| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
-Directed Monoclonal Antibodies Provide Effective Therapy in a Murine Model of Adult T-Cell Leukemia by a Mechanism other than Blockade of IL-2/IL-2R Interaction
Metabolism Branch, Division of Clinical Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892-1374 [K. E. P., B. H., L. A. W., M. S. R., M. Z., C. K. G., T. A. W.], and Protein Design Labs, Inc., Fremont, California 94555 [J. Y. T.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, on their cell surface. Presently,
there is not an accepted curative therapy for ATL. We developed an
in vivo model of ATL in non-obese diabetic/severe
combined immunodeficient (NOD/SCID) mice by introducing cells from an
ATL patient (MET-1) into the mice. The leukemic cells proliferated in
these mice that lack functional T, B, and natural killer (NK) cells.
The MET-1 leukemic cells could be monitored by measurements of both
serum soluble Tac (IL-2R) and soluble human
ß2-microglobulin (ß2µ) by ELISA. The
disease progressed to death in the mice after 
4–6 weeks. The mice
developed grossly enlarged spleens and a leukemia involving ATL cells
that retained the phenotype and the T-cell receptor rearrangement and
human T-cell lymphotrophic virus-I integration pattern of the
patient’s ATL leukemia cells. This model is of value for testing the
efficacy of novel therapeutic agents for ATL. The administration of
humanized anti-Tac (HAT), murine anti-Tac (MAT), and 7G7/B6, all of
which target IL-2R, significantly delayed the progression of the
leukemia and prolonged the survival of the tumor-bearing mice. In
particular, HAT induced complete remissions in 4 of 19 mice and partial
remissions in the remainder. It appears that the antibodies act by a
mechanism that had not been anticipated. The prevailing view is that
antibodies to the IL-2R receptor have their effective action by
blocking the interaction of IL-2 with its growth factor receptor,
thereby inducing cytokine deprivation apoptosis. However, although both
HAT and MAT block the binding of IL-2 to IL-2R of the high
affinity receptor, the 7G7/B6 monoclonal antibody binds to a different
epitope on the IL-2R receptor, one that is not involved in IL-2
binding. This suggested that the antibodies provide an effective
therapy by a mechanism other than induction of cytokine deprivation. In
accord with this view, the MET-1 cells obtained from the spleens of
leukemic mice did not produce IL-2, nor did they express IL-2 mRNA as
assessed by reverse transcription-PCR. Another possible conventional
mechanism of action involves complement-mediated killing. However,
although MAT and 7G7/B6 fix rabbit complement, HAT does not do so.
Furthermore, in the presence of NOD/SCID mouse serum, there was no
complement-mediated lysis of MET-1 cells. In addition, the antibodies
did not manifest antibody-dependent cellular cytotoxicity with NOD/SCID
splenocytes that virtually lack NK cells as the effector cells as
assessed in an in vitro chromium-release assay. However,
in contrast to the efficacy of intact HAT, the F(ab')2
version of this antibody was not effective in prolonging the survival
of mice injected with MET-1 ATL cells. In conclusion, in our
murine model of ATL, monoclonal antibodies, HAT, MAT, and 7G7/B6,
appear to delay progression of the leukemia by a mechanism of action
that is different from the accepted mechanism of IL-2
deprivation leading to cell death. We consider two alternatives: the
first, antibody-dependent cellular cytotoxicity mediated by FcRI- or
FcRIII-expressing cells other than NK cells, such as monocytes or
polymorphonuclear leukocytes. The second alternative we consider
involves direct induction of apoptosis by the anti-IL-2R antibodies
in vivo. It has been shown that the IL-2R is a critical
element in the peripheral self-tolerance T-cell suicide mechanism
involved in the phenomenon of activation-induced cell death. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
HTLV-I was the first retrovirus to be associated with a human
malignancy (4, 5, 6, 7, 8)
. There is monoclonal integration of
HTLV-I in the DNA of ATL cells, and patients with ATL have antibodies
to HTLV-I (7)
. One of the genes encoded by HTLV-I,
tax, is expressed in the early phases of ATL. taxis responsible for the transactivation of the expression of many
viral and cellular genes, including those of IL-2 and the IL-2R,
Tac, the private receptor that is IL-2-specific (9)
. The
malignant cells associated with all phases of ATL express abnormally
high numbers of IL-2R (3–11,000 receptors/cell; Refs.
9, 10, 11
). The up-regulation of these cellular genes by tax
could be responsible for the proliferation of ATL cells in the
premalignant and early leukemic phases of the disease (12
, 13)
. However, neither IL-2 mRNA nor IL-2 protein is expressed in
most cases of acute ATL, and thus autocrine stimulation by IL-2/IL-2R
interaction is not observed in most stages of the disease
(14)
. Therefore, there could be different subsets of ATL
patients that would respond to different strategies to achieve
effective therapy.
The conventional therapies [i.e., multidrug chemotherapies
or zidovudine (AZT) and IFN ] do not appear to prolong life in
patients with ATL, although there is some benefit with AZT/IFN
therapy (15)
. We have targeted the IL-2R expressed by
ATL cells using a murine monoclonal antibody, MAT. Six of 19 treated
patients had a partial (4 patients) or complete (2 patients) remission
(16)
. Our goal was to develop a preclinical in
vivo murine model of the ATL disease, such that new agents could
be tested before conducting patient trials. To establish this
model, cells from a patient (MET-1) with ATL were injected i.p. into
NOD/SCID mice. The human leukemic cells were able to proliferate
in these mice that lack functional B, T, and NK cells
(17)
. The injected mice developed leukemia distributed
widely in the tissues, similar to the leukemia in the patients. The
cells manifested a phenotype (CD3 dim, CD4±,
CD25+, CD7-) and a
molecular integration pattern of HTLV-I and the TCR-ßgene rearrangement as assessed by Southern analysis that were
identical to those of ATL cells immediately ex vivo from
this patient MET-1. The ATL cells infiltrated the spleen, which could
then be used to serially transfer the leukemia to other mice. In
addition, the disease was monitored by measurement of the plasma levels
of the soluble form of human IL-2R or ß2µ
by ELISA. The MET-1 ATL cells obtained ex vivo from the
patient and those harvested from the mouse spleens did not express IL-2
mRNA.
The observation that IL-2R is not expressed by normal resting cells,
but is expressed by ATL cells, provided the rationale for the use of
mAbs directed toward IL-2R as a treatment modality. Malignant T
cells of ATL patients express 3,100–11,400 IL-2R receptors per
cell (11)
. Therefore, antibodies to IL-2R
(CD25), such as HAT, MAT, and 7G7/B6 (non-IL-2 blocking) were evaluated
in the MET-1 model for their efficacy in the therapy of ATL. Each of
the antibodies inhibited the progression of the leukemia and prolonged
the survival of the mice. Because there was no IL-2 mRNA expression or
IL-2 production by the leukemic cells, the prevailing view of the
mechanism for anti-Tac action (i.e., the blockade of IL-2
interaction with its growth factor receptor IL-2R with consequent
cytokine deprivation cell death) was not supported by the data.
Furthermore, we present evidence against complement fixation or ADCC
involving NK cells as the mode of effective therapeutic mAb action.
However, we demonstrated that F(ab')2 HAT in
contrast to intact antibody was not effective in this model. We
consider Fc-dependent antibody-mediated cytotoxicity involving FcRI or
-III-expressing cells, such as monocytes or polymorphonuclear
leukocytes, as one operative mechanism of action of these mAbs in this
model and, by inference, in the IL-2-independent phase of ATL in
patients.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
ATL Transfer.
Both the spleen, if enlarged, or the s.c. tumor from the injection site
were ground between two frosted ends of microscope slides and then
dispersed through a cell strainer (Falcon 2340) into a 50-l centrifuge
tube. The tube was centrifuged at 4°C for 5 min at 1200 rpm. The
cells were then resuspended in 5–10 ml of ACK lysing buffer and
maintained at room temperature for 5 min. The cells were then
centrifuged for 5 min at 1200 rpm and resuspended in RPMI 1640. The
cells were counted using a hemacytometer, and media was added to yield
the appropriate cell concentration for injection (20 x 106 cells/0.5 ml).
Phenotypic Analysis of ATL Cells.
Before transfer, the leukemic cells isolated from mouse spleens
containing ATL cells were analyzed by flow cytometry to insure that the
cells being transferred were pure MET-1 ATL cells. Cells for
phenotyping were washed in FACS staining buffer (100 ml of PBS, 1 g of BSA, and 1 ml of a 10% solution of NaN3).
One million cells/well were stained with FITC-labeled antihuman
CD2, CD3, CD4, CD7, CD8, CD20, CD25, CD28, and class I HLA for 30–45
min on ice in the dark. After staining with antibodies and washing with
buffer, 100 µl of a 1:20 dilution of 0.1 mg/ml propidium iodide was
added to each well as a stain to confirm viability. After a wash, the
viable cells were then enumerated on a Becton Dickinson FACSort Flow
Cytometer. The phenotypic cell subtypes were expressed as a percentage
of live cells.
Mouse Model of ATL.
Female NOD/SCID mice (pathogen-free) were purchased from the Jackson
Laboratory at the age of 4–6 weeks. The mice were used in studies at
the age of 4–12 weeks. Leukemia was established by i.p. injection of
either 15–20 x 106 viably frozen
ATL-NOD/SCID spleen cells or the same number of splenocytes from
NOD/SCID mice with a high (>400,000 pg/ml) soluble serum IL-2R
levels (sIL-2R). Therapy experiments were performed on these mice
when their sIL-2R levels were >1000 pg/ml serum, i.e.,
10–14 days after tumor inoculation.
Measurement of sIL-2R and Soluble
ß2-Microglobulin by ELISA.
Throughout the therapy experiments, the serum concentrations of the
soluble released IL-2R chain (Tac) and soluble
ß2 µ were measured in the mice using ELISA
kits purchased from R&D Systems (soluble Tac; soluble
ß2µ). Mice were anesthetized with metofane
(A.J. Buck, Mundelein, IL) for retro-orbital bleeding. The blood was
collected into Microtainer serum separator tubes (Becton Dickinson).
The tubes were centrifuged at high speed for 4 min in an Eppendorf
centrifuge and the serum was collected. The ELISAs were performed as
indicated in the manufacturer’s kit inserts.
Monoclonal Antibodies.
MAT was produced as described previously (9
, 18)
by
fusion of NS-1 mouse myeloma cells with spleen cells of mice that had
been immunized with a cell line derived from a patient with ATL. Large
quantities of the antibody were produced by inoculating hybridoma cells
into the peritoneal cavity of BALB/c mice and then purifying the mouse
IgG2a anti-Tac from the resulting ascites by DEAE cellulose
chromatography. The final antibody preparation was in saline at pH 7.4
in a concentration of 2 mg/ml HAT (Daclizumab, Zenapax) was obtained
from Hoffmann-La Roche (Nutley, NJ). 7G7/B6 is a mouse IgG2a,
mAb
directed toward an epitope of the IL2-R peptide other than that
identified by anti-Tac (19)
. The 7G7/B6 was purified from
supernatants of a hybridoma (American Type Culture Collection) using
ImmunoPure Protein A columns (Pierce). For the
F(ab')2-HAT studies, the antibody fragments were
generated by a procedure described previously, with the exception that
a monospecific HAT F(ab')2, rather than a
bispecific agent, was generated (20)
. A homodimer-forming
"zipper" peptide was generated with the Jun zipper
linked to the HAT F(ab') portion of the genetically engineered HAT by
gene fusion. The HAT F(ab'-Jun)2 homodimers were
expressed in the mouse myeloma cell line NS0. The resulting end
products were >95% F(ab'-Jun)2 homodimers as
confirmed by acrylamide gel analysis. Humanized Mikß1 (HuMikß1), an
antibody that is directed to the IL-2 and IL-15 binding sites of the
IL-2/15Rß subunit, was obtained from Hoffmann-La Roche. Human IgG,
the negative control antibody, was purified from the serum of a normal
volunteer by DEAE cellulose chromatography. The HuMikß1
F(ab'-Fos)2 antibody fragments were generated as
described previously, with the exception that a monospecific HuMikß1
F(ab')2, rather than a bispecific agent, was
generated.
Comparison of the Avidity of the HAT F(ab'-Jun)2
Homodimers with Intact HAT.
The avidity of the intact HAT monoclonal antibody was compared with
that of HAT F(ab'-Jun)2 by two FACS assays. In
the first assay, CD25+ HuT-102 line cells were
incubated with serially diluted HAT or HAT
F(ab'-Jun)2, washed and stained with
FITC-conjugated goat antihuman antibodies. The potency of each
antibody in shifting the mean channel fluorescence was measured. In the
second assay, a fixed concentration of FITC-conjugated HAT
F(ab'-Jun)2 was incubated with serially diluted
HAT or HAT F(ab'-Jun)2. After incubation and
washing, the mean channel fluorescence at each competitor antibody was
measured. In both cases, HAT F(ab'-Jun)2 appeared
to have an avidity for HuT-102 cells 2-fold higher than that of HAT
(data not shown).
Treatment with Antibodies.
For the complete antibody studies, 100 µg/mouse in a volume of 0.2 ml
was injected once per week for 4 weeks by i.v. injection in the tail
vein. In addition to HAT, MAT, and 7G7/B6, the control treatments at
the same protein concentrations included groups injected with the
vehicle (PBS), normal human IgG (HuIgG), or HuMikß1. The leukemic
cells expressed the IL-2/15Rß peptide identified by HuMikß1 as well
as the IL-2R chain. Therefore, the HuMikß1 antibody was a control
for an effect mediated by any specific binding of a mAb to the cells.
In light of the short survival of
F(ab'-Jun)2-HAT, this antibody fragment was
injected twice daily for 14 days at a molar equivalent dose to that of
weekly administered HAT (6.67 x 10-10 mol; 66.7 µg/mouse/day
F(ab')2-HAT). During this latter study involving
F(ab')2-HAT, the PBS and HAT were administered
once per week for the same 2-week period. Throughout the studies, the
leukemic progression was monitored by ELISA for sIL-2R and
ß2µ in the serum as well as by Kaplan Meier
analysis of the survival of the mice.
Statistics.
The serum levels of sIL-2R and ß2µ at
different time points for the different treatment groups were analyzed
for statistical significance using StatView (Abacus Concepts, Berkeley,
CA). The tests used included ANOVA for significant differences between
groups, and the Fisher’s F procedure for post-hoc comparisons to
determine P. In terms of the mouse survival plots, StatView
was used to generate Kaplan-Meier cumulative survival plots.
IL-2-induced Proliferation of Kit-225 (K6) Cells.
Kit 225 (K6) cells were cultured in RPMI (+ 10% FCS) without IL-2 for
3 days before the assay. Two x 104 cells/well in triplicate in a 96-well
microtiter plate were incubated for 3 days at 37°C with a range of
IL-2 concentrations (1.56, 3.13, 6.25, 12.5, and 25 units) in the
presence or absence of HAT, MAT, or 7G7/B6 (4 µg/well). Six h before
harvest, 1 µCi/well of [3H]thymidine was
added to each well. The data are presented as the proportion of
IL-2-induced proliferation blocked by the antibody.
RT-PCR for IL-2 Message.
RNA was isolated from the spleens of NOD/SCID mice with a high tumor
burden as determined by sIL-2R ELISA. The isolation of total RNA was
performed as described in the PURESCRIPT RNA isolation kit manual
(Gentra Systems, Inc., Minneapolis, MN). The Advantage RT-for-PCR Kit
(CLONTECH Laboratories, Inc., Palo Alto, CA) was used to make cDNA from
the RNA. One µl of oligo-dT primers was added to 1 µg of RNA from
each sample (3 MET-1 NOD/SCID mouse spleens, SP-2/Tac cells, and a
positive control, Jurkat cells stimulated with PHA/PMA, and Jurkat
cells stimulated with Ionomycin/PMA). These mixtures were heated at
70°C for 2 min. Then a mixture of reaction buffer, dNTPs, RNase
inhibitor, and Moloney murine leukemia virus reverse transcriptase was
added while the tubes were on ice. The thermal cycles were as follows:
(a) 42°C for 1 h; and (b) 94°C for 5
min. diethyl pyrocarbonate water was then added and the cDNAs stored at
-70°C. The cDNAs were used for the PCR reaction with primers for
IL-2 or G3PDH (CLONTECH). The thermal cycles for the reaction with
primers were as follows: (a) 1 cycle of 94°C, 4 min;
58°C, 45 s; 72°C, 1 min; (b) 30 cycles of 94°C, 1
min; 58°C, 45 s; 72°C, 1 min; (c) hold at 72°C
for 5 min; and (d) hold at 4°C. The PCR products were then
run at 100 volts on a 1% agarose gel (0.5 g agarose + 50 ml 1 x Tris acetate buffer dissolved, then 5
µl ethidium bromide).
ADCC.
The capacity of the various antibodies to function in ADCC was measured
by a chromium release assay. MET-1-NOD/SCID spleen cells were labeled
with 400 µCi/107 cells of chromium-51
(Amersham, Piscataway, NJ) by incubation at 37°C for 1–2 h. The
cells were washed three times and aliquoted into a flat-bottomed
96-well microtiter plate (1 x 104
cells/well). The antibodies were added at 1 µg/ml to each of the
appropriate wells. Effector cells were spleen cells from either
CB17-SCID (the parental strain of NOD/SCID mice) or naïve
NOD/SCID mice at effector:target ratios of 0:1, 5:1, 10:1, 30:1, 60:1,
100:1 or 200:1. After 4 h of incubation at 37°C, the plates were
centrifuged at 1200 rpm for 2 min. Plates were harvested using the
Skatron harvesting system (Skatron Instruments, Inc.), and the
supernatants were counted in a gamma counter. The data were plotted as
the percentage specific lysis {% SL = [(experimental cpm - spontaneous cpm/maximum cpm - spontaneous cpm)]
x 100}.
Complement Fixation.
A viability assay using trypan blue was used to determine whether the
unmodified antibodies to IL-2R could fix complement as their
mechanism of action. HuT-102 cells (1 x 105 cells/well), which are from an HTLV-I
infected T-cell line typically used as a prototype ATL in
vitro, were incubated with the unmodified antibodies (1 µg/ml)
on ice for 1 h. The cells were washed, and 200 µl/well of a
source of complement were added for 1–2 h at 37°C. The assay
positive control serum source was Rabbit Complement MA (Accurate
Chemical & Scientific, Westbury, NY) at a 1:5 dilution. The
experimental serum source was serum from naïve NOD/SCID mice at
a 1:5 dilution. The data are plotted as the percentage of cells that
remain viable as assessed by trypan blue exclusion.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in meaningful quantities,
activated T cells express large numbers of the IL-2R subunit of the
multisubunit high-affinity receptor for IL-2 (Tac, IL-2R), this
receptor provides a target for rationally designed therapies to target
the cells involved in the leukemia. We developed an in vivo
model of ATL to test the efficacy of potential therapies including
IL-2R-directed approaches for the disease.
Establishment of a Murine Model of ATL.
Peripheral blood lymphocytes from a patient (MET-1) with ATL were used
to develop a model of ATL in mice. NOD/SCID mice were used to establish
this model because the mice lack functional B, T, and NK cells. Once
the initial patient cells were established in NOD/SCID mice, the mice
were used to expand and serially transfer the cells to other NOD/SCID
mice for studies. After the i.p. injection of 20 x 106 MET-1 leukemic cells, there was wide tissue
distribution of human CD3- and CD25-expressing leukemic cells with
marked infiltration of the lungs, spleen, liver, kidney, and lymph
nodes and moderate infiltration of the bone marrow. The spleens of the
mice increased to a size of 3 cm x 1 cm by 4–6
weeks after tumor cell injection. At that time the peripheral WBC
counts included more than 10,000/mm3 leukemic
lymphocytes. This therapeutic model appears to be clinically meaningful
because the cells that proliferate in the mice were characterized and
shown by several methods to be identical to the actual leukemic MET-1
cells obtained ex vivo. Each person with ATL has a certain
pattern of HTLV-I integration and TCR-ß gene rearrangement
that can be used as a "fingerprint" of the leukemic cells. By
Southern blot analysis, we were able to show that the molecular markers
(HTLV-I integration pattern and TCRß gene rearrangement)
present in ex vivo leukemic cells were conserved after the
transfer to the mouse model (data not shown). A second method of
characterization was based upon the fact that the original cells from
MET-1 had a distinct phenotype elucidated by FACS analysis: CD3 dim,
CD7-negative, and CD25-positive. Therefore, before the transfer
of cells, they were analyzed for several surface markers by flow
cytometry. The typical phenotype of MET-1 cells from the mice is as
follows: CD2+, CD3 dim,
CD4±, CD7-,
CD8-, CD20-,
CD25+, CD28+, and HLA class
I+ (Table 1)
. Thus we view the cells in
the murine model as the counterpart of the patient’s circulating ATL
cells. Lastly, it is well known that the soluble portion of the
IL-2R is cleaved and can be found circulating in serum from patients
with ATL. Human IL-2R as well as soluble human
ß2µ could be detected and quantified in the
serum from MET-1 ATL-inoculated mice by an ELISA methodology. The
levels of the soluble IL-2R (Tac) and ß2µ
increased as the disease progressed in the mice (Fig. 1)
. The soluble IL-2R reached levels
of 500,000 to 1,000,000 pg/ml immediately before death. Mice typically
survived 6–10 weeks after leukemic cell inoculation. The ELISAs for
sIL-2R and ß2µ provided a good measure of
the progression of the disease throughout therapy studies.
|
|
and ß2µ were indicative of
the tumor load of ATL in our mouse model. When the levels of sIL-2R
reached 1000 pg/ml, we concluded that the leukemic cells were
proliferating in the mice. At this point the mice were entered into a
therapeutic study. In the trial, groups of 8–10 mice each were treated
with PBS, IgG, HAT, MAT, 7G7/B6, or HuMikß1. The study was performed
on six occasions. In each study, the antibodies at 100 µg in 200 µl
were injected i.v. one time per week for 4 weeks. The HAT, MAT, and
7G7/B6 antibodies, all of which are directed toward the IL-2R on the
leukemic cells, had a therapeutic effect as seen by their effect on the
serum levels of sIL-2R or ß2µ (Fig. 2)
. In comparison with the serum
concentration of IL-2R in the PBS control mice, on day 28 there was
a significant reduction of sIL-2R in the HAT-
(P < 0.01), MAT- (P < 0.01), or 7G7/B6- (P < 0.01) treated
animals compared with the PBS-treated controls. Similarly, the human
ß2µ levels were significantly reduced
(P < 0.01) for the same groups. Furthermore,
there was a significant (P < 0.01)
prolongation of the survival of the mice treated with HAT, MAT, or
7G7/B6 as compared with the PBS control (Fig. 3)
. The median survival duration of the
control group (PBS) was 37 days. All of the mice in this group as well
as the IgG and HuMikß1 groups had died by day 58 of the study. In
contrast, 60% of the 7G7/B6-treated and all of the MAT- and
HAT-treated mice were alive at that time. Comparable results were
observed when the study was repeated five additional times. The graph
shown in Fig. 3
is typical of the six comparable studies performed. An
additional prolongation of survival was observed when mice were treated
continuously weekly with HAT until death. In particular, 90% of the
mice remained alive at 110 days after tumor inoculation in this group,
compared with 60% in mice that had been treated with HAT for only 4
weeks (data not shown).
|
|
epitope than that identified by HAT, one which is
not involved in the binding of IL-2. Furthermore, as shown in Fig. 4
, 7G7/B6 does not block IL-2-induced
proliferation. This suggested that such a blockade of the IL-2 action
was not the mechanism of antibody action. In strong support of this
view, the ATL cells obtained from the mouse spleen did not express IL-2
mRNA as assessed by RT-PCR (Fig. 5)
,
excluding the presence of an IL-2/IL-2R autocrine loop. Furthermore,
there was no evidence of human IL-2 protein in the serum of these
AJ-ATL-bearing mice as assessed by an IL-2-specific ELISA (data not
shown). Moreover, as is true for human cells in general, MET-1 cells
did not proliferate in response to murine IL-2 (data not shown).
Therefore, there appears to be a mechanism of effective therapeutic
action for these antibodies other than the blockade of IL-2/IL-2R
interaction. An alternative theoretical mechanism of action could
involve complement fixation. The assay used to test for this action was
a viability assay using trypan blue exclusion in the presence of the
antibodies and a serum source. The assay positive control involved the
use of rabbit serum (1:5 dilution). With this as the complement source,
MAT and 7G7/B6 were associated with 50% death of the target HuT-102
cells. The other antibodies used, including HAT, did not fix rabbit
complement. Furthermore, when naïve NOD/SCID mice serum was
used as the potential complement source, none of the antibodies were
able to fix complement (data not shown). This was expected because it
had been reported previously that NOD/SCID mice are deficient in
complement-mediated killing (17)
.
|
|
. To define the role of
NK-mediated ADCC in the MET-1 model, the effector cells analyzed were
from naïve NOD/SCID mice spleens because these mice were the
ones used in the in vivo model of antibody therapy for ATL.
Using the same targets (MET-1 NOD/SCID splenocytes containing ATL
cells) and the same antibodies, but different effectors (NOD/SCID
splenocytes), the results were very different. None of the antibodies
caused specific lysis in this ADCC system (Fig. 6A)
. This
observation is in accord with the reported virtual lack of NK-cell
activity in NOD/SCID mice.
|
.
Furthermore, as noted in "Materials and Methods," the avidity of
the F(ab'-Jun)2 homodimer for the IL-2R
receptor on cells was at least as great as that of the intact mAb.
In vivo survival of F(ab'- Jun)2 HAT
antibodies were evaluated in 10 mice using the F(ab'-
Jun)2 homodimers iodinated by iodogen. The
t1/2 of the terminal exponential of decline
of the radiolabeled F(ab'-Jun)2 HAT in these mice
was approximately 7 h, which contrasts with the 3.5–4 days
observed with intact anti-Tac antibodies (data not shown). For the
efficacy studies, the F(ab'-Jun)2 anti-Tac
fragment was administered at 100 µg 2 x a day for 14
days in light of the short survival of F(ab')2
observed in the mice. In parallel, 100-µg doses of intact HAT were
administered weekly for the same 2-week period. However, it must be
noted that the short survival of the fragments may alter the exposure
and binding of these fragments so that the IL-2R is not occupied to
the same extent and time as is true with the intact antibody. There was
a significant increase (P < 0.01) of the
survival of mice with MET-1 tumors that were treated with intact HAT
when compared with the PBS controls (Fig. 8)
. In contrast, the
F(ab'-Jun)2 HAT was ineffective in prolonging the
survival of the tumor-bearing mice, with no significant increase
(P > 0.35) in survival of the tumor bearing
mice. Thus an FcR requiring action of the HAT must be considered for at
least one of the modes of action of HAT, MAT, and 7G7/B6 in the MET-1
mouse model and, by inference, in select human patients with ATL where
the malignant cells do not express IL-2 mRNA nor produce IL-2 protein,
yet undergo remissions after administration of HAT.
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Finally, the MET-1 ATL model presents many features that parallel those
observed in patients with adult T-cell leukemia and thus represents a
valuable model to evaluate the tissue toxicity and efficacy of
therapeutic agents directed toward the treatment of ATL. We are
evaluating an array of such agents including antibodies directed toward
the multiple elements of the IL-2R system, unmodified as well as armed
with toxins and - and ß-emitting radionuclides. Furthermore, the
MET-1 ATL model is being used to test Jak3 inhibitors, rapamycin
analogues and NFB inhibitors. During the present trial, we made the
observation that antibodies directed toward IL-2R provided
meaningful therapy for ATL in this model. This was an unanticipated
observation in light of the fact that the MET-1 ATL cells neither
produced nor required IL-2 for their survival.
The dominant target of our anti-ATL therapeutic program has been the
IL-2R, a subunit identified by the anti-Tac mAb
(23, 24, 25)
. The scientific basis for this approach is that
resting normal cells, including the normal cells of patients with
leukemia, do not express IL-2R in contrast with its expression by
the leukemic cells of patients with HTLV-I-associated ATL. Furthermore,
IL-2R is constitutively expressed by select malignant cells of
patients with cutaneous T-cell lymphoma, hairy cell B-cell leukemia,
and the Reed Sternberg cells of Hodgkin’s disease (26)
.
Moreover, IL-2R is expressed by the abnormal T cells in a variety of
autoimmune disorders as well as on those T cells involved in organ
allograft rejection (26)
. To exploit this difference in
IL-2R expression, we performed therapeutic trials with the mAb
anti-Tac that blocks the binding of IL-2 to IL-2R. On the basis of
successful clinical trials, the humanized form of this antibody has
been approved by the Food and Drug Administration for use to prevent
renal organ allograft rejection and has shown efficacy in the treatment
of such autoimmune disorders as tropical spastic paraparesis and T-cell
mediated uveitis (23
, 27, 28, 29)
. In addition, approximately
one-third of ATL patients treated with MAT developed a partial or
complete remission (24)
. In those cases where an effective
therapeutic response was observed, it had been presumed that the
antibody mediated its effective response by preventing IL-2 from
interacting with its growth factor receptor, thereby leading to
cytokine deprivation apoptosis. Support for this mechanism in select
cases is provided by the observation that the leukemic cells of a
proportion (
15–20%) of patients with ATL are in the autocrine
IL-2/IL-2R phase of their disease (12, 13, 14)
. In such
patients, predominantly those with the smoldering and chronic stages of
ATL, we have demonstrated spontaneous proliferation of their ex
vivo leukemic cells. This spontaneous proliferation could be
inhibited by anti-Tac in the in vitro culture. These
observations in these ATL patients parallel the ones observed
previously in patients with tropical spastic paraparesis/HTLV-I
associated myelopathy (30)
. Although anti-Tac blockade of
IL-2 interaction with IL-2R may play a role in the frequent
therapeutic responses observed in patients with the early stages of
ATL, during the disease progression the malignant cells advance to a
phase where, although they continue to express IL-2R, they no longer
produce nor require IL-2 for their proliferation and survival
(30)
. Nevertheless, the signaling pathway involving Jak1
and Jak3 as well as Stat 5 remains activated (31
, 32)
. A
number of factors underlie this IL-2-independent action. HTLV-I tax
transactivates IL-15, which acts on the private IL-15R and on
IL-2/IL-15ß and 
c shared with IL-2 (33)
. In addition,
in IL-2-independent cell lines, there is a loss of SHP-1, the
phosphatase that normally inactivates Jak3 (34)
. It had
been presumed that ATL in its IL-2-independent phase would no longer
respond to unmodified antibodies such as HAT that are directed to the
private IL-2-specific receptor IL-2R. However, in conflict with this
prediction, IL-2R directed mAbs provided effective therapy in the
MET-1 ATL model by a mechanism other than the blockade of IL-2/IL-2R
interaction. In particular, in each of six separate trials, the 7G7/B6
antibody that binds to an epitope on the IL-2R receptor that is not
involved IL-2 binding provided effective therapy. Furthermore, the
MET-1 cells obtained ex vivo from the spleens of leukemic
mice did not express IL-2 mRNA as assessed by RT-PCR, nor did they
release IL-2 into the media during short-term culture. In clinical
parallel with this observation in the murine model, we have noted at
least one patient who responded to high-dose HAT therapy, although his
leukemic cells did not proliferate spontaneously ex vivo nor
did they produce IL-2 mRNA as assessed by RT-PCR.
We searched for other conventional mechanisms of action to explain the
efficacy of IL-2R-directed antibodies in the MET-1 ATL model.
Complement-mediated cytotoxicity appears to be excluded by the
observation that HAT did not fix rabbit complement, and in the presence
of NOD/SCID mouse serum that is complement-poor there was no
complement-mediated lysis of MET-1 cells with any of the three
anti-IL-2R antibodies examined. Classical ADCC mediated by NK cells
also does not appear to be a likely mode of action. The NOD/SCID mice
were chosen for the transfer of ATL cells because they virtually lacked
functional NK cells. Furthermore, the three antibodies directed toward
IL-2R did not manifest ADCC with NOD/SCID splenocytes as effector
cells as assessed in a chromium-release assay. Although classical
NK-mediated ADCC does not appear to be a dominant factor in the action
of the anti-IL-2R antibodies in the MET-1 ATL model, the
immunoglobulin-Fc receptor does appear to be one of the elements
involved in the action of the antibodies. In contrast to the efficacy
of intact HAT, the F(ab')2 version of this
antibody was ineffective in the present study in prolonging the
survival of the mice injected with MET-1 ATL cells. In these studies,
we treated the mice with the F(ab')2 version of
the antibody twice a day for 14 days, whereas the HAT in this
experiment was used weekly for 2 weeks. The more frequent
administration of the F(ab')2 version was used to
compensate for the short (7 h versus 77 h) terminal
t1/2 of the
F(ab')2 fragment as compared with that of the
intact antibody. However, because of the wide-spread distribution of
leukemic cells and their small number in the body at the time of
therapy, we cannot be assured that the exposure and binding of the
fragments to IL-2R on the tumor cells is equivalent to that provided
by the intact HAT mAb. The effective action of the intact antibody may
involve the FcRIII present on monocytes and macrophages in the
NOD/SCID mice. This conclusion is in accord with the results of
Clynes et al. (35
, 36)
. The FcR common
chain-deficient mice (FcR-/-) they generated
that lack the activation FcRs I and III did not manifest
passive or active protection against pulmonary metastases in the
syngeneic B16 melanoma mouse model. Furthermore, such
FcRIII-deficient mice did not manifest the efficacy in tumor models
observed in wild-type mice when anti-HER2/neu (Trastuzumab, Herceptin)
or anti-CD20 (Rituximab) antibodies were evaluated. Thus Clynes
et al. (35
, 36)
have proposed that although
multiple mechanisms have been suggested for the ability of antitumor
antibodies to mediate their effects in vivo, there is a
dominant and necessary role for FcR-dependent binding for in
vivo activity (35
, 36)
.
A final alternative mechanism, for which we have no evidence,
that might contribute to the efficacy of these IL-2R-directed mAbs
is the induction of apoptotic death through the IL-2/IL-2R-associated
pathway that is involved in AICD. Normally the apoptotic cell
death of mature peripheral T cells occurs after persistent T-cell
receptor stimulation (37, 38, 39)
. After this stimulation,
IL-2 is produced and interacts with IL-2R, ultimately placing the cell
in cycle. The triggering of these cycling cells through the
CD3-TcR pathway leads to the expression of fas ligand or
tumor necrosis factor- that are effector molecules associated with T
cell suicide. IL-2 and IL-2R appear to play critical roles in this
AICD process, in that mice made deficient in IL-2 or IL-2R by gene
targeting manifest lymphocytosis, hyperglobulinemia, and autoimmune
hemolytic anemia. We reason that IL-2-specific AICD might be associated
with IL-2R expression, and that the addition of the three mAbs to
IL-2R may have induced AICD-related cell death in the MET-1 ATL T
cells.
In summary, in our murine model of ATL the mAbs HAT, MAT, and 7G7/B6
appear to delay the progression of leukemia by a mechanism of action
other than the blockade of IL-2/IL-2R interaction. The one clear
mechanism is FcR-mediated death by FcRIII-expressing cells with
monocytes or granulocytes as the potential effector cells. We also
consider induction of AICD by anti-IL-2R antibodies in
vivo as a contributing factor.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 To whom requests for reprints should be
addressed, at Chief, Metabolism Branch, National Cancer Institute, NIH,
Building 10, Room 4N-115, 10 Center Drive, MSC 1374, Bethesda, MD
20892-1374. 
2 The abbreviations used are: ATL, adult T-cell
leukemia; HTLV-I, human T-cell lymphotrophic virus-I; IL-2, interleukin
2; IL-2R, IL-2 receptor ; NK, natural killer; TCR, T-cell
receptor; PBMC, peripheral blood mononuclear cell; FACS,
fluorescence-activated cell sorting; ADCC, antibody-dependent cellular
cytotoxicity; AICD, activation-induced cell death; HAT, humanized
anti-Tac; mAb, monoclonal antibody; MAT, murine anti-Tac; NOD/SCID,
non-obese diabetic/severe combined immunodeficient; RT-PCR, reverse
transcription-PCR.
Received 5/15/00. Accepted 10/31/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-expressing adult T-cell leukemia with Yttrium-90-labeled anti-Tac. Blood, 86: 4063-4075, 1995.B site. Proc. Natl. Acad. Sci. USA, 95: 2452-2457, 1998.This article has been cited by other articles:
|
|
 |
|
  C. Parrula, B. Zimmerman, P. Nadella, S. Shu, T. Rosol, S. Fernandez, M. Lairmore, and S. Niewiesk Expression of Tumor Invasion Factors Determines Systemic Engraftment and Induction of Humoral Hypercalcemia in a Mouse Model of Adult T-cell Leukemia Vet. Pathol., September 1, 2009; 46(5): 1003 - 1014. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chen, M. Zhang, W. Ju, and T. A. Waldmann Effective treatment of a murine model of adult T-cell leukemia using depsipeptide and its combination with unmodified daclizumab directed toward CD25 Blood, February 5, 2009; 113(6): 1287 - 1293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Kasaian, X.-Y. Tan, M. Jin, L. Fitz, K. Marquette, N. Wood, T. A. Cook, J. Lee, A. Widom, R. Agostinelli, et al. Interleukin-13 Neutralization by Two Distinct Receptor Blocking Mechanisms Reduces Immunoglobulin E Responses and Lung Inflammation in Cynomolgus Monkeys J. Pharmacol. Exp. Ther., June 1, 2008; 325(3): 882 - 892. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhang, M. Zhang, K. Garmestani, V. S. Talanov, P. S. Plascjak, B. Beck, C. Goldman, M. W. Brechbiel, and T. A. Waldmann Effective treatment of a murine model of adult T-cell leukemia using 211At-7G7/B6 and its combination with unmodified anti-Tac (daclizumab) directed toward CD25 Blood, August 1, 2006; 108(3): 1007 - 1012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Z. Dewan, J.-n. Uchihara, K. Terashima, M. Honda, T. Sata, M. Ito, N. Fujii, K. Uozumi, K. Tsukasaki, M. Tomonaga, et al. Efficient intervention of growth and infiltration of primary adult T-cell leukemia cells by an HIV protease inhibitor, ritonavir Blood, January 15, 2006; 107(2): 716 - 724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ohsugi, R. Horie, T. Kumasaka, A. Ishida, T. Ishida, K. Yamaguchi, T. Watanabe, K. Umezawa, and T. Urano In vivo antitumor activity of the NF-{kappa}B inhibitor dehydroxymethylepoxyquinomicin in a mouse model of adult T-cell leukemia Carcinogenesis, August 1, 2005; 26(8): 1382 - 1388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zhang, Z. Zhang, C. K. Goldman, J. Janik, and T. A. Waldmann Combination therapy for adult T-cell leukemia-xenografted mice: flavopiridol and anti-CD25 monoclonal antibody Blood, February 1, 2005; 105(3): 1231 - 1236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Linden Remission of a Refractory, Anaplastic Large-Cell Lymphoma after Treatment with Daclizumab N. Engl. J. Med., September 30, 2004; 351(14): 1466 - 1467. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zhang, Z. Zhang, K. Garmestani, C. K. Goldman, J. V. Ravetch, M. W. Brechbiel, J. A. Carrasquillo, and T. A. Waldmann Activating Fc Receptors Are Required for Antitumor Efficacy of the Antibodies Directed toward CD25 in a Murine Model of Adult T-Cell Leukemia Cancer Res., August 15, 2004; 64(16): 5825 - 5829. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. C. Moura, Y. Lepelletier, B. Arnulf, P. England, C. Baude, C. Beaumont, A. Bazarbachi, M. Benhamou, R. C. Monteiro, and O. Hermine A neutralizing monoclonal antibody (mAb A24) directed against the transferrin receptor induces apoptosis of tumor T lymphocytes from ATL patients Blood, March 1, 2004; 103(5): 1838 - 1845. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhang, M. Zhang, C. K. Goldman, J. V. Ravetch, and T. A. Waldmann Effective Therapy for a Murine Model of Adult T-Cell Leukemia with the Humanized Anti-CD52 Monoclonal Antibody, Campath-1H Cancer Res., October 1, 2003; 63(19): 6453 - 6457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhang, M. Zhang, J. V. Ravetch, C. Goldman, and T. A. Waldmann Effective therapy for a murine model of adult T-cell leukemia with the humanized anti-CD2 monoclonal antibody, MEDI-507 Blood, July 1, 2003; 102(1): 284 - 288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zhang, Z. Zhang, K. Garmestani, J. Schultz, D. B. Axworthy, C. K. Goldman, M. W. Brechbiel, J. A. Carrasquillo, and T. A. Waldmann Pretarget radiotherapy with an anti-CD25 antibody-streptavidin fusion protein was effective in therapy of leukemia/lymphoma xenografts PNAS, February 18, 2003; 100(4): 1891 - 1895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zhang, Z. Yao, K. Garmestani, D. B. Axworthy, Z. Zhang, R. W. Mallett, L. J. Theodore, C. K. Goldman, M. W. Brechbiel, J. A. Carrasquillo, et al. Pretargeting radioimmunotherapy of a murine model of adult T-cell leukemia with the alpha -emitting radionuclide, bismuth 213 Blood, June 17, 2002; 100(1): 208 - 216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Pise-Masison, M. Radonovich, R. Mahieux, P. Chatterjee, C. Whiteford, J. Duvall, C. Guillerm, A. Gessain, and J. N. Brady Transcription Profile of Cells Infected with Human T-cell Leukemia Virus Type I Compared with Activated Lymphocytes Cancer Res., June 1, 2002; 62(12): 3562 - 3571. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tan and T. A. Waldmann Proteasome Inhibitor PS-341, a Potential Therapeutic Agent for Adult T-Cell Leukemia Cancer Res., February 1, 2002; 62(4): 1083 - 1086. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
