[CANCER RESEARCH54, 6387-6394, December 15, 1994]
Quantitative Analysis of Protein Synthesis Inhibition by
Transferrin-Toxin Conjugates1
Parvin
T. Yazdi and Regina
Department of Chemical Engineering.
M. Murphy@
University of Wisconsin, Madison, Wisconsin 53706
ABSTRACT
bind to cell membranes in the absence of a targeting agent and thus
has low nonspecific toxicity (20). CRM1O7 is a mutant form of DT3
A mathematical model was developed which relates the time and con
which has comparable toxic activity as DT in cell-free systems but
centration dependence of protein synthesis inhibition of an Immunotoxin
binds with 8000-fold less affinitY to cell surfaces and demonstrates
to the properties of the targeting agent and the conjugated toxin. The role
10,000-fold less toxicity than DT on whole cells (21).
of the targeting agent and that of the toxin in determining the CYtOtOxICItY
We developed a mathematical model which correlates the protein
were separated in this model by describing protein synthesis inhibition as
a function of a cellular trafficking variable, which is calculated from the synthesis inhibition kinetics of transferrin-toxin conjugates with the
cellular trafficking characteristics of transferrin and the translocation
trafficking parameters of the targeting agent, and a protein synthesis
and enzymatic properties of the toxin. An equation was derived which
Inhibition constant, which Is a property ofthe translocatlon and enzymatic
rate constants of the toxin. Transferrin cellular trafficking parameters
relates protein synthesis inhibition to the double integral over time of
were determined experimentally for HeLa and SK-MEL-2 cells. PrOtein the intracellular transferrin concentration and a constant dependent
synthesis inhibition of transferrin-gelonin
and transferrin-CRM1O7
con
only on the toxin properties. We show that all of our protein synthesis
jugates in both cell lines was measured as a function of time and concen
inhibition data can be explained by this model.
tration. Analysis of the data showed that the model was a good represen
tation of the experimental results, and correctly explained cell line
differences in sensitivitY to transferrin-toxin conjugates. The translocation
rate constant for transferrln-CRM1O7 was approximately 3000 tImes MATERIALS AND METhODS
greater than that for transferrin-gelonin. The model may be useful in
Cell Lines. Human melanoma cell line SK-MEL-2 and human cervical
understanding
the factors
that influence
immunotoxin
efficacy
and in
designing more lethal hnmunotoxins.
INTRODUCTION
Immunotoxins, which are plant or bacterial toxins conjugated to
antibodies or ligands that preferentially bind to tumor cells, have been
used in clinical trials, animal experiments, and in vitro culture systems
to specifically attack tumor cells (1—4).Numerous immunotoxins
have been produced, with widely varying efficacies. In order to kill
cells, immunotoxins must direct their toxin moiety into the cytosol,
where the toxin can inhibit protein synthesis. The influence of factors
such as antigen density, binding affinity, internalization rate, degra
dation rate, and intracellular pathway on the cytotoxicity of immuno
toxins has been qualitatively investigated (5—11).Currently, there are
no universally valid methods for quantitatively explaining how the
cellular trafficking pathway of the antibody, coupled with the intrinsic
propertiesof the toxin, dictatethe rate of transportof immunotoxins
from the extracellular medium into the cytosol.
In work presented here, we measured transferrin trafficking kinetics
and the kinetics of protein synthesis inhibition of two transferrin-toxin
conjugates in two human tumor cell lines (HeLa and SK-MEL-2).
Transferrin is an attractive targeting agent for anticancer immunotox
ins because the transferrin receptor is generally expressed at high
densities on malignant cells (12), and drug-resistant tumor cells may
carcinoma cell line HeLa (American Type Culture Collection, Rockville, MD)
were cultured as monolayers in MEM supplemented with 10% FBS, 3.6 mM
L-glutamine, 100 units/mI penicillin, and 100 p@@ml
streptomycin in a humid
ified incubator containing 5% CO2. Medium components were all purchased
from GIBCO (Gaithersburg, MD).
Transfemn. Human difertic transferrin was purchased from Calbiochem
(San Diego, CA). Size-exclusion HPLC analysis of transferrin indicated the
presenceof approximately10% impuritiesin the sample.Transferrinwas purified
on a SephadexG-150(Pharmacin,Piscataway,NJ) columnequilibratedwith PBS.
Purifiedtransferrinwas labeledwith ‘@I
(DuPont-NewEngland Nuclear,Boston,
MA) using lodo-beads (Pierce, Rockford, IL) following the manufacturer's direc
tions. Briefly,
transferrin
was reacted
with Na'@I
(1 mCi/S mg protein)
in the
presence of lode-beads for 10 miii at pH 7. Unreacted Na'@I was removed by
dialysis.Trichloroaceticacid-solubleactivityofthe radiolabeledtransferrinsample
was 3% of total activity. Concentration was determined by absorbance at 280 nan,
using an extinction
coefficient
of 1.41 ml mg'
cm@. The specific
radioactivity
of ‘@I-labeled
transferrinwas 1.5 X 119dpm/pg.
Steady-State Binding and Internalization Assay Cells were harvested
and resuspended in culture medium at a concentration of 40,000 cells/ml. From
this suspension, 6.6 ml were placed in each 60-mm tissue culture dish. After
24 h of culture,
medium
was removed,
and 3.3 ml of a solution
containing
expresshigher surface densitiesof transferrin receptor than drug
1@I-labeledtransferrin in 32% PBS and 68% fresh medium were added to each
dish. Samples were incubated on a shaker at 37°Cfor 1 h. The supernatant was
removed, and the radioactivity was measured in order to determine the con
centration of free transferrin in the medium. The cell monolayer was washed
three times with cold PBS and treated with 2.5 ml ofO.3% Pronase (Boehringer
Mannheim, Indianapolis, IN) in MEM at 4°Cfor 5—10
mm to detach cells. The
entire cell suspension was collected, and the radioactivity was counted to
susceptible
determine the total transferrin associated with cells. One ml of the Pronase cell
cells (13). Immunotoxins
constructed
from transferrin
or
antitransferrin receptor antibodies have been demonstrated to be
effective in inhibiting protein synthesis in several cell lines (14—19).
Two toxins, gelonin and CRM1O7, were chosen for investigation.
Gelonin, a plant-derived ribosome-inactivating protein, is unable to
suspension was incubated at 4°Cfor 30 min to remove surface-bound proteins.
The sample was then centrifuged,
and the supernatant was counted to deter
mine surface-bound transferrin. The cell pellet was washed once with PBS and
measured for internal radioactivity.
Nonspecific binding of ‘@I-labeledtransferrin was measured as described
above in the presence of excess (100-fold) unlabeled transferrin. The radioac
Received 7/21/94; accepted 10/13/94.
tivity measured
Thecostsof publicationof thisarticleweredefrayedin partby the paymentof page
transferrin that was not removed during the washing process. Nonspecifically
charges. This article must therefore be hereby marked advertisement in accordance with
as nonspecifically
bound transferrin
also included
any free
18 U.S.C. Section 1734 solely to indicate this fact.
1
This
work
was
supported
by
National
Science
Foundation
Presidential
3 The abbreviations used are: DT, diphtheria toxin; EF-2, elongation factor 2; FBS,
Young
Investigator Award BCS-9057661.
2 To whom requests for reprints should be addressed,at University ofWisconsin, 1415
Johnson Drive, Madison, WI 53706.
fetal bovine serum; SPDP, N-succinimidyl-3-(2-pyridyldithio)propionate; Me2SO,
dimethyl sulfoxide; ‘fl-Gel,transferrin-gelonin conjugate; Tf-CRM1O7, transferrin
CRM1O7conjugate; CTV, cellular trafficking variable.
6387
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QUANTITATIVEANALYSIS OF IMMUNOTOXINS
bound transferrin did not internalize, as indicated by the complete release of
where [TfRu] @5
the total number of transferrin binding sites on the cell surface.
cell-associated
The total number of transferrin molecules associated per cell, [if,], is expressed
as:
‘@I-labeledtransferrin when Pronase treated.
To determine cell concentration, cells were plated onto culture dishes as
described
above
and cultured
for 24 h. Medium
was removed,
and 33
ml of
leucine-freemedium containing32% PBS were added to each dish. Dishes were
incubated at 37°Cfor various periods of time, cells were harvested, and cell
number was determined using a hemacytometer. A growth curve was fitted
through
the cell growth
data. Cell number
for the steady-state
assay was deter
[TJ] = [TI,,]+ [TI,,] + [Tj@]
All concentrations are in molecules/cell with the exception of [Tf] which is in
M. The following equationsareobtainedif [TJ',,Jand [TI1]are at steadystate:
mined from this growth curve to be 65 X 1Q@
HeLa and 3.7 X 10@SK-MEL-2
T
cells/dish.
of a constant
concentration
of
@I-labeled transferrin
(1 X 108
-
[Tf][TIRJ
[ .1@@]
—
[TfJ+ (k_1+ k.)/k1
Binding and Internalization Kinetics Assay. Monolayersamples were
prepared as described in the steady-state assay and incubated at 37°Cin the
presence
(i)
(1)
- k.[TI,,]
[T.fj---@---
M)
(k)
for varying times (1—30mm). Samples were then processed as described above
to determine
transferrin
acetic
period.
total, surface-bound,
degradation
acid-soluble
These
significantly
incubation
and internal
by cells
were
radioactivity
indicated
not shown).
that
Average
medium
transferrin
cell
number
period of this assay was determined
The kinetics
by measuring
in the incubation
measurements
(data
radioactivity.
determined
a 30-mm
j, and k were fitted to the steady-statebinding and internalizationdata to
not degraded
during
estimate steady-state parameters, whereas Equations e, g, h, and i were fitted
the 30-mm
to the binding and internalization kinetics data to estimate kinetic parameters.
The total number of transferrin binding sites on the cell surface and the
from the cell growth curve
described previously to be 5.7 X i0@and 3.5 X iO@cells/dish for HeLa and
SK-MEL-2, respectively.
Determination
binding,
of Transferrin
internalization,
Trafficking
and exocytosis
Parameters.
of transferrin
A model for
was used
Cellular processing parameters for transferrin were determined by fitting
model equations to transferrin binding and internalization data. Equations e, i,
over
was
of
trichloro
to analyze
nonspecific equilibrium binding constant estimated from the steady-state data
were used in the kinetic parameter estimation. The transferrin concentration in
the medium did not change, within experimental error, during the 30-mm
experimental period (data not shown). The medium contains both diferric and
cellular trafficking data. The model is identical to a model proposed previously
apotransferrin.
(22), with the exceptions that nonspecific binding was explicitly accounted for
1—4%over a 30-mm period. Therefore, the concentration of free diferric
transferrin in the medium was assumed constant over 30 mm and taken as the
and that transport of apotransferrin from internal vesicles to the surface and
subsequent dissociation from surface receptors was lumped into one step.
k—I
k2
Tf + m ;:@Tf,@
(b)
k—2
the model
showed
that [TI] dropped
only
program GREG (23,24) in the multiresponse mode. The program uses a
modified Newton method, starting from user-input initial guesses for the
parameters. Convergence was reached when the Newton corrections of the
estimated parameters were no larger than 0.1 times their respective uncertain
ties. Converged parameter values did not depend on the initial guessed values.
( .,
TI,,
using
average of [TJ] measured for all samples.
Parameter estimation was performed using the general purpose regression
(a)
Tf+TfR@Tf@
Simulations
For
estimation
of kinetic
parameters,
where
the model
equations
consisted
of
TJ@
@c1 ordinary differential equations, the DDASAC software package (25) was
used in combination with GREG. DDASAC uses a predictor-corrector
TI—>TI,+TIR
(d) integration method based on a variable-order, variable step approach to
solve systems of coupled ordinary differential and algebraic equations.
TI represents the concentration of diferric transferrinin the medium. TI,, and
Tf@, denote specifically
tively. TIR represents
and nonspecifically
bound
the unoccupied
transferrin
Construction
surface transferrin, respec
binding sites on the cell
surface. if, and TI0denote intracellular transferrin and dissociated apotrans
SPDP:transferrin.
ferrin, respectively. m represents nonspecific binding sites on the cell mem
brane. Binding assays indicated that nonspecific binding is a nonsaturable
process over the concentration range studied (data not shown). Therefore, m
was assumed constant and incorporated into a pseudo-first order reaction rate
constant, k2' . Experimental data (not shown) indicated that nonspecific binding
measured after varying time periods (1—30
mm) did not change within exper
imental
error. This constant
level of nonspecific
binding
may be due to fast
binding kinetics and/or the presence of residual free transferrin in the samples.
Therefore, Reaction b was assumed to be at equilibrium at all times. The
equilibrium value of [Tf@,]is calculated from:
[TI,,] =
where
C denotes
medium/dish),
cell concentration
and N is Avogadro's
number.
Purified trans
Unreacted
SPDP
was
removed
by passing
the
mixture
through a Sephadex G-25 (Pharmacia) chromatography column equilibrated
with 0.01 Msodium phosphate buffer (pH 7.2) containing 0.05 MNaC1.The
fraction containing SPDP-modified transferrin was collected and passed
through a blue Sepharose CL-6B (Pharmacia)
column equilibrated with the
buffer mentioned above. The effluent peak was collected, concentrated, and
run through a Sephadex G-25 column equilibrated with 0.1 M sodium phos
phate buffer (pH 7.5) containing 0.1 M NaCl, for buffer exchange. The
modification resulted in 0.7 dithiopyridyl groups per transferrin, as determined
by published methods (26).
Gelonin (Pierce) was prepared at 2 mg/mI in equal volumes of PBS and 0.8
(e)
r; [TII
in cellsfL
of Tf-Gel Immunotoxin.
M borate buffer, pH 8.5. This solution was mixed with 0.015 M 2-iminothiolane
N k@
@
and Purification
ferrin (10 mg/ml in PBS) was mixed with 0.02 M SPDP (Pierce) in Me2SO
(Sigma, St. Louis, MO) for 30 mm at room temperature
at a 2:1 molar ratio of
(cell
number/dish/volume
The following
equations
of
were
(Pierce) in borate buffer and incubated for 1 h at room temperature in the
presence of nmtrogen at a 20: 1 molar ratio of 2-iminothiolane:gelonin.
To
remove unreacted 2-iminothiolane, the mixture was run through a Sephadex
G-25 column equilibrated with nitrogen-flushed PBS. The modification re
sulted in the addition of 0.75 sulfhydryl groups per gelonin, as determined by
derived from the above reactions and equilibrium expressions:
published
methods (27). Modified
transferrin
and modified
gelonin were
mixed at a 1:1 molar ratio and incubated at 4°Cin the presence of nitrogen for
@i@1
= @{—k1[TI]([TIR,,]
—
[TI,,])+
d[T.f,j
—di-—
= k1[TI]([T.fRj—
[TI,,])—
(k_1+ k@)[TI,,]
d[TIJ
.—@ii.= k1[TI,,]
—
k0[T.fj
(1)
40 h. This mixture was then treated with 2 mM iodoacetamide (Sigma) for 1 h
(g)
at room temperature in order to block any remaining free sulthydryl groups.
The conjugation mixture was diluted by a factor of 22 in 0.01 M sodium
phosphate buffer (jH 7.2) containing 0.05 M NaCl and run through a blue
Sepharose
CL-6B
column
equilibrated
with the same buffer.
Gelonin-contain
ing components of the mixture were adsorbed to the column while unconju
@h' gated transferrin passed through. Tf-Gel conjugates of varying gelonin-to
‘. I
transferrin
ratios
were
eluted
from
the
column
on
increasing
the
mobile
6388
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phase
QUANTITATIVE
ANALYSIS
NaCI concentration to 0.3 M.Several fractions were collected and analyzed on
SDS-PAGE. The fraction consisting of 1:1 Tf-Gel conjugate and a small
amount of unconjugated gelonin was chosen for further purification. This
fraction was concentrated and passed through a Sephadex G-150 column
equilibrated with PBS to remove unconjugated gelonin. The purity of the final
sample was checked on SDS-PAGE. The final preparation contained only one
species with a molecular weight corresponding to a complex of one transferrin
and one gelonin. Concentration was determined by absorbance at 280 nm using
an extinction coefficient of 1.21 ml mgt cm'.
Construction and Purification of Tf-CRM1O7 Immunotoxin. Purified
transferrin (2 mg/mi in PBS) was modified as described for Tf-Gel immuno
@
toxin, except that the desalting column was equilibrated with 0.1 M sodium
phosphate buffer (jH 7.5) containing 0.1 M NaC1. CRM1O7 (Inland Labs,
Dallas, TX) at 1 mg/ml in PBS was diluted in 0.8 Mborate buffer (pH 8.5) to
a final concentration of 0.67 mg/mi. As described for Tf-Gel, the toxin was
modified, unreacted 2-iminothiolane was removed, modified transferrin and
modified CRM1O7 were reacted, and the remaining free sulfhydryl groups
wereblocked.The conjugationmixturewas passedthrougha SephadexG-200Superfine (Pharmacia) column equilibrated with PBS. Several fractions were
collected and analyzed on SDS-PAGE. The fraction consisting of mainly 1:1
Tf-CRM107 conjugate was chosen for protein synthesis inhibition studies.
Concentration
was determined
by UV absorbance
extinction coefficient of 1.36 ml mg'
@
Cell-free Translation
OF IMMUNOTOXINS
RESULTS
Steady-State Binding and Internalization. Transferrin binding
and internalization at 37°Cwere measured for HeLa and SK-MEL-2
cells as a function of transferrin concentration (Fig. 1). An incubation
period of 1 h was chosen for this assay since kinetic data indicated that
surface-bound and internal transferrin reached steady state before this
time. Model equations were fitted to the experimental data to estimate
steady-state parameters (Table 1). Experimental data and model fits
for the three measured variables are shown in Fig. 1.
HeLa cells express slightly more transferrin receptors on their
surface
compared
receptors
cells, but the difference
is not statis
in SK-MEL-2
cells. The ratio
+ k.)/k1 is a factor of 2
lower for SK-MEL-2 cells than for HeLa cells. According to
Equation j, this ratio influences the surface concentration of trans
at 280 nm using an
cm'.
Assay. The toxicity of Tf-Gel and of intact gelonin
20
‘
was measured in a cell-free translation assay. Varying concentrations of
Tf-Gel or gelonin were mixed with 10 @lrabbit reticulocyte lysate
I
‘
I
‘ I
• 1
A.
(GIBCO) and 3 gil lox translation mixture without leucine (GIBCO) in the
@I
.
___—e
15
presence of 10 mg/ml rabbit globin mRNA (GIBCO) and 80 mMpotassium
acetate at 37°Cfor 15 mm. One @Ciof 40—60Ci/mmol [3H]leucine
(DuPont-New England Nuclear) was added to each sample, and samples
were incubated at 37°Cfor 30 mm. The total volume of the reaction
mixture was 30 p1. Protein synthesis was stopped by placing samples on
ice. To hydrolyze the radioactive aminoacyl-tRNAs, samples were treated
with 0. 1 mg/ml bovine pancreatic RNase A (Boehringer Mannheim) at
37°Cfor 15 mm. Trichloroacetic acid-precipitable radioactivity was then
measured in a scintillation
to SK-MEL-2
tically significant. The ratio k1/k0 is much higher for HeLa cells. At
steady state, this ratio represents internal-to-surface transferrin con
centration. Therefore, at steady state only one third of total cell
associated transferrin is on the surface of HeLa cells, whereas trans
ferrin is roughly evenly distributed between surface and internal
10
5
I-
counter. The protein synthesis inhibitory activity
of Tf-Gel was similar to that of intact gelonin (data not shown).
Competitive
@
@
Inhibition
Assay. The binding affinity of Tf-Gel or Tf
0
CRM1O7was comparedto that of radiolabeledtransferrinin a competitive
inhibition assay. SK-MEL-2 monolayer culture was harvested using 5%
Me2SO in PBS containing 0.53 m@iEDTA (BoehringerMannheim).Cells
were resuspended in culture medium containing a constant concentration of
transferrin in the form of either ‘@l-labeled
transferrin or an equimolar mixture
of WI-labeledtransferrinandTf-Gel or Tf-CRM1O7.Sampleswere incubated
at 4°Cfor 1 h, centrifuged, then washed three times with PBS, after which the
radioactivity associated with the cell pelletwas measured. The results indicated
that WI-labeled transferrin, Tf-Gel, and Tf-CRM1O7all had similar affinities
for the transferrin receptor (data not shown).
Protein Synthesis Inhibition Assay. Cells were harvested and resus
pended in culture medium at a concentration of 40,000 cells/ml. One hundred
of the cell suspension was placed in each well of a 96-well plate. After 24
h of culture, medium was removed, and 50 @d
leucine-free medium containing
32% PBS and varying concentrations of Tf-Gel or Tf-CRM1O7were added to
each well. Plates were placed in the incubator for 2 to 24 h. Samples were then
@10
00
4
2
0
leucine, cell monolayerswere treated twice with 5% trichloroaceticacid at room
temperaturefor 10 mm to remove free [3H}leucine.Cells were then dissolved in
0.1 MKOH, and radioactivitywas measured in a scintillationcounter.
To determine cell concentration in the wells, cells were plated in 60-mm
medium was removed, and leucine-free medium containing 32% PBS was
added to obtain the same cell concentration as that used in the protein synthesis
inhibition assay. Dishes were incubated at 37°Cfor periods of time used in the
8
6
treated with 2 ,i.Ci [3Hjleucine at 37°Cfor 1 h. Before measuring incorporated
tissue culture dishes at the same concentration and cell-to-surface ratio as for
the protein synthesis inhibition assay. Dishes were then cultured for 24 h,
2
4
6
8
10
12
I O•
x Free Tf Concentration (M)
0
1
2345678
I O@x Frs Tf Concentration (M)
Fig. 1. Binding and internalization
of transferrin at steady state. HeLa (A) or SK
MEL-2 (B) cells were incubated with different concentrations of ‘@l-labeled
transferrin
at 37°Cfor 1 h as described in “Materials
and Methods.―
Number of total (•),surface
bound (0), and internal (A) transferrin molecules per cell were determined. Data shown
for HeLa cells are duplicate measurements of a single run, and those shown for SK
MEL-2 cells are single measurements of two runs. Data include both specific and
nonspecific binding. Equations e, i, j, and k and parameter values listed in Table 1 were
used to calculate the number of total (—
assay described above, cells were harvested, and cell concentration was de
termined. Average of all dishes indicated 1.4 ± 0.2 X l0@ HeLa and
7 ±1 X iø@SK-MEL-2 cells per well of a 96-well plate.
-), surface (—),
and intemal (———
transferrin molecules per cell. X-axis values should be divided by 10'@to obtain concen
tration in M. Y-axis values should be divided by iO@ to obtain concentration in molecules!
cell.
6389
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QUANTITATIVE
ANALYSIS
parametersParameter
Table 1 Transferrin steady-state
HeLaSK-MEL-2ITIR,II1,
@
surfacebindingsites/cetl
(k,+k1)/k,
k.Jk@
i0-@a
k@/k2
(M)
5.4±
0.6 X 10@―
6 ±2 X 10_8
2.3±0.1
1.4 ±0.1 x [email protected]
±0.8 X 1O@
3.0 ±0.5 x i0—@
0.7±0.1
7 ±1 X
Reportedrange is the 95% confidenceinterval.
OF
IMMUNOTOXINS
SK-MEL-2. The rate constant of release k0, however, has similar
values for the two systems. To check the consistency of the kinetic
and steady-state results, k/k0 and
+ k1)/k1 were calculated
from the kinetic parameters and compared to the values reported in
Table 1. The two sets of parameters showed statistical agreement
in all cases.
Protein
I
I
I
I
I
A.
3
I
.
.----..—--——
-
2
@
,4
-‘
S
—-‘r_@
I-.
Synthesis
Inhibition.
Unconjugated
transferrin,
gelonin,
or CRM1O7 did not inhibit protein synthesis relative to controls up
to the highest concentration studied for the corresponding immu
notoxin (data not shown). The kinetics of protein synthesis inhi
bition by Tf-Gel were measured for HeLa and SK-MEL-2 cells at
three concentrations of the immunotoxin (Fig. 3). At the lowest
concentration of Tf-Gel (1 X iO@ M), no significant cytotoxicity
was observed. SK-MEL-2 showed a slightly greater sensitivity to
1 X 10_8 M Tf-Gel compared to HeLa. The data indicated a lag
time of approximately 12 h. After 24 h at this concentration,
50—70%
proteinsynthesis
activityremained
relativetocontrols.
In
0
parametersParameterHeLaSK-MEL-rk,
Table 2 Transferrin kinetic
00
0@@-
It
u@.
I
I
I
I
B.
@
@
±0.50.6
±0.050.13
±0.020.13
±0.4 x
±
±
±0.03
aParameter
values
reported
forSK-MEL-2
aretheaverage
ofvalues
estimated
from
._.._____—-----..
I
00
±1 x 107b2.1
(M@ miic')4
[email protected]
0.2k1(miif')1.3
(miir')0.38
0.02k0
(min@')0.18
I
two sets of data.
@0'@
•
b Reported
range
is the 95%
confidence
interval.
I00
1
0
0
5
10
15
20
25
30
10
35
Time (mm)
@
@
Fig. 2. Kinetics of transferrin binding and internalization. HeLa (A) or SK-MEL-2
(B) cells were incubated with ‘@l-labeledtransferrin at 37°Cfor various periods of
time as described in “Materialsand Methods.― Number of total (•),surface-bound
•0
(0), and internal(A) transferrinmoleculesper cell weredetermined.Datashownfor
CC
HeLa cells are single measurements of two runs, and those shown for SK-MEL-2 cells
are duplicate measurements of a single run. Data include both specific and nonspecific
binding. Equations e, g, h, and i, [TfR,,] and k2'/k_2 from Table 1, and the parameters
listed in Table 2 were used to calculate the number of total (— -), surface (—),
and internal (—
—
—)
transferrin molecules per cell. Concentration of free transferrin in
the medium, determined by taking the average of the radioactivity in the supernatant
of all of the samples, was 7.8 X i0@ and 8.6 X i09 Mfor A and B, respectively.
Y-axisvaluesshouldbe dividedby i0@ to obtainconcentrationin molecules/cell.
@
A.
, I
I
@100
0‘.
a.
10
ferrin only when [Tf] is of the same order of magnitude or lower
than (k_1 + k,)/k1. The lower value for SK-MEL-2 suggests higher
surface binding compared to HeLa cells at relatively low trans
ferrin concentrations.
Binding and Internalization Kinetics. The kinetics of trans
B.
ferrin binding and internalization at 37°Cwere measured for HeLa
I
I
I
I
.
• i
i
I
•
.
• I
• • • .
I
.
•
I
and SK-MEL-2 cells (Fig. 2). Transferrin kinetic equations were
0
5
10
15
20
25
fitted to the experimental data to estimate kinetic parameters
Time (h)
(Table 2). Model calculations are presented in Fig. 2 along with the
Fig. 3. Protein synthesis inhibition by Tf-GeI HeLa (A) or SK-MEL-2 (B) cells were
data.
incubated in medium containing 1 X 10@ M(•),1 X iO@ M(0), or 1 X iO@ M(A)
The ratio
@,which represents the association constant for
Tf-Gel at 37°Cfor various periods of time as described in “Materialsand Methods.―
Protein
the binding of transferrin to the transferrin receptor at 37°C, is Controlsampleswerepreparedand treatedsimilarlyin the absenceof ‘Ft-Gel.
synthesis was determined by measuring [3Hlleucine incorporation. Each point shown is
3 ±1 X iO@M1 for both HeLa and SK-MEL-2. The internaliza
the averageof two to four replicateruns.Error bars, SD of replicateruns.Duplicate
measurements were taken in each run.
tion rate constant k. is significantly higher for HeLa compared to
6390
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0@@
QUANTITATIVE
ANALYSIS
OF IMMUNOTOXINS
step and no degradation or loss of toxin in the cytosol, the cytosolic
toxin concentration may be derived:
100
1..@
Tox1—@
Tox),,
10
(1)
[Tox1@j= Ic1@@, [ToxJ dt
(m)
1
@
@
where Tox1 and Tox@@1
denote toxin present in an intracellular vesic
ular compartment and that in the cytosol, respectively, and t, is the
length of time that cells are exposed to the immunotoxin.
Both gelonin and CRM1O7 act enzymatically. Assuming simple
Michaelis-Menten kinetics, the rate of substrate inactivation is given as:
01
•0
.C
CC
@100
@
d[S@@j
—@---
kio
a.
Similarly,
0.1
[Sa01
(n)
inactivation
of EF-2
by DT
in a cell-free
system
was
n was modified
to a form consistent
with these findings
by
assuming that Km >> [Sactl
0.01
0
5
10
15
20
25
d[S@@j —k1@,
—@ii-= [Tox@][S@@j
(0)
Time (h)
Fig. 4. Protein synthesis inhibition by Tf-CRM1O7. HeLa (A) or SK-MEL-2 (B)
cells were incubated in medium containing 3 X 10b0 M (X), 1 X 10b0 M (A),
3 X 10― M (ta), 1 X 10h1 M(•),or 3 X 1012 M (0) Tf-CRM1O7 at 37°Cfor
various periods of time as described in “Materials
and Methods.―Control samples
were prepared and treated similarly in the absence of Tf-CRM1O7. Protein synthesis
was determined by measuring [3Hlleucine incorporation. Each point shown is the
average of two to three replicate runs. Error bars, SD of replicate runs. Duplicate
measurements were taken in each run.
Integrating Equation o gives:
[S@1,], I k f'
-t:@
exp
—
f [Tox@]
dt
)
where [S0] is the initial substrate concentration
[5actl:1 @Sthe concentration
@
+
determined to be first order in the concentration of active EF-2 (29).
Equation
@
Km
where ESact]is the concentration of active substrate (ribosomes for
gelonin, EF-2 for CRM1O7), kcat @5
the rate constant of the reaction,
and Km is the Michaelis constant. Olsnes et a!. (28) studied the
kinetics of ribosome inactivation by two toxins, ricin A and abrin A,
in a cell-free system. The results indicated that the rate of ribosome
inactivation was first order in the concentration of active ribosomes.
I
@
=
of active
substrate
of ribosomes
at time
and
t.. Protein
synthesis is directly proportional to the concentration of intact
ribosomes (30) or active EF-2 (29). Using this proportionality and
the presenceof 1 x i07 MTf-Gel, however,HeLacellsdemonstrated substituting Equation m into Equation p yields:
a stronger sensitivity to the immunotoxin compared to SK-MEL-2 cells.
With a lag time of5 h, Tf-Gel inhibited 90% ofprotein synthesis in HeLa
PS = ex@(_
f@ i@: [Tox1]dt dt' )
(q)
cells after 24 h. Under the same conditions, SK-MEL-2 cells showed
33% protein synthesis after 24 h with a lag time of 10 h.
Kinetics of protein synthesis inhibition by Tf-CRM1O7 were meas
where PS is the fraction of protein synthesis relative to control measured
ured for HeLa and SK-MEL-2 cells at five concentrations (Fig. 4).
at time t1.The variables of integration t and t' represent time.
The lowest immunotoxin concentration(3 X 10 12M) demonstrated
no significant protein synthesis inhibition activity. At a concentration
of 1 x 10—
‘
‘M, the immunotoxin inhibited protein synthesis in
SK-MEL-2 cells by 50% after 24 h of incubation with a lag time of 10
h. Under the same conditions, protein synthesis in HeLa cells was
inhibited by only 20%. At the higher concentrations, HeLa cells were
slightly more sensitive to the immunotoxin than SK-MEL-2 cells.
Protein synthesis inhibition increased with increasing immunotoxin
concentration and incubation period. Lag time decreased with
increasing immunotoxin concentration. At the highest immuno
toxin concentration studied (3 X 10 10 M), the time required to
reach 50% protein synthesis inhibition in HeLa and SK-MEL-2
cells was only 2 and 3 h, respectively.
Correlating Protein Synthesis Inhibition with Cellular Traf
ficking. In order for protein synthesis inhibition to occur, the toxin
moiety must transbocate across the membrane of an intracellular
compartment
into the cytosol
(3). Assuming
a first-order
To determine [Tox1],we assumed (a) that the toxin remained associ
ated with transferrin throughout the intracellular pathway, and (b) that the
cellular trafficking characteristics of transferrinwere not modified by its
association with the toxin. Given these assumptions, then [Tox,]
[Tf@].
Justification for these assumptions is described in “Discussion.―
By making the substitution [Tox1] = [Tf1]in Equation q, the double
integral in that equation now represents a quantity that is a function of
transferrin cellular trafficking parameters only. This quantity will be
referred to as the CTV. Substituting kra@k@jKmwith a single param
eter, called the protein synthesis inhibition constant K@1, yields:
PS = exp(—K@1C1V)
(r)
where
translocation
Ii
1
cTv=
[Tf,]dtdt'
0
0
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(s)
QUANTITATIVEANALYSIS OF IMMUNOTOXINS
inhibition by gelonin in a cell-free system indicated a kesjKm value
equal to approximately 6 X 108 M1 min' (data not shown), sug
gesting similar enzyme kinetics for gelonin, ricin A, and abrin A
Using K@51= 3.6 X 10_12 cell molecule@ min2 for Tf-Gel,
k@,,JKm 7 X 108 M1 mm',
and a cell volume of 4.2 X 1012
@,
I20
I00
80
ktrans
was
constant
60
calculated
to
be
approximately
is seven orders of magnitude
1 X
smaller
1O_8
min'.
This
than k0, confirming
rate
the
assumption that the rate of translocation is negligible compared to the
rate of transferrin release from the cells and thereby justifying the
neglect of the translocating species in material balance and rate
equations. kcalKm for DT was calculated to be 8 X 108 M1 mn@
from the data obtained in a cell-free system by Moynihan and Pap
penheimer (29). Using this value and K@1 = 8.8 X i0@ cell mole
cule1 min2 for Tf-CRM1O7, k@ was calculated to be approxi
mately 3 X iO@5 min'. Thus, the translocation rate constant for
CRM1O7 conjugates is 3000-fold greater than that for gelonin. Even
in this case, however, k@rans
is nearly four orders of magnitude smaller
than k0.
40
20
0
DISCUSSION
80
60
40
20
0
0
1
2
1 0.8 x civ
3
4
5
6
7
(Molecule-min2/CeII)
Fig. 5. Protein synthesis in the presence of immunotoxins as a function of the CTV of
transferrin. C7V was calculated using Equations f, g, h, and s, [TfR@] reported in Table
1, and the parameters listed in Table 2. Protein synthesis in the presence ofTf-Gel (A) or
Tf-CRMIO7(B)was
plotted versus CfVfor HeLa(•)and SK-MEL-2(O)cell
lines. Data
An enormous variety of immunotoxins have been constructed and
tested with varying degrees of success. Factors related to the binding
and intracellular pathway of the targeting agent, including antigen
density, binding affinity, internalization rate, and degradation rate,
have been identified as influencing cytotoxicity (1). hnmunotoxin
efficacy is also affected by the choice of toxin (3). A quantitative
knowledge of the relative role of these factors is essential in order to
design more effective immunotoxins.
In work presented here, we have attempted to set up a flexible
framework for interpreting and predicting immunotoxin efficacy. To
our knowledge, this represents the first attempt to quantitatively relate
protein synthesis inhibition kinetics of an immunotoxin to cellular
processing of the corresponding targeting agent. Braham et aL (31)
developed a mathematical analysis of protein synthesis inhibition
kinetics and used the model to interpret data for a single immunotoxin.
points corresponding to protein synthesis values less than 1% of control were excluded.
@
The model neglected exocytosis and assumed that the toxin was
Values calculated using Equation r are represented for HeLa (—) and SK-MEL-2
consumed during the enzymatic reaction. There was no attempt to
(—
——).
To account for the 1-h incubation period in the presence of Tf-Gel and
independently measure the kinetics of antibody processing or to relate
[3H]leucine,30 mm were added to each time interval for C7V calculations. X-axis values
shouldbe dividedby 10―(A) and 10_B(B) to obtainCTVin molecule.min2/cell.
that to the resultant cytotoxicity.
The relationship between the transferrin cellular trafficking param
eters and the kinetics of protein synthesis inhibition by transferrin
cm, for transferrin was calculated by numerical integration of toxin conjugates described in Equation r is clearly representative of
Equations f, g, h, and s using the parameters listed in Table 2 and the
the experimental data. The model adequately describes the time and
number of surface transferrin binding sites presented in Table 1. The
concentration dependence of protein synthesis inhibition in each cell
conditions of the protein synthesis inhibition assay including cell
line (Fig. 5). The two cell lines demonstrated different transferrin
concentration, immunotoxin incubation period, and immunotoxin
processing characteristics and immunotoxin sensitivities. The rela
concentration were used in these calculations. Protein synthesis in the
tionship between PS and CTV for a given toxin is identical, however,
presence of Tf-Gel or Tf-CRM1O7 was then plotted versus CTV, and
suggesting that the differences in cellular trafficking parameters of
Equation r was fit to the data by nonlinear least-squares regression
transferrin between the two target cells quantitatively account for their
(Fig. 5). Data points corresponding to protein synthesis less than 1% different sensitivities to transferrin-toxin conjugates.
of control were excluded from the fit since below this level protein
A concentration-dependent lag time has been reported in several
studies of protein synthesis inhibition kinetics by immunotoxins (16,
synthesis is practically zero, and a plateau is reached in the plot of
protein synthesis versus CTV. Fig. 5 clearly shows that all of the data
32, 33). The cytotoxicity kinetics of Tf-Gel and Tf-CRM1O7 on HeLa
for a given transferrin-toxin conjugate collapse onto a single curve
and SK-MEL-2 cells were also characterized by concentration-depen
dent lag times (Figs. 3 and 4). The lag time disappears when protein
which is adequately described by Equation r. K@51determined from
Equation r is 3.6 ±0.4 X 10 12 cell molecule
min2 for Tf-Gel
synthesis data are plotted versus CTV (Fig. 5). The lag time is simply
and 8.8 ±0.9 x 108 cell molecule'
min2 for Tf-CRM1O7. These
due to the slow rate of appearance of the toxin in the cytosol at lower
results indicate that K@1 is a function only of the toxin, and is concentrations.
In developing our model, we made the critical supposition that the
independent of the trafficking parameters or the cell line.
concentration of internalized toxin equals that of internalized immu
Olsnes et a!. (28) estimated kk,,,t/KmtO be 7 X 108 M â€m̃m ‘
for
ribosome inactivation by ricin A and abrin A in a cell-free assay.
notoxin. This equivalence requires that two assumptions are valid: (a)
Preliminary data collected in our laboratory on protein synthesis
that the toxin remains associated with transferrin throughout the entire
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QUANTITAtiVE ANALYSIS OF IMMUNOTOXIN5
intracellular pathway or at least until it translocates, and (b) that the
cellular trafficking kinetics of the transferrin-toxin conjugate are the
same as those of the unconjugated transferrin. Several investigators
showed that the disulfide linkage between the targeting agent and the
toxin remained intact within cells (34—36),in support of the first
assumption. In particular, transferrin linked via SPDP to ricin A was
shown to remain associated throughout the intracellular pathway (36).
@
A different study, however, demonstrated intracellular release of ricin
A from an immunotoxin, although it was not clear whether the toxin
release was associated with translocation (9). Cleavage of the disul
fide bond between the two components immediately before, during, or
after toxin translocation does not conflict with the assumption above
due to the relatively negligible number of transbocating toxins.
The assumption that the cellular trafficking of transferrin-toxin was
the same as that of radiolabeled transferrin is also critical to our
model. We showed by competitive inhibition assays that the equilib
rium binding properties of transferrin were not altered upon conjuga
tion to gelonin or CRM1O7. Trafficking kinetics of Tf-Gel or Tf
CRM1O7 were not directly measured. However, in a careful study,
Raso et a!. (36) showed that receptor binding, intracellular localiza
tion, iron release, and exocytosis for transferrin-ricin A conjugates
was the same as that for unconjugated transferrin. Sasaki et a!. (37)
demonstrated that the differences in binding, internalization, and
intracellular processes between transferrin and transferrin-drug con
jugates were insignificant at 1:1 drug:transferrin molar ratios but
became more pronounced as the molar ratio increased (37). The
preparation used in these experiments was carefully synthesized and
purified such that it contained conjugates of 1:1 (toxin:transferrin)
molar ratio almost exclusively. This degree of conjugation should
result in negligible differences in the cellular trafficking characteris
tics of transferrin-toxin
conjugates compared to unconjugated
transferrin.
Two additional assumptions in the model require brief comment.
(a) Transferrin degradation was neglected based on experiments con
ducted for 30 mm. Over the 24-h incubation period of the protein
synthesis inhibition experiments, it is likely that some degradation in
fact occurs. This reduces the extracellular concentration of transferrin
able to bind to the cells. However, apotransferrin released into the
medium is also incapable of rebinding to the cell due to its signifi
canfly lowered affinity for the transferrin receptor at neutral pH. Thus,
transferrin that cycles through the cell, whether it be released as
apotransferrin or degraded, is removed from the pool in the extracel
lular space. (b) We assumed no intracellular degradation of the toxin.
Fragment A of diphtheria toxin is very resistant to degradation in the
cytosol (38). No comparable data are available, to our knowledge, on
the cytosolic stability of gelonin, although the toxin is stable to SDS,
urea, acid, and base and is resistant to proteolysis (20). It is yet
unknown whether the stability of a toxin is altered by chemical
conjugation. Finally, we note that protein synthesis inhibition does not
necessarily correlate with cell death (39).
The effect of individual trafficking parameters on the efficacy of
immunotoxins has been investigated (5—1
1). However, the sensitivity
of a target cell to an immunotoxin is determined by an interplay of the
targeting agent trafficking parameters. This can be seen by analyzing
the role of different transferrin trafficking parameters in the cytotox
icity of Tf-Gel. HeLa and SK-MEL-2 cells were similarly sensitive to
Tf-Gel at 1 X 108 M, whereas HeLa cells showed more sensitivity
to the immunotoxin at 1 X i0@ M compared to SK-MEL-2 cells
@
effect of kjk0. The steady-state equations (Equations j and k) provide
a simple, approximate description of how [Tf1] depends on the trans
ferrin trafficking parameters and the transferrin concentration in the
medium. The equations show, for example, that the internalization
rate constant
and therefore
of transferrin
rate constant
k. appears in both the numerator and the denominator,
the sensitivity of [Tf1]to k. depends on the concentration
in the medium, the affinity (k1/k1), and the association
k1.
The rateof translocationis by far the sloweststepin the processof
protein synthesis inhibition. For Tf-Gel, Ittransin approximately
1 X 10_8 min ‘,
seven orders of magnitude lower than k0. In other
words, for every 10 million Tf-Gel molecules that are exocytosed by
the cell, only 1 molecule of gelonin actually enters the cytosol. This
is an exceptionally inefficient process. It has been suggested that
retrograde transport through the Golgi stacks to the endoplasmic
reticulum is necessary for translocation of ribosome-inactivating tox
ins (40). The transferrin pathway intersects the secretory pathway at
the trans-Golgi network (41), although it is unclear if all transferrin or
just a small fraction cycles through this organelle. About 5—15%of
transferrin receptors are cycled via a slow route through the trans
Golgi (42). Pulse-chase experiments conducted in our laboratory (data
not shown) did not show any evidence for a measurable degree of
routing of transferrin via a slow pathway. The value that we deter
mined for k@05for Tf-Gel may be a direct measure of transport across
an endosomal or trans-Golgi membrane, or the combined retrograde
transport
up the secretory
pathway
followed
by translocation
through
the endoplasmic reticulum membrane. Toxins such as diphtheria toxin
mutants which contain transbocation domains (43) should be substan
tially more efficient. This is borne out by our estimate of ktrans for
Tf-CRM1O7 conjugates, which is 3000-fold higher than that for
Tf-Gel. Furthermore, translocation of DT requires a low pH environ
ment (43). The intracellular pathway of transferrin includes exposure
to a low pH endosome in order to release iron; thus, it is likely that
for CRM1O7 is a direct measure of the rate of crossing the
endosomal membrane.
For the direct comparison of trafficking kinetic data with protein
synthesis inhibition studies, it was important to keep the cells under
the same conditions in all experiments. All assays were done in the
presence of 6.8% FBS since FBS improved the accuracy of protein
synthesis measurements by enhancing cell attachment to culture
plates. The cellular trafficking parameters of transferrin reported here
may be influenced by the presence of bovine serum transferrin. The
conclusions reached, however, still hold since assay conditions were
identical in all experiments. Pulse-chase assays, in which the cells are
first cooled to 4°Cand then warmed to 37°C,were not used to derive
the kinetic parameters, because microtubule depolymerization, which
occurs with cooling to 4°C,can decrease transferrin internalization
rates (44). Metabolic inhibitors such as monensin are useful tools for
elucidating the intracellular pathways taken by ligands. These inhib
itors were not used in this work, however, because they do not always
completely eliminate a single pathway,and can have multiple con
founding effects on intracellular routing. Rather, use of appropriate
mathematical tools allowed us to estimate cellular processing param
eters of transferrin from simple experiments which did not require
temperature changes or metabolic inhibitors. Parameter values for
transferrin trafficking were generally of similar orders of magnitude
as those reported for other transformed cells in culture. Literature
(Fig. 3). The difference in cell line sensitivity at the higher immuno
values culled from a nonexhaustive list (22, 44—47)show the follow
toxin concentration is due to the greater numerical value of kjk0 for ing ranges of variables: 1 X 10@to 3 X 106 transferrin surface binding
HeLa. At the lower immunotoxin concentration, however, sites/cell, k1 of 1 X 106 to 1 x i0@ M1 min', k_1 of 0.01 to 0.09
1
k1)/k1
also
becomes
an important
parameter
in determining
min',
Ka ( k1/k_1) of 1 X i07 to 8 X 108 M', k. of 0.2 to 0.6
[Tfj; the lesser value of this ratio for SK-MEL-2 cells counteracts the
min @,
and k0 of 0.05 to 0.23 min'. The major difference is that our
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QUANTITATIVE
ANALYSIS
rate constants for association and dissociation from the surface are
somewhat higher than other literature values.
The model we have proposed here provides a flexible framework
for interpreting experimental data on in vitro immunotoxin efficacy.
The structure of the model is such that the properties of the toxin and
the properties of the targeting agent are described by two independent
variables, K@@1
and CTV. This is a useful way to separate the influence
of the two components of an immunotoxin, allowing a clearer inter
pretation of the relative role of each in the overall efficacy. The
protein synthesis inhibition constant K@@1
is primarily a function of the
toxin and describes the intrinsic enzymatic inactivation kinetics as
well as the translocation efficiency. Therefore, given a toxin (K@@1),
one can conceivably predict the cytotoxicity of an immunotoxin from
the parameters of the targeting agent (Cry). Alternatively, a param
eter sensitivity analysis of CTV for a given targeting agent would
show which steps in the trafficking patterns are most crucial to
enhancing cytotoxicity. This analysis could predict the optimum con
ditions (immunotoxin concentration, incubation time, cell concentra
tion) for the most effective use of the immunotoxin. The analysis may
also suggest which steps in the trafficking patterns may be most
profitably manipulated. Finally, the theoretical framework used to
derive Equation r can flexibly incorporate different cellular processing
pathways or different enzymatic expressions that describe other sys
tems of antibody, toxin, and cell, thus expanding the general utility of
the model.
REFERENCES
1. Blakey, D. C., waw@ncaI@k, E. J., wallace, P. M., and Thorpe, P. E. Antibody toxin
conjugates: a perspective. In: H. Waldmann (ed.), Progress in Allergy (Monoclonal
Antibody Therapy), pp. 50—90.Basal, Switzerland: Karger, 1988.
2. Pai, L. H., and Pastan, I. lmmunotoxin therapy for cancer. JAMA, 269: 78—81,1993.
3. Pastan, I., Willingham, M. C., and FitzGerald, D. J. P. Immunotoxins. Cell, 47:
641—648,1986.
4. Vitetta, E. S. Immunotoxins: new therapeutic reagents for autoimmunity, cancer, and
AIDS. J. Clin. Immunol. (Suppl.) 10: 15S-18S, 1990.
5. May, R. D., Wheeler, H. T., Finkelman, F. D., Uhr, J. W., and Vitetta, E. S.
Intracellular routing rather than cross-linking or rate of internalization determines the
potency of immunotoxins directed against different epitopes of sIgD on murine B
cells. Cell. Immunol., 135: 490—500, 1991.
6. Godal,
A., Oystein,
F., and Pihi, A. Kinetics
of uptake
and degradation
of an abrin
immunotoxin by melanoma cells and studies of the rates of cellular intoxication. Int.
J. Cancer, 42: 400—404, 1988.
7. Wiedlocha, A., Sandvig, K., Waizel, H., Radzikowsky, C., and Olsnes, S. Intemal
ization and action of an immunotoxin containing mistletoe lectin A-chain. Cancer
Res., 5!: 916—920,1991.
8. Byers, V. S., Pawluczyk, I. Z. A., Hooi, D. S. W., Price, M. R., Carroll, S., Embleton,
OF IMMUNOTOXINS
17. Theuer, C. P., Kreitman, R. J., FitzGerald, D. i., Pastan, I. Immunotoxins made with
a recombinant form of Pseudomonas Exotoxin A that do not require proteolysis for
activity. Cancer Res., 53: 340—347, 1993.
18. MatteD, L A., Agrawal, A., Ross, D. A., and Muraszko, K. M. Efficacy of transfernn
receptor-targeted immunotoxins in brain tumor cell lines and pediatric brain tumors.
Cancer Res., 53: 1348—1353,1993.
19. Cazzola, M., Bergamaschi, G., Dezza, L., D'Uva, R., Ponchio, L., Rosti, V.,
and Ascari, E. Cytotoxic activity of an anti-transferrin receptor immunotoxin
on normal and leukemic human hematopoietic progenitors. Cancer Res., 51:
536—541, 1991.
20. Stirpe, F., Olsnes, S., and PihI, A. Gelonin, a new inhibitor of protein synthesis,
nontoxicto intactcells.J. Biol.Chem.,255:6947—6953,
1980.
21. Youle, R. J. Mutations in diphtheria toxin to improve immunotoxin selectivity and
understand toxin entry into cells. Scm. Cell Biol., 2: 39—45,1991.
22. Ciechanover, A., Schwartz, A. L, Dautry-Varsat, A.. and Lodish, H. F. Kinetics of
internalization and recycling of transfemn and the transferrin receptor in a human
hepatoma cell line. J. Biol. Chem., 258: 9681—9689,
1983.
23. Stewart, W. E., and Sørensen,J. P. Bayesian estimation of common parameters from
multiresponse data with missing observations. Technometrics, 23: 131—141,
1981.
24. Stewart, W. E. Multiresponse parameter estimation with a new and nomnformative
prior. Biometrika, 74: 557—562,1987.
25. Caracotsios, M., and Stewart, W. E. Sensitivity analysis of initial value problems with
mixed ODES and algebraic equations. Comp. Chem. Eng., 9: 359-365,
1985.
26. Carlsson, J., Drevin, H., and Axen, R. Protein thiolation and reversible protein-protein
conjugation. Biochem. J., 173: 723—737,
1978.
27. Ellman, 0. L. Tissue sulthydryl groups. Arch. Biochem. Biophys., 82: 70—77,1959.
28. Olsnes, S., Sandvig, K, Refsnes, K, and PihI, A. Rates of different steps involved in
the inhibition of protein synthesis by the toxic lectins abrin and ricin. J. Biol. Chem.,
257: 3985-3992, 1976.
29. Moynihan, M. R., and Pappenheimer, A. M., Jr. Kinetics of adenosinediphosphori
bosylation of elongation factor 2 in cells exposed to diphtheria toxin. Infect. Iminun.,
32: 575—582,1981.
30. Olsnes, S., Fernandez-Puentes, C., Carrasco, L, and Vazquez, D. Ribosome macti
vation by the toxic lectins abrin and ricin. Eur. J. Biochem., 60: 281—288,
1975.
31. Braham, K., Junqua, S., Tursz, T., Le Pecq, i-B., and Lipinski, M. Kinetic analysis of
choriocarcinoma cell intoxication induced by ricin and ricin A chain immunotoxin.
Cancer Res., 48: 806-811, 1988.
32. Bau, M-Y., and Draper, R. K. Ricin intoxicates End4 mutants that have an aberrant
Golgi complex. J. Biol. Chem., 268: 19939—19942, 1993.
33. Youle, R. J., Uckun, F. M., Vallera, D. A., and Colombatti, M. Immunotoxins show
rapid entry of diphtheria toxin but not ricin via the T3 antigen. J. Immunol., 136:
93—98,
1986.
34. Ravel, S., Colombatti, M., and Casellas, P. Intemalization and intracellular fate of
anti-CD5 monoclonal antibody and anti-CD5 ricin A-chain immunotoxin in human
leukemic T cells. Blood, 79: 1511—1517,1992.
35. Calafat, J., Molthoff, C., Janssen, H., and Hilkens, J. Endocytosis and intracellular
routing of an antibody-ricin A chain conjugate. Cancer Res., 48: 3822—3827,1988.
36. Raso, V., Watkins, S. C., Slayter, H., and Fehrmann, C. Intracellular pathways of ricin
A chain cytotoxins. Ann. N. Y. Acad. Sci., 507: 172—186,1987.
37. Sasaki, K., Kohgo, Y., Kato, J., Kondo, H., and Niitsu, Y. Intracellular metabolism
and cytotoxicity of transferrin-neocarzinostatin
conjugates of differing molar ratios.
Jpn. J. Cancer Rca., 84: 191—196,
1993.
38. Yamaizumi, M., Uchida, T., Takamatsu, K., and Okada, Y. Intracellular stability of
diptheria toxin fragment A in the presence and absence of anti-fragment A antibody.
Proc. NatI. Acad. Sd. USA, 79: 461—465,1982.
39. Sung, C., Wilson, D., and Youle, R. J. Comparison of protein synthesis inhibition
kinetics and cell killing induced by immunotoxins.J. Biol. Chem., 266:
immunotoxin-791T/36-RTAby tumor cells in relation to its cytotoxic action. Cancer
Res., 51: 1990—1995,1991.
14159—14162,1991.
40. Pelham, H. R. B., Roberts, L M., and Lord, J. M. Toxin entry: how reversible is the
secretory pathway. Trends in Cell Biology, 2: 183-185, 1992.
9. Wargalla, U. C., and Reisfeld, R. A. Rate of intemalization of an immunotoxin
41. Stoorvogel, W., Geuze, H. J., Griffith, J. M., and Strous, G. J. The pathways of
correlates with cytotoxic activity against human tumor cells. Proc. NatI. Acad. Sci.
endocytosed transferrin and secretory protein are connected in the trans-Golgi
reticulum. J. Cell Biol., 106: 1821—1829,
1988.
M. J., Garnett, M. C., Berry, N., Robins, R. A., and Baldwin, R. W. Endocytosis of
USA, 86: 5146—5150, 1989.
10. Ramakrishnan, S., and Houston, L. L. Comparison of the selective cytotoxic effects
of immunotoxins containing ricin A chain or pokeweed antiviral protein and anti-Thy
1.1 monoclonal antibodies. Cancer Res., 44: 201—208,1984.
11. Laurent, G., Kuhlein, E., Casellas, P., Canat, X., Carayon, P., Poncelet, P., Correll, S.,
Rigal, F., and Jansen, F. K. Determination of sensitivity of fresh leukemia cells to
immunotoxins. Cancer Res., 46: 2289—2294, 1986.
42. Snider, M. D., and Rogers, 0. C. Intracellular movement of cell surface receptors
after endocytosis: resialylation of asialo-transferrin receptor in human erythrolcuke
mia cells. J. Cell Biol., 1(X):826—834,1985.
43. Sandvig, K@,and Olsnes, S. 1980. Diphtheria toxin entry into cell is facilitated by low
pH. J. Cell Biol., 87: 828—832, 1980.
44. un, M., and Snider, M. D. Role of microtubules in transferrin receptor transport from
the cell surface to endosomes and the Golgi complex. J. Biol. Chem., 268:
18390—18397,1993.
12. Trowbridge, I. S., and Omary, M. B. Human cell surface glycoprotein related to cell
proliferation is the receptor for transferrin. Proc. NatI. Acad. Sri. USA, 78:
3039—3043,1981.
13. Barabas, K., and Faulk, w. P. Transferrin receptors associate with drug resistance in
cancer cells. Biochem. Biophys. Res. Commun., 197: 702—708,
1993.
45. Van der Ende, A., du Maine, A., Schwartxz, A. L, and Strous, G. J. Modulation of
transferrin-receptor activity and recycling after induced differentiation of BeWo
choriocarcinoma cells. Biochem. J., 270: 451—457, 1990.
14. O'Keefe D. 0., and Draper, R. K. Characterization
toxin
46. Sainte-Marie, J., Vidal, M., Bette-Bobillo, P., Philippot, J. R., and Bienvenue, A. The
15. Johnson, V. 0., wilson, D., Greenfield, L., and Youle, R. J. The role of the diphtheria
endocytosis cycle kinetics of its receptor. Eur. J. Bmochem.,201: 295—302,
1991.
47. Klausner, R. D., van Renswoude, J., Ashwell, G., Kempf, C., Schechter, A. N., Dean,
of a transfemn-diphtheria
conjugate. J. Biol. Chem., 260: 932—937,
1985.
influence of transferrin binding to L2C guinea pig leukemic lymphocytes on the
toxin receptor in cytosol translocation. J. Biol. Chem., 263: 1295-1300, 1988.
16. Rybak, S. M., Saxena, S. K., Ackerman, E. J., and Youle, R. J. Cytotoxic potential of
A., and Bridges, K. R. Receptor-mediated endocytosis of transferrin in 1362 cells.
J. Biol.Chem.,258:4715—4724,
1983.
ribonuclease hybrid proteins. J. Biol. Chem., 266: 21202—21207, 1991.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research.
Quantitative Analysis of Protein Synthesis Inhibition by
Transferrin-Toxin Conjugates
Parvin T. Yazdi and Regina M. Murphy
Cancer Res 1994;54:6387-6394.
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