Vol. 183, No. 2, 1992 March 16, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 758-766
THE SPECIFIC CYTOTOXICITY OF IHMDNOCONJDGATES CONTAINING BLGCXEDBICIN IS DEPENDENT ON THE RESIDUALBINDING CAPACITY OF BLOCKED BICIN: EVIDENCETHAT TBE BINDING AND A-CHAIN TRANSLGCATIONACTIVITIES
NENBMNE
OF RICIN CANNOTBE
SEPARATED Victor
ImmunoGen,
Received
John
S. Goldmacheri,
January
Lambert
and Walter
A. Blattler
Street,
Cambridge,
MA 02139
148 Sidney
Inc.,
27,
M.
1992
Recently we have developed blocked ricin, a derivative of native ricin in which the galactose-binding sites of the B-chain are blocked by covalent modification with affinity ligands. This modification impedes the binding function of the B-chain, while sparing its abilLty to facilitate the entry of the toxic subunit of ricin, the A-chain, into the cytoplasm. Immunotoxins prepared with blocked ricin approach the cytotoxic potency of native ricin with antibodydependent specificity. Here we report that the high cytotoxic potency of these immunoconjugates, which is attributed to the preserved translocation function of of the ricin B-chain, is dependent on the minimal residual lectin activity blocked ricin. Our findings support the notion that two functions of ricin. membranebinding and translocation. cannot be separated. o 1992 ~~~~~~~~ press, lllc.
The
high
cytotoxicity
researchers
of
to develop
immunotoxins
of ricin
with
that
had
to be overcome
was the
terminated
oligosaccharides
which
Ricin linked is
is
the
ricin,
in the
strategy
chain alone this
type
was,
proved
supported
lTo whom correspondence 0006-291X/92
hoped
that
to remove
cytotoxic
is
should
evidence
be sent.
$1.50
758
the potency
A major
of
ricin
the entire
to galactose-
native that
B-chain
ricin,
activity
addition
B-chain.
The
and to use the Amost
mainly into
in
cells.
of two disulfidein the
However,
obstacle
of mammalian
composed
activity
ricinA-chainmoiety previous
to combine
inspired
The ribosome-inactivating
(1,2,5). than
beans,
(l-6).
on surfaces
sugar-binding
immunotoxins
castor
binding
are ubiquitous
and the
intransferringtheir
findings
they
indiscriminate
therefore,
to be less
from
antibodies
and the B-chain.
A-chain
in constructing
inefficiency These
in which
protein
the A-chain
contained
lectin
of monoclonal
a ribosome-inactivating
subunits,
initial
selectivity
a
immunotoxins because
the cytoplasm to binding
of
of their (6,7). to
the
Vol.
183,
cell
No.
2,
surface,
cytoplasm since
BIOCHEMKAL
1992
B-chain
(8-10).
also
This
less
A successful
blocking
modification
with
Immunotoxins cytotoxic potency
than
towards
B-chain
containing operates
of ricin
were
blocked
ricin, of
galactose-binding
A-fragment,
was achieved
capacity
of
than
cells,
the its
a
analog were
lOO-
by covalent
(14).
(13).
thousand-fold
while
cells
we address
independently
the
specific,
an A-chain
glycopeptides
more
reducedbinding
report
to
A-chain
gelonin,
triantennary
antigen-positive
In this
A-chain
(11,12).
sites
ricin
a greatly
the
toxin
antigen-negative
towards
despite
function.
immunoconjugates
ricin
containing
blocked
ricin
ricin,
translocation
residual
with
with
diphtheria
native
of the binding
ricin
of native
than
ligands
made
the blocked
ricin
cytotoxic
or with
of
COMMUNICATIONS
seems to be ricin
of the B-chain
multiflorum
RESEARCH
translocation function
conjugates
from seeds of gelonium
BIOPHYSKAL
facilitates
translocation
disulfide-linked
to lOOO-fold
AND
approaching
the
indicated
that
hadmaintained
its
This
affinity,
less
the question
of whether,
translocation
function
lectin
blocked
function ricin
or is
in the of
the
whether
of
the
functional
significance.
Methods Preparation of Blocked Ricin and its Conjugates with Antibodies The immunoconjugates between blocked ricin (13) and the murine monoclonal antibodies anti-B4, recognizing the CD19 molecule (15), andN901, recognizing the CD56-molecule (16), were prepared as described previously (14). These conjugates were tested for their activity as inhibitors of protein synthesis in a cell-free system and their affinity towards their respective antigen-positive cells (14), and it was found that neither the ricin A-moiety nor the antibody were inactivated in the process of the preparation of the immunotoxins. Cell
Lines The human lymphoid cell line Namalwa (ATCC CRL 1432) and the human small cell lung carcinoma line SW2 (17) were maintained in exponential growth phase in culture medium consisting of RPMI-1640 medium supplemented with 10% heat-treated (30 min at 56 "C) fetal calf serum and 2 mM L-glutamine. For experiments shown in Fig. 2, some cell cultures were grown for at least three weeks in medium that contained 30 PM swainsonine (CalBiochem Corp., La Jolla, CA). This treatment was non-toxic for the cells and did not affect their proliferation rate. Binding Assays Binding of ricin and anti-B4-blocked ricin to Namalwa cells was analyzed by indirect immunofluorescence. Cells (1 x lo6 per sample) were incubated successively for 30 min at 0 OC with various concentrations of ricin or anti-B4blocked ricin in AB-buffer [(2.5% pooled human AR serum (PelFreeze Biologicals, Rogers, AR) in Minimal Essential Medium (Cellgro, Herndon, VA), containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.0)], then with rabbit anti-ricin immunoglobulin (Sigma Chemical Co.), and finally with fluorescein-labelled goat anti-rabbit IgG antiserum (Sigma), with a buffer wash in between each incubation. Cells were then analyzed on a flow cytometer (FACScan, Becton-Dickinson), and the mean fluorescence values were recorded. Cytotoxicity Assays Test samples were washed
fractions
and
placed
by a direct
incubated into
fresh
cytotoxicity
with medium
cells for
assay 759
at 37 "C for the
24 h. The cells
determination
as described
of
previously
the
were surviving
(18,19).
then
Vol. 183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Results We reported that
(13,14)
could
be
that
blocked
inhibited
by
demonstrated
directly
for
much greater
affinity
of the
inhibitory
effect
initial Namalwa or
cells
in
without
experiments
24 h prior
were
(several
treated
with
lactose
in
sensitive ricin
the
a very
immunoconjugate lactose
(Fig.
fraction
of
with
0.1)
concentrations involved
of
in the
the
process anti-B4
To demonstrate
cells
containing
that
swainsonine
and prevents terminated 'We thank International
maturation complex Dr.
the
antibody
Philip Meeting
blocked had
lactose
and then without
of cells
being
of anti-B4-blocked fraction
of
anti-B4-blocked
the
ricin
continuous
presence
10% of cells
cytotoxic these
cells,
cells
action
(surviving
of
conditions.
of
saturating
Apparently,
which
can be inhibited
ricin
immunoconjugate
an
by lactose
is
intoxication.
sugars
containing
in
duration
cultures
(surviving
the
with
Namalwa
99.999%
M, and about
under
with
in a more direct
to galactose-terminated Namalwa
cytotoxic
of the anti-B4-blocked
by adding
immunotoxins
less
immunotoxin
of
control
of cells
contrast,
to
absence
more than
ricin
of cells
the entire
concentrations
In
resistant
of
with
of 3.7 x lo-l1
immunotoxin
The cytotoxicity blocked
an IC,,
to the culture
anti-B4-blocked for
(14),
after
of cytotoxicity
cultures,
the
any subpopulation 1).
being
of the
24 h in
of saturating
was significantly l),
interaction
if
(Fig.
the
to us2 important
the 24 h incubation
As expected
of 1.5 x 10-llM,
minor
resistant
with
medium
intoxication
(30 mM) was added
cells
in medium
fresh
was suggested
results
(lactose-free)
for
medium. action
the
These
B4-positive
in
of
of the immunotoxin
In control
immunotoxin
XC,, values
and only
removal
weeks).
the cytotoxic
to
10-5)
the
(14).
functionally
process
here
lactose
of the
It
be
had a slight
24 h either
be still
the
in the medium during
lactose-free
showed
in which
treatment
and after
assay
maintained
5
to the
and maintained
immunotoxin
of the
cells
lactose
for
not
due to the
in which
capacity.
We report
could ricin
and cultured
might
activity
conjugates
ricin
during
the medium.
Namalwa
However,
washed
sugars points
activity
experiments
then
binding
blocked
of these
proliferative
time
from
with
immunotoxin the
their
at later
of lactose
medium
and
galactose-terminated occur
removal
component.
anti-B4-blocked
lactose
to assay
thatbindingto and might
binding
cytotoxicity
incubatedwith
cell
containing
made in cytotoxicity
containing
lactose
This
antibody
specific
were
were
medium
lactose.
retainedweak
immunoconjugates
on the
observations
ricinhad
been (30 @i).
at a concentration way that ricin,
maintained
(20).
Thorpe for this on Immunotoxins
conducted
for
Swainsonine
at
least
has
been
shown
ricin
cytotoxicity
three
with
assays
weeks
in
(20)
during discussions FL, 1990.
on
medium
of a-mannosidase
of glycoproteins
suggestion in Orlando, 760
high
of blocked
cytotoxicity
is an inhibitor
a-glycans It
binding
to achieve
we
of high-mannose IJ-glycans
the residual
was necessary
was completely
of 1 x 10m6 M (14).
II,
to galactosethat
long-term at the
2nd
Vol.
183,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
+ 30mM Lactose
--.No Lactose --.. ----io)-4r~o)-
I
0.2
I
I
I
I
0.4
06
0.8
10
anti-B4-Blocked
,
%
Rich (nM)
Figure 1. Inhibitory effect of lactose on the cytotoxicity of anti-B4-blocked o-lactose (30 mK) was added to some Namalwa ricin conjugate for Namalwa cells. cultures 24 h prior to the exposure to anti-B4-blocked ricin. This concentration of lactose was maintained in the medium during the 24 h incubation of cells with the immunotoxin and after the removal of the immunotoxin for the entire duration of the assay (2-3 weeks). In control cultures, Namalwa cells were treated with the immunotoxin for 24 h in the absence of lactose and then maintained in lactose-free medium. Surviving fractions of cells were determined in a direct cytotoxicity assay as described by Goldmacher et al. (18,19). Points in parentheses represent test cultures in which the surviving fraction of cells was below the detection limit of the test. These points show the upper limit of the surviving fraction of cells.
treatment
of cells
of cells
which
with
renders
Pre-treatment their
of
interaction
affinity
2a)
from
Namalwa
ricin
the
surface
for
4 x lo-l2
the
conjugate
swainsonine-treated immunoconjugate.
While
cells.
latter
value
This
corresponding cell
lung
2b).
of
the
resistant
of 1.6 x lo-"Mwas
is
in
the
A-chain SW2 that
range
of
conjugate expresses 761
the
with
the
M for on
was observed (Fig.
2a)
However, action
as the
of the
on non-treated the
IC,,
of the anti-B4-
cytotoxic
cytotoxicity
(14).
the
CD19 antigen
cells
measured
on lower
to 4 x lo-lo
CD19 antigen.
to the
effect
with
binding
swainsonine-treated
expression highly
surface
lo-fold
of the
similar
of 4 x 10-a M was obtained
line
expected
the behavior epitope
As expected, and
the were
anti-B4-ricin carcinoma
Next we tested to a protein
(15,21).
an IC,,
IC,,
the
cells
binds
on the
approximately
of non-treated
alter
higher
had
with
M in cultures
cells
a 2500-fold
swainsonine bound
some loo-fold
(Fig.
cells
sites
was reduced
that
not
binding
to ricin.
with Ricin
on non-treated
does
ricin
cytotoxicity
cells conjugate
of Namalwa
swainsonine
cells
ricin.
and its
swainsonine-treated blocked
depletes
them more resistant
with
(Fig.
increasing
swainsonine
cells,
swainsonine-treated observed
Similarly, CD56 antigen,
the
for
the
human small became
more
Vol.
BIOCHEMICAL
183, No. 2, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 2. Effects of svainsonine on the binding and cytotoxicity of ricin and Prior to the experiments some anti-B4-blocked ricin conjugate for Namalwe cells. Namalwa cultures were grown for at least three weeks in culture medium that contained 30 m swainsonine. This treatment was non-toxic for the cells and did not affect their proliferation rate. Panel (A), binding of ricin and anti-B4-blocked ricin to Namalwa cells. Swainsonine-treated (open symbols) or non-treated (filled symbols) cells (lx lo6 per sample) were incubated successively for 30 min at 0 "C with various concentrations of ricin (@,O) or anti-B4-blocked ricin (A, & in AB-buffer (see Experimental Procedures), thenwith rabbit anti-ricin antiserum, and finally with fluorescein-labelled goat anti-rabbit IgG antiserum, with a buffer wash in between each incubation. Cells were then analyzed on a flow cytometer, and the mean fluorescence values for the cell samples were obtained. and anti-B4-blocked ricin (A ,a ) Panel (B), cytotoxicity of ricin (0 ,O) for swainsonine-treated (open symbols) or non-treated (filled symbols) Namalwa cells. Test samples of a toxin were incubated with cells at 37 'C for 24 h. The cells were then washed and placed into fresh medium for the determination of the surviving fractions by a direct cytotoxicity assay as described by Goldmacher et al. (18,19).
resistant
to ricin
conjugate
(Fig.
As was
the
treatment
(data 3) after
case of
not
with
and to an anti-CD56
culturing the
SW2 cells
shown)
the
cells
expression
did
not
of
alter
antibody-blocked
in medium
containing
CD19 on Namalwa
the
level
of
ricin
swainsonine.
cells,
swainsonine
CD56 expression
(data
not
shown). These
experiments
terminated ricin
sugars
d
alone,
B-chain
with
translocation
cellular cytotoxicity
be more potent
that
the
blocked
This of the
interaction A-chain
with appears
to the
immunoconjugates
the can
reason
still
for
be
avidity
necessary
of
containing
immunoconjugates containing
interact
a much lower to
of galactose-
the cytotoxicity
thanimmunoconjugates
B-chain
albeit
amount
reduces
of
that
ricinto
is
in the
membranes
therefore,
oligosaccharides, (14).
the reduction
We conclude,
blocked
terminated
that
antigen-mediated
ricin.
containing A-chain
associated
the
blocked
demonstrate
with than for
ricin
galactosenative an
ricin
efficient
cytoplasm.
Discussion Native surfaces
ricin through
achieves its
B-chain
its
high and then
cytotoxicity efficiently 762
by
tightly
transferring
binding
to
the catalytically
cell
Vol.
183,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Swamsome-treated cells
lo-5 1 * 2 1 N901 -Blocked Ricin (nM)
0
3
Figure 3. Effect of swainsonine on the cytotoxicity of and N901-blocked ricin conjugate for SW2 cells. Prior to the experiments some SW2 cultures were grown for at least three weeks in culture medium that contained 30 PM swainsonine. This treatment was non-toxic for the cells and did not affect their proliferation rate. Swainsonine-treated (0) or non-treated (0) SW2 cells were incubated with the immunotoxin at 37 'C for 24 h. In order to demonstrate the specificity of the cytotoxic action of N901-blocked ricin, non-treated control cultures of SW2 cells were incubated with the immunotoxin in the presence of 1 x 10e6 M N901 antibody (A). The cells were then washed and placed into fresh medium for the determination of the surviving fractions by a direct cytotoxicity assay as described by Goldmacher et al. (18,19).
active
A-chain
necessary
for
results
of
blocked
ricin
manner
to
demonstrate
phosphate
terminated
Thorpe
and
immunoconjugates Conjugation
an
ricin
hindrance
by the
Youle
B-chain
must
interact
located
to (6).
be The
containing in
on cell
a specific
membranes
to
had come to a similar
ricin
taken
linked
another
to
for
affinity
approach galactose
antibodies
galactose-affinity
a mannose-6-
to
as showing by the
galactose, it. that
is
species due
cytotoxicity
that
to steric of
these
of the B-chain
that
independent
of
the experiments described here, we have also prepared of an altered ricin that has a greatly reduced affinity for 763
(23,24).
by fractionation
yielded
the property
preparing
residues
apparently
The high
A-chain
to
followed
chromatography for
cross-linked
translocation
appears
the cytoplasm
and colleagues
to monoclonal
interpreted
B-chain
(22).
have
reduced
antibody
membrane In
the
N-acetylimidazole-treated
ricin by
with
has been
facilitates
function.
the
immunoconjugates
oligosaccharides
colleagues
conjugates
immunotoxin
that
with
of ricinhavingimpairedbinding
contained conjugates
of
of the A-chain to here
oligosaccharide
of native
binding.
further
translocation using
presence
presented
galactose-terminated
its
the
The
introduction
experiments
conclusion
of
cytoplasm.
the efficient the
with
fulfill
the
galactose a potent galactose
Vol.
183,
No.
by virtue from
BIOCHEMICAL
2, 1992
of chemically
our
results
the efficient
that
of cells
in
How does
blocked
ricin,
achieve
B-chain
its
contains
specific
by the
This
that
for of
reagent, reacts
of
weak
with
not
interact
speculate
that
all
the
the
interactions favored
with by the
facilitate
through may further
the binding
Considering fact
that
the the
cytotoxicity lactose
functionally
important
inside
a cell
of their
late
of blocked of
interaction the
moiety
process
ricin of
the
of its 764
and locate
B-chain
blocked
intoxication.
interact
the interaction
with
certain
of
the
can be replaced
by
may be
by binding via
of
the
antigen-mediated vesicles
galactoside
and
ligands.
immunoconjugates
ricin
the
that
surface
the
simply
such interactions
immunoconjugates one might cells,
treated
site
they
to cellular
6-
cross-linking
in intracellular of
the
oligosaccharide
to
ligand
cell
that
be
Alternatively,
but
cases,
still
Analysis
Internalization
cytotoxicity
exposure in
on the
B-chain
assumes
each
underway.
that
the conjugate
antigen-specific
by prolonged
Inboth
antibody.
concentrate
the
affinity
units
would
of ricin
ricin, of
enough
achieved the
sites
inblocked
galactosides.
concentrations
residue.
residues
of low
cellular
immunoconjugate endocytosis
are
N-
binding
are currently
sugar-binding
galactose
sites
in
triantennary
chloride
a sugar
lysine
ligands
terminal
binding
cyanuric
with
on the protein
the two covalently-linked some of
the
of
galactose-
The two ligands
of the triantennary
carries
bind
an explanation
B-chain
This
is
Binding
galactose
the
six
sugars.
provides
sugars.
branch
(13,26).
one of these
terminal
to the has
hypothesis
containing of
in the
ligands
galactose.
sites
that
homology This
in ricin
cell-surface
accessible
shows
B-chain
of these
towards
specifically
a sterically
one might
affinity
affinity
specificity,
sugars
two unaltered
third
high
ricin
the
to more than
binding
also
that
glycopeptides
only
the
that
of cross-linking
of
of
cannot
galactose-terminated
galactoside
do simple
ricin
from
of the
positions
affinity
for
functions
linked
(25).
sites
of blocked
ligand,
does
than
to the high
interaction
the
with
the
galactose-containing
sugars
derived
methylamino-galactoside moiety
necessary
of A-chain
show structural
multivalent
contain two
Therefore,
that
galactose-binding
are
with
speculate
affinity
contribute
oligosaccharides
available
that
affinity
ricin
(13).
that
of native
affinities
terminated
of six
the residual
linked
is
transport
with
sites
varying
findings
could
concept
blocked
of
galactose
units
therefore,
analysis
therefore
much higher
multi-branched binding
It is clear
B-chain.
two covalently
domains
might
sites
with
COMMUNICATIONS
galactose
interaction
additional
One
galactose-binding
with
contains
crystallographic
four
domains.
ricin
for
of transmembrane
which
X-ray
strengthened
on the ricin
affinity
to the two high-affinity binding
binding
for
ligands
RESEARCH
ricin.
oligosaccharides? addition
BIOPHYSICAL
and we conclude,
and facilitation
be separated ligands,
affinity
the weak residual
killing
galactose-binding
to
linked
AND
and the
is only inhibited even argue that the moiety
happens
only
Vol.
183,
No.
2, 1992
We conclude cytotoxic with
that
potency
cytotoxicity
BIOCHEMICAL
blocked
due to a lowered,
can be restored
a cell
ricin
surface
antigen.
and CRM107 of diphtheria
AND
BIOPHYSICAL
behaves
like
but
abolished,
not
by conjugation It
toxin
joins for
in
which
RESEARCH
a mutant
toxin
binding
to a monoclonal its very
COMMUNICATIONS
behavior
antibody
results
reduced
affinity,
the point
similar
with
mutants
have been
whose reacting CRM103 reported
(27). Acknowledgments ricin
-
We thank
conjugate
and Sherrilyn
for
technical
Martell
superb
Dr.
Albert
B. Cook, assistance
Collinson
for
preparing
Nancy Cochran, in the cytotoxicity
Beth Levine
the N901-blocked and Bridget
A.
andbindingtestings.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Jansen, F. K., Blythman, H. E., Carriere, D., Casellas, P., Gros, O., Gros, J. C., Paolucci, F., Pau, B., Poncelet, P., Richer, G., Vidal, P ., Laurent, H ., and Voisin, G. A. (1982) Immunol. Rev. 62, 185-216. Thorpe, P. E. (1985) In Monoclonal Antibodies'84: Biological and Clinical Applications (A. Pinchera, G. Doria, F. Dammacco, and A. Bargellesi, Eds.), pp. 475-506, Kurtis, Milano. Frankel, A. E., Houston, L. L., and Issell, B. F. (1986) Annu. Rev. Med. 37, 125-142. Pastan, I., Willingham, M. C., and FitzGerald, D. J. (1986) Cell 47, 641648. Vitetta, E. S., Fulton, R. J., May, R. D., Till, H., and Uhr, J. W. (1987) Science 238, 1098-1104. Blattler, W. A., Lambert, J. M., and Goldmacher, V. S. (1989) Cancer Cells 1, 50-55. Blakey, D. C., Wawrzynczak,, E.J., Wallace, P. M., and Thorpe, P. E. (1988) In Monoclonal antibody therapy (H. Waldmann, Ed.), pp. 50-90, Karger, Basel. D. M. (1982) J. Biol. Chem. 257, 1598-1601. Youle, R. J., and Neville, McIntosh, D. P., Edwards, D. C., Cumber, A. J., Parnell, G. D., Dean, C. J., J. A. (1983) FEBS Lett. 164, 17-20. Ross, W. C., and Forrester, Vitetta, E. S., Cushley, W., and Uhr, J. W. (1983) Proc. Natl. Acad. Sci. USA 80, 6332-6335. Goldmacher, V. S., Blittler, W. A., Lambert, J. M., McIntyre, G., and Stewart, J. (1990) Mol. Pharmacol. 36, 818-822. Youle, R. J., and Neville, D. M. (1979) J. Biol. Chem. 254, 11089-11096. Lambert, J. M., McIntyre, G., Gauthier, M. N., Zullo, D., Rao, V., Steeves, R. M., Goldmacher, V. S., and Blattler, W. A. (1991) Biochemistry 30, 32343247. Lambert, J. M., Goldmacher, V. S., Collinson, A. R., Nadler, L. M., and Blittler, W. A.(l991) Cancer Res. 51, 6236-6242. Nadler, L. M., Anderson, K. C., Marti, G., Bates, M. P., Park, E., Daley, J. F., and Schlossman, S. F. (1983) J. Immunol. 131, 244-250. Griffin, J. D., Hercend, T., Beveridge, R., and Schlossman, S. F. (1983) J. Immunol. 130, 2947-2951. Smith, A., Waibel., R., Westera, G., Martin, A., Zimmerman, A. T., and Stahel, R. A. (1989) Br. J. Cancer 59, 174-178. Goldmacher, V. S., Anderson, J., Blittler, W. A., Lambert, J. M., and Senter, P. D. (1985) J. Immunol. 135, 3648-3651. Goldmacher, V. S., Tinnel, N. L., and Nelson, B. C. (1986) J. Cell Biol. 102, 1312-1319. Foddy, L., Feeney, J., and Hughes, R. C. (1986) Biochem. J. 233, 697-706. Goldmacher, V. S., Scott, C. F., Lambert, J. M., McIntyre, G. D., Blittler, W. A., Collinson, A. R., Stewart, J. K., Chong, L. D., Cook, S., Slayter, H. S., Beaumont, E., and Watkins, S. (1989) J. Cell. Physiol. 141, 222-234. 765
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
22. Youle, R. J., Murray, G. J., and Neville, D. M. (1981) Cell 23, 551-559. 23. Thorpe, P. E., Ross, W. C. J., Brown, A. N. F., Myers, C. D., Cumber, A. J., Foxwell, B. M. J., and Forrester, J. T. (1984) Eur. J. Biochem. 140, 63-71. 24. Wawrzynczak, E. J., Watson, G. J., Cumber, A. L., Henry, R. V., Parnell, G. D., Rieber, E. P., and Thorpe, P. E. (1991) Cancer Immunol. Immunother. 32, 289-295. 25. Rutenber, E., Ready, H., and Robertus, J. D. (1987) Nature 326, 624-626. 26. Baenziger, J. U., and Fiete, D. (1979) J. Biol. Chem. 254, 9795-9799. 27. Greenfield, L., Johnson, V. G., and Youle, R. J. (1987) Science, 238, 536539.
766
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 758-766
THE SPECIFIC CYTOTOXICITY OF IHMDNOCONJDGATES CONTAINING BLGCXEDBICIN IS DEPENDENT ON THE RESIDUALBINDING CAPACITY OF BLOCKED BICIN: EVIDENCETHAT TBE BINDING AND A-CHAIN TRANSLGCATIONACTIVITIES
NENBMNE
OF RICIN CANNOTBE
SEPARATED Victor
ImmunoGen,
Received
John
S. Goldmacheri,
January
Lambert
and Walter
A. Blattler
Street,
Cambridge,
MA 02139
148 Sidney
Inc.,
27,
M.
1992
Recently we have developed blocked ricin, a derivative of native ricin in which the galactose-binding sites of the B-chain are blocked by covalent modification with affinity ligands. This modification impedes the binding function of the B-chain, while sparing its abilLty to facilitate the entry of the toxic subunit of ricin, the A-chain, into the cytoplasm. Immunotoxins prepared with blocked ricin approach the cytotoxic potency of native ricin with antibodydependent specificity. Here we report that the high cytotoxic potency of these immunoconjugates, which is attributed to the preserved translocation function of of the ricin B-chain, is dependent on the minimal residual lectin activity blocked ricin. Our findings support the notion that two functions of ricin. membranebinding and translocation. cannot be separated. o 1992 ~~~~~~~~ press, lllc.
The
high
cytotoxicity
researchers
of
to develop
immunotoxins
of ricin
with
that
had
to be overcome
was the
terminated
oligosaccharides
which
Ricin linked is
is
the
ricin,
in the
strategy
chain alone this
type
was,
proved
supported
lTo whom correspondence 0006-291X/92
hoped
that
to remove
cytotoxic
is
should
evidence
be sent.
$1.50
758
the potency
A major
of
ricin
the entire
to galactose-
native that
B-chain
ricin,
activity
addition
B-chain.
The
and to use the Amost
mainly into
in
cells.
of two disulfidein the
However,
obstacle
of mammalian
composed
activity
ricinA-chainmoiety previous
to combine
inspired
The ribosome-inactivating
(1,2,5). than
beans,
(l-6).
on surfaces
sugar-binding
immunotoxins
castor
binding
are ubiquitous
and the
intransferringtheir
findings
they
indiscriminate
therefore,
to be less
from
antibodies
and the B-chain.
A-chain
in constructing
inefficiency These
in which
protein
the A-chain
contained
lectin
of monoclonal
a ribosome-inactivating
subunits,
initial
selectivity
a
immunotoxins because
the cytoplasm to binding
of
of their (6,7). to
the
Vol.
183,
cell
No.
2,
surface,
cytoplasm since
BIOCHEMKAL
1992
B-chain
(8-10).
also
This
less
A successful
blocking
modification
with
Immunotoxins cytotoxic potency
than
towards
B-chain
containing operates
of ricin
were
blocked
ricin, of
galactose-binding
A-fragment,
was achieved
capacity
of
than
cells,
the its
a
analog were
lOO-
by covalent
(14).
(13).
thousand-fold
while
cells
we address
independently
the
specific,
an A-chain
glycopeptides
more
reducedbinding
report
to
A-chain
gelonin,
triantennary
antigen-positive
In this
A-chain
(11,12).
sites
ricin
a greatly
the
toxin
antigen-negative
towards
despite
function.
immunoconjugates
ricin
containing
blocked
ricin
ricin,
translocation
residual
with
with
diphtheria
native
of the binding
ricin
of native
than
ligands
made
the blocked
ricin
cytotoxic
or with
of
COMMUNICATIONS
seems to be ricin
of the B-chain
multiflorum
RESEARCH
translocation function
conjugates
from seeds of gelonium
BIOPHYSKAL
facilitates
translocation
disulfide-linked
to lOOO-fold
AND
approaching
the
indicated
that
hadmaintained
its
This
affinity,
less
the question
of whether,
translocation
function
lectin
blocked
function ricin
or is
in the of
the
whether
of
the
functional
significance.
Methods Preparation of Blocked Ricin and its Conjugates with Antibodies The immunoconjugates between blocked ricin (13) and the murine monoclonal antibodies anti-B4, recognizing the CD19 molecule (15), andN901, recognizing the CD56-molecule (16), were prepared as described previously (14). These conjugates were tested for their activity as inhibitors of protein synthesis in a cell-free system and their affinity towards their respective antigen-positive cells (14), and it was found that neither the ricin A-moiety nor the antibody were inactivated in the process of the preparation of the immunotoxins. Cell
Lines The human lymphoid cell line Namalwa (ATCC CRL 1432) and the human small cell lung carcinoma line SW2 (17) were maintained in exponential growth phase in culture medium consisting of RPMI-1640 medium supplemented with 10% heat-treated (30 min at 56 "C) fetal calf serum and 2 mM L-glutamine. For experiments shown in Fig. 2, some cell cultures were grown for at least three weeks in medium that contained 30 PM swainsonine (CalBiochem Corp., La Jolla, CA). This treatment was non-toxic for the cells and did not affect their proliferation rate. Binding Assays Binding of ricin and anti-B4-blocked ricin to Namalwa cells was analyzed by indirect immunofluorescence. Cells (1 x lo6 per sample) were incubated successively for 30 min at 0 OC with various concentrations of ricin or anti-B4blocked ricin in AB-buffer [(2.5% pooled human AR serum (PelFreeze Biologicals, Rogers, AR) in Minimal Essential Medium (Cellgro, Herndon, VA), containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.0)], then with rabbit anti-ricin immunoglobulin (Sigma Chemical Co.), and finally with fluorescein-labelled goat anti-rabbit IgG antiserum (Sigma), with a buffer wash in between each incubation. Cells were then analyzed on a flow cytometer (FACScan, Becton-Dickinson), and the mean fluorescence values were recorded. Cytotoxicity Assays Test samples were washed
fractions
and
placed
by a direct
incubated into
fresh
cytotoxicity
with medium
cells for
assay 759
at 37 "C for the
24 h. The cells
determination
as described
of
previously
the
were surviving
(18,19).
then
Vol. 183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Results We reported that
(13,14)
could
be
that
blocked
inhibited
by
demonstrated
directly
for
much greater
affinity
of the
inhibitory
effect
initial Namalwa or
cells
in
without
experiments
24 h prior
were
(several
treated
with
lactose
in
sensitive ricin
the
a very
immunoconjugate lactose
(Fig.
fraction
of
with
0.1)
concentrations involved
of
in the
the
process anti-B4
To demonstrate
cells
containing
that
swainsonine
and prevents terminated 'We thank International
maturation complex Dr.
the
antibody
Philip Meeting
blocked had
lactose
and then without
of cells
being
of anti-B4-blocked fraction
of
anti-B4-blocked
the
ricin
continuous
presence
10% of cells
cytotoxic these
cells,
cells
action
(surviving
of
conditions.
of
saturating
Apparently,
which
can be inhibited
ricin
immunoconjugate
an
by lactose
is
intoxication.
sugars
containing
in
duration
cultures
(surviving
the
with
Namalwa
99.999%
M, and about
under
with
in a more direct
to galactose-terminated Namalwa
cytotoxic
of the anti-B4-blocked
by adding
immunotoxins
less
immunotoxin
of
control
of cells
contrast,
to
absence
more than
ricin
of cells
the entire
concentrations
In
resistant
of
with
of 3.7 x lo-l1
immunotoxin
The cytotoxicity blocked
an IC,,
to the culture
anti-B4-blocked for
(14),
after
of cytotoxicity
cultures,
the
any subpopulation 1).
being
of the
24 h in
of saturating
was significantly l),
interaction
if
(Fig.
the
to us2 important
the 24 h incubation
As expected
of 1.5 x 10-llM,
minor
resistant
with
medium
intoxication
(30 mM) was added
cells
in medium
fresh
was suggested
results
(lactose-free)
for
medium. action
the
These
B4-positive
in
of
of the immunotoxin
In control
immunotoxin
XC,, values
and only
removal
weeks).
the cytotoxic
to
10-5)
the
(14).
functionally
process
here
lactose
of the
It
be
had a slight
24 h either
be still
the
in the medium during
lactose-free
showed
in which
treatment
and after
assay
maintained
5
to the
and maintained
immunotoxin
of the
cells
lactose
for
not
due to the
in which
capacity.
We report
could ricin
and cultured
might
activity
conjugates
ricin
during
the medium.
Namalwa
However,
washed
sugars points
activity
experiments
then
binding
blocked
of these
proliferative
time
from
with
immunotoxin the
their
at later
of lactose
medium
and
galactose-terminated occur
removal
component.
anti-B4-blocked
lactose
to assay
thatbindingto and might
binding
cytotoxicity
incubatedwith
cell
containing
made in cytotoxicity
containing
lactose
This
antibody
specific
were
were
medium
lactose.
retainedweak
immunoconjugates
on the
observations
ricinhad
been (30 @i).
at a concentration way that ricin,
maintained
(20).
Thorpe for this on Immunotoxins
conducted
for
Swainsonine
at
least
has
been
shown
ricin
cytotoxicity
three
with
assays
weeks
in
(20)
during discussions FL, 1990.
on
medium
of a-mannosidase
of glycoproteins
suggestion in Orlando, 760
high
of blocked
cytotoxicity
is an inhibitor
a-glycans It
binding
to achieve
we
of high-mannose IJ-glycans
the residual
was necessary
was completely
of 1 x 10m6 M (14).
II,
to galactosethat
long-term at the
2nd
Vol.
183,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
+ 30mM Lactose
--.No Lactose --.. ----io)-4r~o)-
I
0.2
I
I
I
I
0.4
06
0.8
10
anti-B4-Blocked
,
%
Rich (nM)
Figure 1. Inhibitory effect of lactose on the cytotoxicity of anti-B4-blocked o-lactose (30 mK) was added to some Namalwa ricin conjugate for Namalwa cells. cultures 24 h prior to the exposure to anti-B4-blocked ricin. This concentration of lactose was maintained in the medium during the 24 h incubation of cells with the immunotoxin and after the removal of the immunotoxin for the entire duration of the assay (2-3 weeks). In control cultures, Namalwa cells were treated with the immunotoxin for 24 h in the absence of lactose and then maintained in lactose-free medium. Surviving fractions of cells were determined in a direct cytotoxicity assay as described by Goldmacher et al. (18,19). Points in parentheses represent test cultures in which the surviving fraction of cells was below the detection limit of the test. These points show the upper limit of the surviving fraction of cells.
treatment
of cells
of cells
which
with
renders
Pre-treatment their
of
interaction
affinity
2a)
from
Namalwa
ricin
the
surface
for
4 x lo-l2
the
conjugate
swainsonine-treated immunoconjugate.
While
cells.
latter
value
This
corresponding cell
lung
2b).
of
the
resistant
of 1.6 x lo-"Mwas
is
in
the
A-chain SW2 that
range
of
conjugate expresses 761
the
with
the
M for on
was observed (Fig.
2a)
However, action
as the
of the
on non-treated the
IC,,
of the anti-B4-
cytotoxic
cytotoxicity
(14).
the
CD19 antigen
cells
measured
on lower
to 4 x lo-lo
CD19 antigen.
to the
effect
with
binding
swainsonine-treated
expression highly
surface
lo-fold
of the
similar
of 4 x 10-a M was obtained
line
expected
the behavior epitope
As expected, and
the were
anti-B4-ricin carcinoma
Next we tested to a protein
(15,21).
an IC,,
IC,,
the
cells
binds
on the
approximately
of non-treated
alter
higher
had
with
M in cultures
cells
a 2500-fold
swainsonine bound
some loo-fold
(Fig.
cells
sites
was reduced
that
not
binding
to ricin.
with Ricin
on non-treated
does
ricin
cytotoxicity
cells conjugate
of Namalwa
swainsonine
cells
ricin.
and its
swainsonine-treated blocked
depletes
them more resistant
with
(Fig.
increasing
swainsonine
cells,
swainsonine-treated observed
Similarly, CD56 antigen,
the
for
the
human small became
more
Vol.
BIOCHEMICAL
183, No. 2, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 2. Effects of svainsonine on the binding and cytotoxicity of ricin and Prior to the experiments some anti-B4-blocked ricin conjugate for Namalwe cells. Namalwa cultures were grown for at least three weeks in culture medium that contained 30 m swainsonine. This treatment was non-toxic for the cells and did not affect their proliferation rate. Panel (A), binding of ricin and anti-B4-blocked ricin to Namalwa cells. Swainsonine-treated (open symbols) or non-treated (filled symbols) cells (lx lo6 per sample) were incubated successively for 30 min at 0 "C with various concentrations of ricin (@,O) or anti-B4-blocked ricin (A, & in AB-buffer (see Experimental Procedures), thenwith rabbit anti-ricin antiserum, and finally with fluorescein-labelled goat anti-rabbit IgG antiserum, with a buffer wash in between each incubation. Cells were then analyzed on a flow cytometer, and the mean fluorescence values for the cell samples were obtained. and anti-B4-blocked ricin (A ,a ) Panel (B), cytotoxicity of ricin (0 ,O) for swainsonine-treated (open symbols) or non-treated (filled symbols) Namalwa cells. Test samples of a toxin were incubated with cells at 37 'C for 24 h. The cells were then washed and placed into fresh medium for the determination of the surviving fractions by a direct cytotoxicity assay as described by Goldmacher et al. (18,19).
resistant
to ricin
conjugate
(Fig.
As was
the
treatment
(data 3) after
case of
not
with
and to an anti-CD56
culturing the
SW2 cells
shown)
the
cells
expression
did
not
of
alter
antibody-blocked
in medium
containing
CD19 on Namalwa
the
level
of
ricin
swainsonine.
cells,
swainsonine
CD56 expression
(data
not
shown). These
experiments
terminated ricin
sugars
d
alone,
B-chain
with
translocation
cellular cytotoxicity
be more potent
that
the
blocked
This of the
interaction A-chain
with appears
to the
immunoconjugates
the can
reason
still
for
be
avidity
necessary
of
containing
immunoconjugates containing
interact
a much lower to
of galactose-
the cytotoxicity
thanimmunoconjugates
B-chain
albeit
amount
reduces
of
that
ricinto
is
in the
membranes
therefore,
oligosaccharides, (14).
the reduction
We conclude,
blocked
terminated
that
antigen-mediated
ricin.
containing A-chain
associated
the
blocked
demonstrate
with than for
ricin
galactosenative an
ricin
efficient
cytoplasm.
Discussion Native surfaces
ricin through
achieves its
B-chain
its
high and then
cytotoxicity efficiently 762
by
tightly
transferring
binding
to
the catalytically
cell
Vol.
183,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Swamsome-treated cells
lo-5 1 * 2 1 N901 -Blocked Ricin (nM)
0
3
Figure 3. Effect of swainsonine on the cytotoxicity of and N901-blocked ricin conjugate for SW2 cells. Prior to the experiments some SW2 cultures were grown for at least three weeks in culture medium that contained 30 PM swainsonine. This treatment was non-toxic for the cells and did not affect their proliferation rate. Swainsonine-treated (0) or non-treated (0) SW2 cells were incubated with the immunotoxin at 37 'C for 24 h. In order to demonstrate the specificity of the cytotoxic action of N901-blocked ricin, non-treated control cultures of SW2 cells were incubated with the immunotoxin in the presence of 1 x 10e6 M N901 antibody (A). The cells were then washed and placed into fresh medium for the determination of the surviving fractions by a direct cytotoxicity assay as described by Goldmacher et al. (18,19).
active
A-chain
necessary
for
results
of
blocked
ricin
manner
to
demonstrate
phosphate
terminated
Thorpe
and
immunoconjugates Conjugation
an
ricin
hindrance
by the
Youle
B-chain
must
interact
located
to (6).
be The
containing in
on cell
a specific
membranes
to
had come to a similar
ricin
taken
linked
another
to
for
affinity
approach galactose
antibodies
galactose-affinity
a mannose-6-
to
as showing by the
galactose, it. that
is
species due
cytotoxicity
that
to steric of
these
of the B-chain
that
independent
of
the experiments described here, we have also prepared of an altered ricin that has a greatly reduced affinity for 763
(23,24).
by fractionation
yielded
the property
preparing
residues
apparently
The high
A-chain
to
followed
chromatography for
cross-linked
translocation
appears
the cytoplasm
and colleagues
to monoclonal
interpreted
B-chain
(22).
have
reduced
antibody
membrane In
the
N-acetylimidazole-treated
ricin by
with
has been
facilitates
function.
the
immunoconjugates
oligosaccharides
colleagues
conjugates
immunotoxin
that
with
of ricinhavingimpairedbinding
contained conjugates
of
of the A-chain to here
oligosaccharide
of native
binding.
further
translocation using
presence
presented
galactose-terminated
its
the
The
introduction
experiments
conclusion
of
cytoplasm.
the efficient the
with
fulfill
the
galactose a potent galactose
Vol.
183,
No.
by virtue from
BIOCHEMICAL
2, 1992
of chemically
our
results
the efficient
that
of cells
in
How does
blocked
ricin,
achieve
B-chain
its
contains
specific
by the
This
that
for of
reagent, reacts
of
weak
with
not
interact
speculate
that
all
the
the
interactions favored
with by the
facilitate
through may further
the binding
Considering fact
that
the the
cytotoxicity lactose
functionally
important
inside
a cell
of their
late
of blocked of
interaction the
moiety
process
ricin of
the
of its 764
and locate
B-chain
blocked
intoxication.
interact
the interaction
with
certain
of
the
can be replaced
by
may be
by binding via
of
the
antigen-mediated vesicles
galactoside
and
ligands.
immunoconjugates
ricin
the
that
surface
the
simply
such interactions
immunoconjugates one might cells,
treated
site
they
to cellular
6-
cross-linking
in intracellular of
the
oligosaccharide
to
ligand
cell
that
be
Alternatively,
but
cases,
still
Analysis
Internalization
cytotoxicity
exposure in
on the
B-chain
assumes
each
underway.
that
the conjugate
antigen-specific
by prolonged
Inboth
antibody.
concentrate
the
affinity
units
would
of ricin
ricin, of
enough
achieved the
sites
inblocked
galactosides.
concentrations
residue.
residues
of low
cellular
immunoconjugate endocytosis
are
N-
binding
are currently
sugar-binding
galactose
sites
in
triantennary
chloride
a sugar
lysine
ligands
terminal
binding
cyanuric
with
on the protein
the two covalently-linked some of
the
of
galactose-
The two ligands
of the triantennary
carries
bind
an explanation
B-chain
This
is
Binding
galactose
the
six
sugars.
provides
sugars.
branch
(13,26).
one of these
terminal
to the has
hypothesis
containing of
in the
ligands
galactose.
sites
that
homology This
in ricin
cell-surface
accessible
shows
B-chain
of these
towards
specifically
a sterically
one might
affinity
affinity
specificity,
sugars
two unaltered
third
high
ricin
the
to more than
binding
also
that
glycopeptides
only
the
that
of cross-linking
of
of
cannot
galactose-terminated
galactoside
do simple
ricin
from
of the
positions
affinity
for
functions
linked
(25).
sites
of blocked
ligand,
does
than
to the high
interaction
the
with
the
galactose-containing
sugars
derived
methylamino-galactoside moiety
necessary
of A-chain
show structural
multivalent
contain two
Therefore,
that
galactose-binding
are
with
speculate
affinity
contribute
oligosaccharides
available
that
affinity
ricin
(13).
that
of native
affinities
terminated
of six
the residual
linked
is
transport
with
sites
varying
findings
could
concept
blocked
of
galactose
units
therefore,
analysis
therefore
much higher
multi-branched binding
It is clear
B-chain.
two covalently
domains
might
sites
with
COMMUNICATIONS
galactose
interaction
additional
One
galactose-binding
with
contains
crystallographic
four
domains.
ricin
for
of transmembrane
which
X-ray
strengthened
on the ricin
affinity
to the two high-affinity binding
binding
for
ligands
RESEARCH
ricin.
oligosaccharides? addition
BIOPHYSICAL
and we conclude,
and facilitation
be separated ligands,
affinity
the weak residual
killing
galactose-binding
to
linked
AND
and the
is only inhibited even argue that the moiety
happens
only
Vol.
183,
No.
2, 1992
We conclude cytotoxic with
that
potency
cytotoxicity
BIOCHEMICAL
blocked
due to a lowered,
can be restored
a cell
ricin
surface
antigen.
and CRM107 of diphtheria
AND
BIOPHYSICAL
behaves
like
but
abolished,
not
by conjugation It
toxin
joins for
in
which
RESEARCH
a mutant
toxin
binding
to a monoclonal its very
COMMUNICATIONS
behavior
antibody
results
reduced
affinity,
the point
similar
with
mutants
have been
whose reacting CRM103 reported
(27). Acknowledgments ricin
-
We thank
conjugate
and Sherrilyn
for
technical
Martell
superb
Dr.
Albert
B. Cook, assistance
Collinson
for
preparing
Nancy Cochran, in the cytotoxicity
Beth Levine
the N901-blocked and Bridget
A.
andbindingtestings.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
22. Youle, R. J., Murray, G. J., and Neville, D. M. (1981) Cell 23, 551-559. 23. Thorpe, P. E., Ross, W. C. J., Brown, A. N. F., Myers, C. D., Cumber, A. J., Foxwell, B. M. J., and Forrester, J. T. (1984) Eur. J. Biochem. 140, 63-71. 24. Wawrzynczak, E. J., Watson, G. J., Cumber, A. L., Henry, R. V., Parnell, G. D., Rieber, E. P., and Thorpe, P. E. (1991) Cancer Immunol. Immunother. 32, 289-295. 25. Rutenber, E., Ready, H., and Robertus, J. D. (1987) Nature 326, 624-626. 26. Baenziger, J. U., and Fiete, D. (1979) J. Biol. Chem. 254, 9795-9799. 27. Greenfield, L., Johnson, V. G., and Youle, R. J. (1987) Science, 238, 536539.
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