Download Potentiation With Lysosomotropic Amines

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T-Lymphocyte
Killing
by TiOl-Ricin
Potentiation
By Pierre
To maximize
T-lymphocyte
cyte immunotoxin
monoclonal
antibody
the
influence
the
that
much
less
RTA
IT.
This
which
of
blood
susceptible
narrow
R
than
become
with
only
to
pH
ICIN
(TiOl
lymphocytes
T
when
pH
A-chain
immunotoxins
unit
phytotoxin
have
which
ricin
already
display
(RTA).
variability
in their
enhancers,
such
ionophores,
cell killing
Their
for the
in vitro
to the
above
However,
are
used
NH4CI.
to the
fact
currently
procedures
A-chain
subunit
biological
preby
couof the
properties
these
conjugates,
present
a marked
when
assisted
or
by IT
canboxylic
exhibit
a highly
ITs have great
specific
clinical
of selected
popula-
cell
tions.
They
could
be useful
in the treatment
of autologous
bone marrow
grafts
for eliminating
infiltrated
leukemic
cells
before
tnansplantation.7’8
In this context,
we showed
that an
anti-CD5
immunotoxin
ammonium
chloride,
with
than six orders
leukemic
cells,
more
genic
progenitor
stem
were preserved.8
for
the
graft-v-host
(T101-RTA
IT), when
associated
could
achieve
a cytoneduction
of
of magnitude
of CD5-positive
while
CD5-negative
cells
cells, required
for the marrow
engraftment,
The same IT could also potentially
be used
removal
of T cells
disease
(GVHD)
However,
the treatment
ing to modalities
in T-lymphocyte
sensitive
to the
clonoincluding
defined
depletion
T101-RTA
from donor
marrow
to prevent
after allogeneic
bone marrow
of human
indicating
IT than
increase
T-lymphocyte
killing
effect
of numerous
parameters.
key role of pH in the expression
MATERIALS
T lymphocytes
for leukemic
T cells
ineffective
cells are
T cells.
less
To
with the IT, we examined
In this study,
we show
of IT cytotoxicity.
the
the
AND
that these
malignant
accord-
was
METHODS
Products.
Ammonium
chloride and methylamine
hydrochloride
were purchased
from Merck
(Paris-France).
Monensin,
PIPES
(piperazine-N,
N’-bis-(2-ethane-sulfonic
acid),
HEPES
(N-2hydroxyethyl-piperazine-N’-2-ethanesulfonic
acid), and glycylglycine were
obtained
from Calbiochem
Behring
(San Diego).
3Hthymidine
(20 mCi/mmol)
was obtained
from New England
Nuclear
(Boston).
SPDP (N-succinimidyl-3(-2-pynidyl-)dithio-propionate)
was supplied
by Pharmacia
(Sweden)
and THAMACETAT
(Trishydroxymethylaminomethane)
by Roger Bellon Labonatories (France).
RTA was purified from nicin (extracted
from seeds
Blood.
Vol 72, No4(October),
1988:
pp 1197-1202
more
that
effective
an optimal
and
enhancing
F(ab’)2
than
From
K. Jansen,
Franz
effective
IgG counterpart.
vitro
extremely
is due
amines
elimination
an
is the
showed
for
neutraliby
NH3
also
are
TiOl-
Derocq,
We
much
pH-Dependent
Amines
that
We
of the
activation
main
lysosomotropic
IT.
IT when
width.
most of these conjugates
potency.”
Such activated
potential
the
to the
Briefly,
specificity,
potency.3”
as
that
T cells
(ITs)
been
reported.”2
stringent
binding
have
which
within
pared
according
to standardized
monoclonal
antibodies
to the
pling
to
pH rose
IT
effect
of 0.7
to
we
enhancement
the
of
all-or-nothing
IT).
lymphocytes.
However.
process
A-chain
of parameters
sensitive
window
ricin
-RTA
extent
malignant
NH4CI.
occurred
an
T
highly
pH-sensitive
led
the
sensitivity
could
IT by NH4CI
TiOl
and
peripheral
in conjunction
ty.
(MoAb)
Lysosomotropic
anti-pan-T-lympho-
by linking
Immunotoxin:
Bernard
J.P. Bourri&
Jean-Marie
Guy Laurent, and Pierre Gros
Ravel.
with
prepared
nature
With
Sophie
killing
(IT).
established
showed
Casellas,
A-Chain
those
these
specific
component
or Fab
produced
data.
of
containing
using
we defined
elimination
NH4CI.
IT were
the
whole
a procedure
of T lymphocytes
in
10
mol/L
at pH 7.8 in the presence
of NH4CI for two
hours.
This
peripheral
blood cell processing
elicited
an abrogation
of
three logs of functional
T-cell response.
Under the same
conditions,
there
was no reduction
in the number
of
marrow
hematopoietic
precursor
granulocyte-macrophage
colony-forming
units (CFU-GM).
C 1988
by Grune
& Stratton,
Inc.
by treating
them
with
(Fab)T1O1-RTA
at
of Ricinus
communis
sanguineus)
as previously
described.’2
AntiRTA serum was produced
in goats immunized
with RTA mixed with
complete
Freund’s
adjuvant.
Goat anti-RTA
antibodies
were punfled by affinity chromatography
on a column
of RTA coupled
to
CNBr-activated
Sepharose
(Pharmacia).
Monoclonal
antibodies.
The mouse MoAb
TlOl , purchased
from Hybritech
(San Diego), is an IgG2a that recognizes
the CD5
antigen (TI , P-67, gp67, T-65) expressed
on all peripheral
blood T
cells, chronic
B lymphocytic
leukemias,
and some T-cell--derived
hematological
malignancies.’3
The affinity constant
of this MoAb is
in the range of 10)0 mol/L.
This same antigen
is also recognized
by
MoAbs Leu-1, 10.2, H65, and OKTI.
The mouse MoAb lO-3D2,
generously
donated
by Dr Edginton
(Research
Institute
of Scripps
Clinic, La Jolla, CA),
is an IgG2a which recognizes
mammary
carcinoma
cells.” The MoAbs were purified from ascitic fluid by
affinity chromatography
on Staphylococcus
aureus
protein A, coupled to Sepharose.
F(ab’)2
and Fab fragments
were produced
by
digestion
with pepsin and papain, respectively.
F(ab’)2 was purified
by gel filtration
(ACA
44, IBF) and Fab by ion exchange
on
DEAE-Trisacnyl
(LKB).’5
Immunotoxins.
Details of the procedure
used to link purified
RTA to MoAb have previously
been reported.ZI((
Briefly, activated
disulfide radicals were introduced
into the MoAbs by treatment
with
SPDP.
Following
dialysis,
activated
MoAbs
were reacted
with
excess RTA, which resulted
in the formation
of a disulfide linkage
between the two proteins.
The resulting
IT molecules
were purified
by gel filtration
chromatography.
Different
ITs were produced
using
the TlOl and the lO-3D2 antibody
molecules
as a whole and their
respective
F(ab’)2 and Fab fragments.
Purity ofthe ITs was checked
by sodium dodecyl sulfate (SDS) polyacrylamide
gel electrophoresis
using a 2% to 16% gradient
gel. The ITs contained
an average of 1.5
From
the
Department
oflmmunology.
Sanofi-Recherche,
pellier. France.
Submitted
August
13, 1987; accepted
June
1, 1988.
Address
reprint
requests
to Pierre
Casellas,
PhD,
Recherche
Centre de Montpellier,
rue
du Professeur
J.
34082 Montpellier.
Cedex, France.
The publication
costs ofthis article were defrayed
in part
charge payment.
This article
must therefore
be hereby
“advertisement”
in accordance
with 18 U.S.C. section 1 734
indicate this fact.
C 1988 by Grune
& Stratton.
Inc.
0006-4971/88/7204-0012$3.00/0
Mont-
SanofiBlayac,
by page
marked
solely to
1197
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CASELLAS
1 198
to 2 RTA per IgG, I to 2 RTA per Fab’2 and I .5 RTA per Fab,
established
as previously
described)6
Cells.
Peripheral
blood
mononuclear
cells (PBMCs)
were
obtained
from heparinized
blood by centnifugation
(400 x g, 30
minutes)
on Ficoll-Hypaque
(Pharmacia).
Cells were washed three
times in RPMI solution (M#{233}nieux,Lyon, France)
before treatment.
T cells which represent
an average of 75% of PBMCs,
express an
average
number of 40,000 determinants
of P-67 per cell.’7”#{176}
Treatment
of Cells.
All cell treatments
were performed
at a
defined pH using RPMI-based
medium
containing
10% fetal calf
serum (FCS) (Flow Laboratories,
McLean,
VA), streptomycin
(0.1
mg/mL),
penicillin
(100 U/mL),
and buffered with either PIPES,
HEPES,
or glycylglycine
at 30 mmol/L
final concentration.
The pH
of the mediums
was adjusted
at 37#{176}C
with NaOH
1 mol/L.
PIPES,
HEPES,
and glycylglycine-buffered
mediums
were used for the pH
ranges 6 to 7, 7 to 7.5, and 7.5 to 8, respectively.
Cells (10 cells/mL)
resuspended
in the appropriate
medium
at the pH indicated
in the
figures and tables, were treated
with the IT at 37#{176}C
for four hours or
as indicated.
Treated cells were washed twice and resuspended
in the
culture medium (RPMI
1640 containing
antibodies,
acid carbonate
and 10% FCS) for the subsequent
in vitro assay.
Mitogenic
stimulation
of T lymphocytes.
The proliferation
of
normal
polyclonal
T cells was promoted
by PHA stimulation.
In
short, IT-treated
PBMCs
(10 cells/well)
were cultured
in 96-well
flat-bottomed
micnotiter
plates in 0.2 mL culture medium,
containing 1% phytohemagglutinin
(PHA, Difco Chemical
Co. Detroit)
for
two days at 37#{176}C,
in an atmosphere
of 5% CO2. The cultures
were
pulsed with I Ci 3H.TdR
and harvested
after eight hours. 3H-TdR
incorporation
was quantitated
by standard
scintillation
counting
techniques.
For each treatment
the mean response
from quadruplicate wells was expressed
as the percentage
of control responses
as
follows:
cpm treated
-
cpm untreated
PBMC
PBMC
cpm of the background
-
cpm of the background
-
Effect
ofpH
to TiOl
The background
level was obtained
using cells treated
with i0#{176}
mol/L nicin, which ensured a complete
inhibition
of growth.
Ammonia
determination.
Ammonia
in solution
exists as an
equilibrium
between
free ammonia
(NH3)
and ammonium
ions
(NH4)
and the percentage
of each molecular
species in the equilibnium is pH-dependent.
The NH3 concentration
was calculated
using the following
formu-
of 50 cells or more were
on
the
(IgG)-RTA
sensitivity
IT.
ofperipheral
PBMCs
blood
were
treated
activity
of the
determined
IT toward
human
by measuring
peripheral
the
blood
inhibition
T cells
with
(IgG)-RTA
IT at a dose of lO_8 mol/L
for four
37#{176}C
at various
pH levels ranging
from 6 to 8. The
of the
TlOl
hours
at
cytotoxic
T cells
was
proliferation
of these
cells in response
to the polyclonal
mitogen
PHA.
Figure
1 illustrates
the effect of the IT treatment
at various
pHs
on immunocompetent
T cells.
From
pH 7 to pH 8, the IT
was completely
unable
to damage
cells;
reduced
to below neutrality,
cells became
tive
to the
IT.
However,
synthesis
When
never exceeded
the treatment
mmol/L
NH4C1,
was obtained
the
a similar
from
extent
An
inhibition
activation
pH
of inhibition
was
sensi-
of DNA
combined
of PHA
with
10
responsiveness
pH 6 up to 7.2 to 7.3. The
all-or-nothing
range of 0.7 pH
with ammonium
when
the
increasingly
80% of control.
with
IT was
T cells then sharply
decreased
beyond
the promoting
effect of NH4C1
was
proliferation
of
pH 7.3, indicating
that
a pH-sensitive
process.
of the
IT
occurred
unit, from 7.4 to 8.1 . Treatment
chloride
alone
did not have
within
a
of the cells
a toxic effect.
The treatment
of PBMCs
under
similar
conditions
with
irrelevant
IT that did not bind T cells (10-3D2
(IgG)-RTA),
an
gave
at
50%
nonspecific
pH 8 (Table
era!
xlOO.
Aggregates
RESULTS
Effect
of controls
Percentage
1 4 using an inverted
microscope.
considered
as colonies.
ET AL
blood
of
toxicity
in the
presence
of NH4C1,
1).
NH3
concentration
T cells
to TiOl
on
the
sensitivity
(IgG)-RTA.
of
Ohkuma
periph-
and
Poole
established
that the active component
of NH4C1,
acting
as a
lysosomotropic
amine,
is the free base NH3)9’30
If the active
species
of NH4C1
as an IT-enhancer
is the free base as well,
this
may
account
for the
pH response
curve
7
8
for activation
as
:
;1
la:
lO’5”#{176}
(NH3)
- (NH4CI)
x
1
+
where pKa - 8.89 at 37#{176}C,
NH4CI
is the NH4C1 concentration
introduced
and pH is the pH value in the medium at 37#{176}C.
Colony
assay for hematopoietic
progenitors
(CFU-GM).
Bone
marrow samples were obtained
from different
healthy bone marrow
donors and collected
in hepanized
syringes.
Bone marrow
mononuclear cells were separated
on Ficoll-Hypaque,
adjusted
to 2 x iO
cells/mL,
and incubated
with the IT for four hours at 37#{176}C
in the
presence
of 20 mmol/L
ammonium
chloride,
at four different
pH
levels (pH 7.2 and 7.5 were obtained
with HEPES,
pH 7.8 and 8.2
with THAMACETAT).
As a control,
a sample
was processed
according
to routine conditions
(alpha
I x medium
alone).
After
treatment
and washing,
cells were plated (1.5 x l05/ml)
in 35-mm
Petri dishes in alpha I x medium
containing
1.8% agar and 20%
FCS over and underlayer
with stimulated
PBMCs
from normal
donors. Each culture point was plated in triplicate
and incubated
at
37#{176}C
in an atmosphere
of 5% CO2. The colonies were scored on day
6
pH
Fig 1 .
Inhibition
of mitogen-induced
proliferation
of human
peripheral
bicod
T cells by TiOl
(lgG)-RTA-lT.
PBMCs
were
incubated
for four hours at 37#{176}C
with TiOl
(IgG)-RTA
IT at a
concentration
of 10’
mol/L.
at the pH indicated
on the abscissa.
in the presence
(S-U)
or absence
(-)
of 10 mmol/L
NHCI.
The pH, which was monitored
in samples
carried
out in parallel,
was stable ( ±0.05 pH unit) throughout
treatment.
The percentage
of H-thymidine
incorporation
assayed
48 hours after PHA stimulation was calculated
as described
in Materials
and Methods.
Each
data point was performed
in quadruplicate
and represents
the
mean
of three
independent
experiments.
In the controls.
the
amount
of DNA
synthesized
by PHA-induced
clonogenic
cells
varied
little
(<1 5%) within
the pH range used after four hours of
incubation
either in the presence
or absence
of NH4CI.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PH DEPENDENT
Table
POTENTIATION
1 . Effect
1 199
OF IT
of pH on the
Non-specific
Cytotoxicity
of ITs
100
3H-TdR
1O-3D2
pH
(%)
Incorporation
(lgG)-RTA
-NH4CI
10-3D2
+NH4CI
F(ab’)2-RI’A
-NH4CI
10
+NH4CI
I
6.3
99
97
98
97
7.0
100
98
99
100
7.2
97
95
99
97
7.5
97
70
97
95
8.0
98
PBMCs
were
concentration
of 1O
NH4CI for four
‘
50
exposed
to
mol/L
hours
97
10-3D2-RTA
in the presence
(irrelevant
absence
at 37#{176}C.3H-thymidine
±
80
ITs
ITs)
at
of 10 mmol/L
incorporation
was
assayed
48 hours after PHA stimulation.
process,5
above.
Indeed,
enhancement
with the pKa of the ammonia
working
pH
described
variation
tion.
(pKa
25#{176}C, 8.89
in pH
causes
a major
To examine
this
hypothesis,
ments
was
TiOl
performed.
(IgG)-RTA
trations,
NH4C1
T
a fixed
pH
the
by
the
The
NH3
consequently,
the
related
tions.
From
were
pH
tion
treated
with
curve
actual
was
the same
NH3
results
strongly
for IT enhancement,
for
resulting
and
NH4C1
NH3
Effect
latter
could
results,
a similar
be expected
for other
acting
as NH4C1.
The results
confirmed
what
was expected
of PHA
inhibition
7.2 to 7.8.
ionophone
Table
response
was
concentra-
IT-enhancer
2.
Influence
monensin,
amines
NH4CI
the
on TiOl
the
influence
of pH
range
NH3’
Percentage
(imel/L)
Stimulationt
200
92
10
752
27
7.9
10
928
10
8.0
8.1
10
1.25
6.25
7.5
PBMCs
mol/L,
with
were
either
varying
thymidine
exposed
with
to T101
concentrations
incorporation
of
tCalculated
the IT.
from
controls
40
872
10
(IgG)-RTA
effect
in Material
treated
at a concentration
pH levels
four
for
48 hours
blood
T
effect
at
pH
the pH-response
7.8.
curve
Compared
was
with
significantly
Tl01
shifted
proliferation
ITs, compared
was
achieved
with
F(ab’)2
with the IgG counterpart.
on Fab
On the
of
hours
after
‘
at
of 108
at pH 8.1
37 #{176}C.
3H-
PHA stimulation.
and Methods.
in the
of peripheral
and TlOl
(Fab)-RTA.
absence
of a potentiating
IT (Fig 1). When
NH4C1 was added,
its promotstarted
to appear
when the pH was raised
to 7.2,
a maximal
(IgG)-RTA,
of T-cell
containing
1.5
at various
NH4CI
was assayed
#{176}Calculatedas described
698
(IgG)-RTA
NH4CI
in comparison
we investigated
3.5
100
1,141
174
1,047
10 mmol/L
sensitivity
to
agent,
both ITs only reduced
PHA-induced
T-cell
prolifenation when pH was below
neutrality,
as observed
with TiOl
64
8.1
on the
cells vis-#{224}-visT101
(F(ab’)2)-RTA
Results
are shown
in Fig 3. In the
T cells
found that the
cells when
pre-
(lgG)-RTA
10
8.1
blood
We recently
on leukemic
weakly
T Cells
7.8
5
ofperipheral
ITs.
active
was
inhibi-
7.2
8.1
cells
to a lower pH region
by 0.3 pH unit. Similar
results
were
observed
in three independent
experiments
on PBMCs
from
different
donors.
Thus, at pH 7.8, a tenfold
higher
inhibition
of the canboxylic
Mature
(mmol/L)
IT
of IT-treated
pared
either
with F(ab’)2
on Fab fragment,
with the whole
IgG molecule.2’
Therefore,
ing
methylamine
all-or-nothing
to be in a pH
presence
of NH3 Concentration
pH of Treatment
with
An
at 50 nmol/L
on Human
for
lysosomotropic
shown
in the
Cytotoxicity
sensitivity
obtained
(Fig 2).
was
In contrast,
pH
response
by the pH (Fig 2).
ofpH
on sensitivity
F(ab’)2 or Fab-containing
T101-RTA
IT is more
at a fixed
of NH3
similar
at
pH. These
pH-response
to the
levels
of the mitogenic
affected
and Methods.
Results
of DNA
synthesis
ofT
form
8
Fig 2.
Effect
of pH on T101
(IgG)-RTA
IT cytotoxicity
to
peripheral
blood
T cells in the presence
of methylamine
or
monensin.
PBMCs
were
treated
as in Fig 1 in the presence
of
either
1 0 mmol/L
methylamine
(-)
or 50 nmol/L
monensin
M-).
In the pH range
examined.
neither
methylamine
nor
monensin
affected
the 3H-thymidine
incorporation
assayed
48
hours after PHA stimulation.
with
the
activation
from
concentra-
set of experi-
concentration
was
of the
is the active
that
indirectly
in NH3
different
final
IT-treatment
regardless
that
the
7
37#{176}C);a subtle
following
as indicated
in Materials
in Table
2. The inhibition
concentrations
suggest
variation
lymphocytes
on with
concentration.
calculated
are shown
at
IT in the presence
ofvanious
NH3 conceneither
with
different
NH4C1
concentra-
achieved
at
tions
cells
at
= 9.2
is a dose-dependent
being far above
6
a
same
conditions,
except
for
61
7.2
7.6
64
6.8
7.2
76
pH
Fig 3.
Influence
of pH on inhibition
of mitogen-induced
proliferation
of human
peripheral
blood T cells by T1O1 -RTA IT composed of F(ab’)2 or Fab fragments.
PBMCs were incubated
for four
hours at 37#{176}C
at the pH indicated
with: (A) TiOl
(F(ab’)2)-RTA
at a
concentration
of 10’ mol/L,
in the presence
(-U)
or absence
(#{149}-#{149})of 10 mmol/L
NHCI.
(B) TiOl
(Fab)-RTA
at a concentration of 10-a mol/L
in the presence
(-U)
or absence
(I-S)
of
10 mmol/L
NH4CI. The percentage
of 3H-thymidine
incorporation
was assayed
48 hours after
PHA stimulation.
Each assay
was
performed
in quadruplicate.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CASELLAS
1200
A
B
The
and
extent
99.9%
of inhibition
with TlOl
respectively,
thus
of the
(IgG)
PHA
RTA
confirming
responsiveness
and T101
the
these two conjugates.
In all the above
experiments
ET AL
was 99%
(Fab)RTA,
difference
in
potency
between
I
mediated
0
measuring
0
stimulation.
latter step
2
3
Time
(hours)
cells
hand,
and
the
irrelevant
lO-3D2(F(ab’)2)-RTA
the viability
of T cells,
throughout
the pH range
ficity
of the
inhibition
the
TIOI-RTA
varying
TlOl
of
(Fab)-RTA
by
the
PBMCs
time
with
were
treated
either
ITs associated
T101
with
cated
significant
that
changes
two
hours
Table
were
3.
sufficient
for
at pH
±
(Fab)-RTA
GM even
99%.
does
not
±
recovery
was
performed
compared
1),whichgave56
with
for
four
a control
± 11 colonies.
hours
These
T cells
that
during
T1O1-RTA
The
IT
CFU-
selectivity
determined
cells using
marrow
results
of
by assaying
the in vitro
mononuclear
cells
were
at pH
human
in Table
3.
of IT at various
The
pH
of colonies
or T101
inhibit
the growth
of CFUthat inhibited
T cells by more
confirm
either
the
treat-
ITfor
cell-type
IT and ammonium
chloride,
7.2 to 8.2, and cultured
for
lower
T
by
IT became
indicating
Results
are shown
cells in the absence
that
the
this
depleting
number
precursor
method
or the
functional
cells.
as a function
two hours;
no
This
promoting
mdieffect.
Progenitors
effect
process:
only
After
Treatment
at Various
of
19.1
55.8
±
NH4C1
a 0.7
TiOl
With
pH
±
(IgG)-RTA
72
±
of CFU
processed
presence
according
Eachvalueisthemeanofatriplicateassay.
killing
range,
and
or TIOl
(Fab)-RTA
pH-sensitive
could
(b)
the
pH7.8
28
36.6
±
threshold
pH8.2
21
28
(65)
17
44.5
±
36
56.1
±
11.6
32.8
NH4CI.
conditions
Numbers
(alpha
24
±
16.5
(58)
28.2
51.2
±
24.5
(91)
(100)
of 20 mmol/L
±
(50)
(79)
to routine
be achieved
pH
-GM Colodss
(128)
at 37#{176}C
in the
a stringent
pH Levels
(97)
25.9
T-cell
unit
(99)
54.6
was
(a) an all-or-nothing
within
immunotoxin
to MoAb
T101 has
in vitro T-lympho-
response.
We demonstrated
that major T-cell
be achieved
with the T1O1-RTA
IT when it
with NH4CI.
However,
we showed
that the
pH7.5
sample
of
of hematopoietic
cyte mitogenic
removal
could
was associated
In
(135)
The treatment
toxicity
Bone
greatly
competence
(105)
76
the
did not significantly
under
pH conditions
than
PHA
that killed
measured
levels caused
a moderate
decrease
in the number
at pH 7.8 and 8.2. The presence
ofTiOl
(IgG)-RTA
or
NH4C1.
59 ± 12.7
T101(Fab)-RTA
entered
T101-RTA
ITs was
to plunipotent
stem
precursors.
of marrow
by
a 48-hour
in Fig 4, the
antibody
progenitors.
with T10l-RTA
ranging
from
myeloid
treatment
(82)
T101(IgG)-RTA
on the
assay.
treated
values
No.
46.2
actually
ofpll
IT-
hours
with anti-RTA
antithe number
of surviving
As seen
anti-RTA
hematopoietic
CFU-GM
IT at two
evaluating
In this report,
the anti-pan-T-lymphocyte
made by linking
the nicin A-chain
subunit
been examined
for its ability
to prevent
pH7.2
None
the
the different
their
toxicity
IT
±
after
the
treatment
the question
as to whether
or not this
for the expression
of IT activity.
To
stimulation.
to
Effect
8 over
an optimal
of CFU-GM
Recovery
(1O#{176}mol/L)
7.4
IT
DISCUSSION
Immunotoxins
CFU-GM
of T cells
prepared
10 mmol/L
thereafter.
PHA
IT had
GM
not
(IgG)-RTA
occurred
after
active
ment.
of T lymphocytes
Fig 4, the mitogenic
response
of T cells declined
of the duration
of IT treatment
for the first
further
PBMCs,
after
of NI-I4CI
the speci-
TlOl-IT
(Table
1).
time on the killing
IT.
periods
IT did
even in the presence
used, demonstrating
of T cells
with antibody
fragments
Effect ofincubation
by
We raised
was necessary
insensitive
Optimal
duration
of treatment.
(A) PBMCs
(2 x i0
cells/mi)
were
treated
with
T101
(lgG)-RTA
(-)
or T101
(Fab)-RTA
(N-S)
at 10’
mol/L,
in NHCI 10 mmol/L.
at a final pH
8. At sach time
interval
cells were
washed,
resuspended
in
RPMI-FCS
plus 1 % PHA. and seeded
at 10 cells/mi
for 48 hours
before measurement
of H-thymidine
uptake.
(B) PBMCs
(2 x 1 0
cells/mi)
were treated
with T101 (lgG)-RTA
or TiOl
(Fab)-RTA
at
10’
mol/L.
in NH4CI 10 mmol/i
at a final pH 8. At the end of a
two-hour
incubation
period.
purified
anti-RTA
antibodies
(1O
mol/i
final)
were added to the assay for 40 minutes
at 4#{176}C
before
washing.
Cells were resuspended
in RPMI-FCS
plus 1 % PHA and
seeded
at
cells/mi
for 48 hours
before
measurement
of
‘H-thymidine
uptake
(hashed
histograms).
In controls.
cells were
treated
at 4#{176}C
for one hour in NHCI 10 mmol/i
at a final pH 8 with
either
TiOl
(lgG)-RTA
or TiOl
(Fab)-RTA
at 10’
mol/i
and with
anti-RTA
(i0
mol/i
final).
Cells were
then incubated
for two
hours at 37#{176}Cbefore
being washed
and assayed
for PHA responsiveness
as above (wide histograms).
These cells recovered
100%
of the response,
demonstrating
the complete
capacity
of the
anti-RTA
to neutralize
the IT.
affect
assessed
synthesis
blocking
cell surface
body, and subsequently
4
4.
other
DNA
involving
was
answer
this, we determined
if the IT molecules
cells were endocytosed
at two hours.
This was
I
Fig
cytotoxicity
in parentheses
1 x medium
represent
the percentage
plus NH4CI and without
of
IT; final pH.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
H
DEPENDENT
POTENTIATION
required
for
mature
T cells
The
abolished
fact
that
activation
by ammonium
by lowering
the pH, which
in turn
ammonia
the
an
1201
OF IT
effective
of the
fact
proliferation
and
of the
mining
factor
protonated
the
IT
who
are
was
the
NH3
pH
RTA-IT.
Methylamine,
which
The
is
NH3
concentration
or
the
NH4C1
with
has
those
of
biological
proliferation
functions
only
and
on the
precluding
entered
the
This
not
required
tion with NH4C1,
a pH response
lymphocytes
(data
not shown).
ability
clinical
reported
using
ricin
amines.2225
activity
the possibility
A-chain
ITs
affected
by the
A comparison
had
on
counterpart,
activity
of the carboxylic
ionophore
a different
mode
of action,
was
T
lymphocytes
showed
activation
pH
unit
was
lower
shifted
value
leading
to an
optimal
removal
that
with
the
using
the
pH
enhancement
either
effect
F(ab’)2
of
threshold
to a significant
for ITs containing
of T101-RTA
a twofold
lower
required
ITs,
the
for
conjugate
at
pH
7.8.
Thus,
this
molecule
clinical
use for cx vivo removal
of T cells
in the following
conditions:
T101(Fab)-RTA
in the presence
of NH4C1,
for
37#{176}C.
For an additional
security,
tion
chosen
was 20 mmol/L.
two hours
the final
Under
these
was
from
conditions,
was
and
a
the
the
selected
donor
at iO
at pH
NH4C1
Fc
that
cause
than
recent
for
to the
anti-RTA
involved
used
since
PHA
the ITstimula-
clinical
cx vivo
occurs,
was
we
had
stimulastep was
evidence
abrogate
described
(submitted).
of enhancing
success
to abrogate
for the
T cells
here,
factors
clinical
IT in patients
might
A-chain
therefore
mediated
in a
has
such
as
through
its
mannose
receptors
of the
residues
RES,
might
use of lysosomotropic
bone
be explained
cytotoxicity
mannose
GVHD
after
by the reticuloendothelial
capacity
of the antibody
A-chain,
As fan as the
this
antibodies,
in cell killing
Complete
an anti-CD5
by a ricin
enhancers
dose and
contrast,
yielded
could
T101-RTA
IT to be effective,
a specific
would not be expected
after in vivo adminis-
of the target
cells
The
opsonization
and reproducible
0.3
antibody
fragment
IT generated
conjugates
NH3
concentration
to
to produce,
IgG
maximal
response
similar
to that of the IgG counterpart.
Because
the IT prepared
with the Fab fragment
most active
at a pH close to the physiological
value
easiest
The
a
determination
stimulation
IT cytotoxicity.
administrating
or
assay
of TlOl-RTA
IT to specifically
situation
using
the procedure
tration.
have
However,
through
no exogenous
row transplantation27
pH of the medium.
of the effect that
as others,
cell during
IT treatment,
before
PHA
strongly
suggested
that the stimulation
NH3
for the
cytotoxic
effect
monweakly
was
of T-cell
measure-
which
analysis,
of a direct
Using
blocking
all the IT molecules
for
the
of
the viability
of IT-treated
T
only over a course
of several
possibility
been recently
demonstrated
Because
of the requirement
elimination
lysosomotnopic
Our data strongly
suggest
that a pH-dependent
should
also be expected
in these cases.
In contrast,
the IT-promoting
ensin,
which
has
Fab,
of T-lymphocyte
associated
with
We,
of this
in which
tion.
also
determination
measure,
by FACS
IT treatment.
was visualized
predictability
questionable.
showed
that
for T
have
the
death
after
cytotoxicity
principle
as NH4CI,2#{176}also induced
a pH-sensitive
activation
of Tl01-RTA
IT. Using
an anti-CD7-RTA
IT in conjunccurve was also found
Different
laboratories
numbers)7
assay simply
because
expected
to decline
the
tion,
same
death
T-cell
assess
biological
cells was
conditions
the
cell
assay
based
on the functional
ability
after
PHA
stimulation,
assessed
by
reliably
hours,26
diffuse
for
synthesis.
This latter
with that obtained
of cell
induced
Under
number
CFU-GM.
used
ment of DNA
data comparable
is the deterand that the
no effect;
precursors
procedure
the concen-
that
medium
in cell,
medium
of
could
be reproducibly
depleted.
there
was no reduction
in the
hematopoietic
properly
function
showed
in the
latter
the free base NH3, which
is lipophilic,
can rapidly
across
the plasma
and lysosomal
membnanes.(9a
This pH effect
was not just restricted
to NH4C1
T101
T lymphocytes
same
conditions,
for inhibition
consistent
previously
NH4
a
the
free base NH3 in the
for base accumulation
species
the
This
as
either
findings
Poole,
tration
that
curves
when
varying
These
Ohkuma
IT on
chloride
was
lowered
the free
suggests
identical
obtained
by
concentration.
that
by
were
generated
(IgG)-RTA
in IT activation.
by the
concentration
of TiOl
medium,
component
T-cell
was
effect
8. 1.
content
demonstrated
of
optimal
was
otherwise
(eg,
by uptake
system
alone
[RESI).
recognized
be taken
amines
by
mar-
into
on
the
by
the
account.
as immunotoxin
in viyo is concerned,
the requirement
of both high
a slightly
alkaline
pH preclude
their use in vivo. By
activation
with
canboxylic
ionophores,
such
as
monensin,
which
NH4C1
and
here, makes
which
them
demands
one
is active under
choice
reagents
million
lower
dose
than
physiological
pH as shown
for in vivo use.28
marrow
mol/L
7.8 and
concentra-
ACKNOWLEDGMENT
at
thank
Dns H.E.
corrections
and A. Garcia
We
3 logs of
Blythman
for typing
and C. Bouloux
the manuscript.
for
English
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1988 72: 1197-1202
T-lymphocyte killing by T101-ricin A-chain immunotoxin: pH-dependent
potentiation with lysosomotropic amines
P Casellas, S Ravel, BJ Bourrie, JM Derocq, FK Jansen, G Laurent and P Gros
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