-gelonin and
folate-S-gelonin. It has recently been shown, contrary to popular
belief, that folate linked to proteins via the - or -carboxyl
group retain both receptor affinity and the ability to trigger
endocytosis of the FR (27). Additionally, removal of the remaining
unconjugated carboxyl group on folate-protein conjugates had no effect
on their cell binding or uptake (27). Therefore folate is a versatile
molecule that can withstand various conjugation chemistries. Such
versatility may be advantageous for the formation of alternative
folate-targeted proteins for use as cancer therapies.
The effect of the two conjugation procedures on the activity of
folate-gelonin in a cell-free translational assay is striking. Folate-S-gelonin exhibits the same IC50 as unmodified
gelonin, implying that the presence of folate on the toxin has no
detrimental effect. Since carbohydrate modification of plant toxins
have not been reported before it is difficult to conclude the reasons
for the retention of toxin activity, however, it is possible that the
small size of folate is a factor. For example, it has recently been
shown that the attachment of antibodies via terminal galactose residues
of cobra venom factor leads to a 25% reduction in cobra venom factor
activity (20). The large size of the antibody (150 kDa) compared with
cobra venom factor (137 kDa) may lead to steric hindrance or physical
obstruction of the cobra venom factor activity. Thus folate may not be
large enough to exert steric hindrance on the toxin. Alternatively the
folate may be located in a region of the toxin that is not dependent on
activity, although the conjugation of a larger ligand to the same site
could lead to steric hindrance.
Conjugation of folate to gelonin via amino modification results in a
225-fold decrease in activity of the toxin. The effect of amino
modification of gelonin on the ribosomal inactivating properties of the
toxin has been previously reported, with the attachment of conconavilin
A (3) or amino modification of the toxin (13) both leading to decreased
ribosomal inactivating properties. The ribosomal inactivating
properties of the toxin are extremely dependent on lysine residues
within the protein (13). Since NHS-folate reacts with lysines it is
likely that this is the reason for the loss of toxin activity in the
folate-CO-gelonin conjugate. Trypsin digest of folate-CO-gelonin
followed by mass spectroscopy analysis revealed no evidence of folate
binding to a single amino residue (data not shown). Therefore, it is
likely that folate is linked to a number of sites within the toxin,
resulting in a heterogeneous conjugate population. Whether the activity of folate-CO-gelonin at 107 M (Fig. 4) is due
to a small population of conjugates that retain 100% activity or an
overall reduction in activity of the total population has yet to be
established. The activity of folate-CO-gelonin at 107
M may also be due to the presence of unmodified gelonin
within the sample.
It has been previously shown that gelonin is relatively non-toxic to
HeLa cells (3), and we show that 107 M
gelonin does not lead to decreased protein synthesis in this cell line.
However, the ovarian cancer cell line Skov3 is more sensitive to
gelonin, with decreased protein synthesis apparent at 107
M of toxin. These differential effects may be due to
altered uptake of the unconjugated toxin or reflect its differential
intracellular trafficking within these cell lines. An unexpected effect
of unmodified gelonin in both cell lines is the increase in protein
synthesis above the control following toxin challenge. This could be
due to increased translation of mRNA already present in the
cytoplasm in an attempt to equilibrate cellular protein levels.
Alternatively, it could reflect an increase in stress response factors
stimulated by the presence of the toxin or its downstream by-products.
This effect is also seen with folate-S-gelonin, although it is most prominent in the HeLa cell line.
The prolonged protein synthesis inhibition exhibited by
folate-S-gelonin in both cell lines shows that the conjugation of folate to the toxin is having an enhanced effect on the toxin activity.
This may reflect increased uptake and cytoplasmic delivery of
folate-S-gelonin compared with unmodified gelonin. Alternatively the
half-life of folate-S-gelonin may be increased upon conjugation to
folate, perhaps due to the folate moiety inhibiting cellular protein
degradation processes. HeLa and Skov3 cell lines exhibit the same level
of uptake of folate-protein conjugates over 8 h.2 If this is also true of
folate-S-gelonin it suggests that the increased protein inhibition in
Skov3 compared with HeLa cells is due to the increased sensitivity of
Skov3 to the toxin, rather than increased uptake of the conjugate.
In a clinical setting it is desirable to administer the lowest level of
drug possible to achieve a therapeutic effect, thus decreasing any side
effects to the patient and the overall cost of the treatment. There are
several approaches that may be useful for increasing the potency of
folate-S-gelonin. Conjugation of folate to multiple carbohydrate
residues on gelonin is possible by increasing the stringency of the
sugar oxidation reaction. Alternatively, the use of a branched peptide,
such as di- or tri-lysine, would present several connecting points
allowing the grafting of 2 or more folates per sugar residue. Such
approaches may increase the avidity of folate-S-gelonin for the FR, as
demonstrated by the attachment of multiple folate moieties to liposomes
(8). We have recently shown that the uptake of folate-albumin-FITC conjugates in tumor cells freshly isolated from the ascitic fluid of
ovarian cancer patients is higher than that seen in both HeLa and Skov3
cell lines (up to 22-fold higher (19)).2 Therefore, the
level of toxin required to inhibit protein synthesis in tumor cells
within a clinical setting may be much lower than the levels used in
this study. This, along with the optimization of the folate-S-gelonin
conjugate, may enable the administration of much lower levels of the
toxin to patients to achieve a therapeutic effect.
Deglycosylated plant toxins exhibit increased half-lives in the blood
(28-30), probably due to decreased phagocytosis by scavenging receptors in the liver (28, 29, 31). Therefore it is possible that the
conjugation of folate to gelonin carbohydrate residues will lead to an
increase in the blood circulation of the conjugate compared with toxin
alone. Intravenous administration of folate-targeted conjugates to mice
has been shown to lead to tumor accumulation of the conjugates, with
minimal accumulation in other tissues (24, 25). Therefore
folate-S-gelonin may be a suitable candidate as an intravenous
administered cancer therapy. Alternatively, the conjugate could be
administered by intraperitoneal or intratumoral injection, thus
providing an additional means of selectivity. The use of the conjugate
as an intraperitoneal administered therapy is promising since it has
already been shown that folate conjugates are internalized by tumor
cells isolated from ascitic fluid of ovarian cancer patients (19).
We have shown that the conjugation of folate to gelonin carbohydrate
residues enables targeting of active toxin to intact cells. The
application of this method of ligand conjugation to other plant toxins
or glycoproteins may enable the formation of more potent anti-cancer
therapies. Additionally, where similar chemistries are possible the
conjugation of alternative ligands to glycoproteins may enable protein
targeting via other cellular receptors up-regulated on cancer cells. It
will be interesting to see if this method of conjugation can be
translated to peptides capable of binding and internalizing via cell
surface receptors (22). The small size of these targeting ligands and
the recent increase in the availability of such peptides may allow the
development of therapeutic proteins targeted to a variety of cell
types. Such studies are currently under investigation within our laboratory.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Immunology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, United Kingdom. Tel.:
44-161-446-3157; Fax: 44-161-446-3109;
E-mail:wardcm@hotmail.com.
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