Clinical and Immunological Effects of Treatment with ...Vol. 1.481-491, May 1995 Clinical Cancer Research 481 Clinical and Immunological Effects of Treatment with Murine Anti-CD3 Monoclonal
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Vol. 1. 481-491, May 1995 Clinical Cancer Research 481
Clinical and Immunological Effects of Treatment with Murine
Anti-CD3 Monoclonal Antibody along with Interleukin 2 in
Patients with Cancer’
Jacquelyn A. Hank,2 Mark Albertini, Osvaldo H.
Wesly, Joan H. Schiller, Aggie Borchert, Karen
Moore, Robin Bechhofer, Barry Storer, Jacek
Gan, Carlo Gambacorti, Jeffrey Sosman, and
Paul M. Sondel
Departments of Human Oncology [J. A. H., M. A., J. H. 5, A. B.,K. M., R. B., B. S., J. G., P. M. S.], Pediatrics [P. M. S.], and
Medical Genetics [P. M. S.], University of Wisconsin-Madison,
Madison, Wisconsin 53792; Memorial Regional Cancer Center/SIUMedical School, Springfield, Illinois 62794-9248 [0. H. W.]; IstitutoNazionale Tumori, Milan, Italy 20133 [C. G.]; and Loyola University
Medical Center, Maywood, Illinois 60153 [J. S.]
ABSTRACT
Anti-CD3 mAb and interleukin 2 (IL-2) were used in aPhase I study to treat 29 patients with cancer. The anti-CD3was given as an i.v. bolus infusion over 10 mm followed bytwo i.v. 96-h continuous infusions of IL-2 at 3 x 106 units/
m2/day with a 3-day rest between the IL-2 infusions. Fourpatients were treated with 6, 18, 60, and 300 �g/m2 anti-
CD3. One patient received 3000 �.ag/m2 anti-CD3. This pa-tient developed profound hypotension and the IL-2 infusions
were delayed for 2 weeks. Two patients were treated at anintermediate dose of 600 �iWm2. These patients developeddose-limiting toxicities including hypotension, dyspnea and
increased blood urea nitrogen, creatinine, and bilirubin.They were unable to complete their first course of therapy.
In an effort to achieve a dose of anti-CD3 which wouldactivate T cells in vivo, pentoxifylline was given to blunt thetoxicities seen with anti-CD3 thought to be due predomi-
nantly to the cytokine syndrome and tumor necrosis factorrelease. Four patients received p.o. pentoxifyffine to cover ananti-CD3 dose of 600 pig/m2. The IL-2 infusion was initiated1 week after the mAb. While there was an anti-CD3 dose-dependent increase in serum tumor necrosis factor level 1 hafter mAb infusion, pentoxifylline did not reduce the serumtumor necrosis factor level. There was also an anti-CD3dose-dependent increase in the serum soluble IL-2 receptorlevels. Other immune parameters monitored, including in
vitro cytotoxic and proliferative responses and lymphocytecount, were similar to treatment courses with IL-2 alone.Fourteen of 26 patients examined developed human anti-
murine antibodies following a single dose of anti-CD3. There
were no objective antitumor responses. We conclude that in
vivo treatment with anti-CD3 did not enhance T cell activityor expansion with subsequent IL-2 infusion and that thecombination of anti-CD3 followed by IL-2 did not improve
upon the antitumor activity previously seen with IL-2 alone.
INTRODUCTION
IL-2,3 a polypeptide produced by activated T lymphocytes,
was first described as a T cell growth factor and utilized in the
propagation and long-term in vitro culture of T lymphocytes (1).
IL-2 can enhance the proliferative and cytotoxic activity of both
T lymphocytes and NK cells in vivo and in vitro (2-4). Most
fresh, unprimed T lymphocytes do not respond well to IL-2,
since a prior encounter with specific antigen or exposure to
mitogen is necessary for a proliferative response (5). However,
some lymphocytes in fresh peripheral blood specimens can
respond directly to IL-2, and these IL-2-activated cells are able
to recognize and lyse tumor cells (6, 7). In addition, clinical
trials using IL-2 as anticancer therapy have demonstrated that
the systemic administration of IL-2 can activate and preferen-
tially expand NK cells in vivo (8-12). Although systemically
administered IL-2 increases the absolute number of CD3� lym-
phocytes, the relative frequency of these cells as a fraction of the
circulating leukocyte population and their immunological func-
tion is suppressed (13, 14).
The CD3 complex of the T cell antigen receptor complex is
able to be targeted by mAbs (15). Depending on the adminis-
tration dose, anti-CD3 mAb is able to function as either an
immunostimulating or an immunosuppressive agent. At very
high doses, anti-CD3 mAb depletes CD3-bearing cells and has
been successfully used to inhibit antigen-specific T cell-medi-
ated graft rejection in the transplant setting (16, 17). Treatment
with low doses of anti-CD3 (approximately 1/100th of the
immunosuppressive dose) can trigger T cell activation through
the CD3 T cell receptor complex and induce expression of the a
chain of the IL-2 receptor on these T cells (18). Thus, admin-
istration of low doses of anti-CD3 have demonstrated immuno-
stimulatory activity, both in vitro and in vivo (19, 20). Animal
models have shown that an immunostimulatory dose of anti-
CD3 can inhibit growth of immunogenic malignant tumors (21,
22). As T cell activation with anti-CD3 mAb induces IL-2
receptor expression, the combination of anti-CD3 mAb and
Received 11/16/94; accepted 2/17/95.
1 This research was supported by NIH Grants and Contracts CA-53441,
CA-05436, CA-32685, CM-87290, CA-14520, CA-13539, CM-47669,HL-02143, RR-03186 and by American Cancer Society Grant CH-237.2 To whom requests for reprints should be addressed, at K4/454 CSC,600 Highland Avenue, Madison, WI 53792.
3 The abbreviations used are: IL-2, interleukin 2; NK, natural killer;
a IL-2 was given as a 96-h continuous infusion of 3 X 106 units/m2/day each week for courses 1, 2, and 3. For courses 1 and 3, the IL-2 was
initiated I h after the anti-CD3 infusion. For group 9, IL-2 was initiated 1 week after the mAb. Anti-CD3 was given as an iv. bolus infusion over
10 mm. PTX, 2 g/day (400 mg p.o. 5 times per day for 3 days), was given the day before, the day of, and the day after each dose of the anti-CD3infusion. In this group, IL-2 was not initiated until 1 week after anti-CD3, and was given as a 96-h continuous infusion for 2 weeks of each treatmentcourse.
b Ab, antibody.
IL-2 in vitro leads to an augmentation of the T cell response to
IL-2 and increased activation and expansion of peripheral blood
lymphocytes over that seen following IL-2 treatment alone (23,
24). We thus performed the clinical trial described here, which
combined treatment with anti-CD3 mAb and IL-2 in vivo. The
goal of this treatment protocol was to augment LAK cell func-
tion via activation of CD3 bearing T cells through the CD3
molecule itself. Furthermore, the in vivo administration of anti-
CD3 could potentially expand putative endogenous T cells able
to selectively recognize autologous tumor.
MATERIALS AND METHODS
Patients. Twenty-nine patients were enrolled in this
Phase I trial. All patients had refractory cancers for which other
proven effective treatments were not available, and all patients
signed approved informed consent forms. Patients had an East-
ern Cooperative Oncology Group performance status of 0-1
(Karnofsky, 80-100) and a life expectancy of >4 months.
Eligibility criteria included normal hematological parameters
Decline in ECOG” performance statusof3(0-*3) 1 2 0
Weight gain �10% of total body weight 4 3 3Central venous catheter thrombosis 1 1 1Dyspnea or hypoxemia requiring oxygen 2 5 0Central nervous system (moderate to 3 4 0
severe transient headache)
a ECOG, Eastern Cooperative Oncology Group.
cluding hypotension, dyspnea or hypoxemia requiring oxygen,
as well as elevations in blood urea nitrogen, serum creatinine,
and bilirubin. One patient did not receive any of the scheduled
IL-2, while the second patient received 54 h ofthe planned 96-h
IL-2 infusion for week 1 and no IL-2 was given in the second
week of course 1 . This patient started the second course of
treatment at week 5 with an IL-2 dose reduction of 50%.
Following a completed 96-h IL-2 infusion, the second week of
course 2 was not given due to disease progression. The MiD
was thus defined as 300 p.g/m2.
PTX Treatment. Since T cell activation and expansion
had not been noted in patients treated thus far, an additional
group of four patients received p.o. PTX to blunt the cytokine
syndrome (26, 27-31). Common toxicities associated with the
cytokine syndrome include elevations in serum creatinine, Se-
rum bilirubin, blood urea nitrogen, and aspartate aminotransfer-
ase and changes in blood pressure (32, 33). The change between
the baseline value and the maximum or minimum post-anti-CD3
value for these parameters were compared for patients treated
with anti-CD3 at 300 and 600 �i.Wm2 (n 6) and anti-CD3 at
600 �.Wm2 with PTX (n = 4). The differences observed between
these two groups were not significant.
Immunological Monitoring. Statistical analyses of lab-
oratory data analyzed by anti-CD3 dose using Student’s t test
revealed that there was no significant anti-CD3 mAb dose effect
detected on the variables of cell surface phenotype, in vitro
proliferative and in vitro cytotoxic responses (data not shown).
Therefore, these data are pooled from the 16 patients receiving
the 6-, 18-, 60-, and 300-p.g/m2 doses anti-CD3 (groups 1-4).
At the higher doses of anti-CD3 mAb, many of the clinical
blood samples were not available at the appropriate sampling
times due to modifications in the treatment schedule resulting
from anti-CD3-induced toxicities; thus, paired comparisons
were not possible for laboratory data from these patients.
Cell Surface Phenotype. The cell surface phenotypepresented in Table 4 shows that in the 16 patients receiving the
combined IL-2 plus anti-CD3 mAb treatment, there was a sig-
nificant drop in the percentage of CD3� T lymphocytes with a
corresponding increase in CD16�/CD56� NK cells. A signifi-
cant increase in the percentage of cells expressing the CD25 �
486 Anti-CD3 mAb and IL-2 Therapy in Cancer Patients
Table 4 Cell s urface phenotype
% of PBMCs positivea
Sample time CD3 CD5 CD3 + CD5 CD16 CD2S CD56 MHC class II
Groups 1-4 (anti-CD3 + IL-2), n = 16On study 67Week 1 65
7065
7259
1622
616”
2034”
18351)
Week 2 55C 55C 52d 31h 20” 44” 26�
Group 8 (IL-2 alone), n = 4
On study 68 70 NA� 20 13 22 20Week 1 56 56 NA 31 18 44 41Week 2 45 44 NA 42 18 56 44
Group 9 (anti-CD3 + PTX), n = 4
On study 62 22 59 12 16 18 16
Week 2 35 40 27 35 13 58 40
Week 3 37 42 24 41 18 62 32
(I PBMCs were isolated by density gradient centrifugation and directly labeled with FITC or phycoerythrin-conjugated antibodies. The percentageof cells positive was based on PBMCs gated for lymphocytes.
I) p < 0.001 based on change from on study.
‘. P < 0.01.dp < 005�
.. NA, not applicable.
Table 5 in vitro proli ferative respo nses in patients receiving IL-2 and anti-CD3”
a PBMCs were isolated from patients prior to (on study) and 24 h after each 96-h continuous infusion of IL-2 in course 1. Quadruplicate cells
(1 X i0� cells/well) were stimulated with the indicated stimuli including irradiated PBMC from allogeneic control donors X and Y (Xx, Yx) andcultured for 54 h, labeled with [3H]thymidine for 18 h, and harvested. Values represent the mean of 16 patients treated in groups 1-4. n = 16, groups1-4; n = 4, group 8.
1) P < 0.001 based on change from on study.
�P < 0.01.dp < 005�
(IL-2 receptor) and MHC class II activation antigens was ob-
served. These values are comparable to those seen in previous
studies of patients receiving IL-2 alone (4, 8) and also are not
statistically different from comparable values obtained from the
four patients in group 8, who received only IL-2 in their first
course of treatment and the four patients in group 9 who re-
ceived PTX with the anti-CD3 infusion (Table 4). In addition,
there was no change in the -1’-S T cells, which remained stable at
a mean of 2% throughout the study.
In Vitro Proliferative Responses. The in vitro prolifer-
ative responses are shown for patients in groups 1-4 receiving
anti-CD3 and IL-2 and group 8 receiving only IL-2 in course 1
(Table 5). While there was not a significant change in the
response to the mitogen phytohemagglutinin, there was a sig-
nificant enhancement of the proliferative response to IL-2. This
was noted for the patients in groups 1-4 following the first and
second week of IL-2 and was also significant in both the 3- and
6-day proliferative assays. The in vitro proliferative response to
anti-CD3 mAb stimulation was depressed following both 1 and
2 weeks of the combined therapy and was noted in both the 3-
and 6-day assays. These enhanced proliferative responses to
IL-2 and depressed responses to irradiated allogeneic lympho-
cytes are similar to the responses seen in patients in group 8,
course 1, receiving only IL-2 (Table 5). In addition, they are
Fig. 1 Anti-CD3 dose-dependent increase in sIL-2Ra level noted in serum samples obtained on day 6 of course 1 for the 16 patients (groups 1-4)receiving 6-300 p.g/m2 anti-CD3. Regression slope is significant at P < 0.005.
A
A
A
10 100 1000
anti -CD3 DOSE (ug/m sq)
488 Anti-CD3 mAb and IL-2 Therapy in Cancer Patients
E
This increase was noted in serum obtained on day 6. The rise in
the sIL-2R is thought to reflect the mass of systemic lymphoid
activation in patients treated with IL-2 alone and is due to
secretion of the receptor, primarily by T cells in IL-2-treated
patients (34, 35). The increased activation, determined by higher
sIL-2Ra levels with greater doses of anti-CD3, may well reflect
augmented activation of IL-2Rcs release by anti-CD3-activated
T cells, as noted in vitro (36) and in mice (20, 21). The four
patients receiving PTX in conjunction with anti-CD3 showed a
similar increase in the sIL-2Ra levels, demonstrating that the
PTX dose and administration schedule did not inhibit the im-
mune stimulation in these patients.
Serum TNF Induction. An increase was noted in serum
TNF in samples obtained 1 h following the mAb infusion for the
25 patients treated in groups 1-8. There was a highly significant
dose-dependent relationship between the dose of anti-CD3 and
490 Anti-CD3 mAb and IL-2 Therapy in Cancer Patients
circulation may be similar; the more activated lymphocytes are
likely not in the peripheral circulation (8, 41). Furthermore, the
few residual CD3� cells in the circulation did show surface
murine antibody 10 and 60 mm after mAb treatment, indicating
that the murine antibody effectively reached CD3� cells in vivo.
Since T cell activation and expansion were not seen at the
lower doses of anti-CD3, and the highest dose given was quite
toxic, an intermediate dose of anti-CD3 was combined with
PTX administration. PTh is a methylxanthine which reduces
TNF secretion seen with administration of anti-CD3 (27, 28)
and the capillary leak syndrome seen with administration of
IL-2 (29-31). Four patients were treated with PTX the days
before, of, and after anti-CD3 infusion. Unlike the severe tox-
icity in both patients treated with 600 p.g/m2 of anti-CD3
without PTX, only one of the four patients in the PTX-treated
group experienced marked side effects requiring intensive care
monitoring (Table 3). This single patient developed nausea and
vomiting during the 3 days of p.o PTX treatment, and as a result
may not have been able to achieve the same systemic level of
PTX. The other three patients tolerated this dose of anti-CD3
well. Thus, the inclusion of PTX appeared to provide a subtle
degree of protection from the toxicities of anti-CD3, based
strictly on subjective clinical impression. However, neither the
serum TNF levels nor other objective toxicity parameters mea-
sured for the patients receiving the FiX were significantly
different from those seen in the patients receiving the 300- and
600-�j.g/m2 dose of anti-CD3 without the PTX. The levels of
sIL-2Ra measured in patients receiving PTX were not de-
pressed compared to values from patients not receiving PTX,
suggesting that the PTX did not inhibit the ability of the mAb to
exert an immunostimulatory effect (42).
While it is clear from animal studies that anti-CD3 can be
effective as an antitumor agent in immunogenic and weakly
immunogenic tumor models (19-22), our current study com-
bining anti-CD3 with IL-2 in a small number of patients and a
previously reported study (43) did not demonstrate any addi-
tional clinical benefit over that previously reported for IL-2
alone. Increases in two immunological parameters, the sIL-2R
and TNF, indicated that anti-CD3 induced a dose-dependent
immunological response, although it is difficult to determine if
these parameters are relevant to the induction of an antitumor
response. Furthermore, laboratory data from these patients do
not support any selective in vivo T cell expansion using anti-
CD3 mAb as a broad, antigen nonspecific activation of T cells.
It is possible that repeated anti-CD3 infusions may activate T
cells in vivo; however, we were not able to examine this possi-
blity, since the first two patients who were to receive the MiD
for 5 days did not tolerate more than 2 days of anti-CD3.
Whether this agent, in combination with other immunomodula-
tors, could help induce the generation of specific antitumor
immunity in humans remains unknown. As other more direct
approaches are now being pursued attempting to directly acti-
vate specific T cell populations with tumor-related vaccines, we
feel that the immunological and clinical data obtained from this
study are not significantly encouraging to continue evaluation of
soluble anti-CD3 antibody as an immunostimulatory agent. The
potential use of anti-CD3 in bifunctional antibodies or as means
to expand (in vivo or in vitro) antigen-reactive T cells for
adoptive transfer requires further clinical analyses.
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