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Vol. 1, 607-614, June 1995 Clinical Cancer Research 607
Autologous Peripheral Blood Stem Cell Transplantation and
Adoptive Immunotherapy with Activated Natural Killer
Cells in the Immediate Posttransplant Period’
John Lister,2 Witold B. Rybka,
Albert D. Donnenberg,
Margarida deMagalhaes-Silverman,
Steven M. Pincus, Elana J. Bloom,
Elaine M. Elder, Edward D. Ball, and
Theresa L. Whiteside
Division of Hematology/Bone Marrow Transplantation, Department
of Medicine [J. L., W. B. R., A. D. D., M. d-S., S. M. P., E. J. B.,
E. D. B.], and Departments of Pathology and Otolaryngology
[E. M. E., T. L. W.], Pittsburgh Cancer Institute, University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213-2582
ABSTRACT
Relapse after high-dose chemotherapy supported by pe-ripheral blood stem cell transplantation (HDC-PBSCT) is the
main cause of therapeutic failure in patients with lymphomaand breast cancer. Adoptive immunotherapy with activatednatural killer (A-NK) cells and interleukin 2 might eliminate
surviving residual tumor without adding to toxicity.Eleven patients with relapsed lymphoma and one with
metastatic breast cancer were entered on a pilot clinical trial
of HDC-PBSCT followed on day 2 after transplant by infu-sion of cultured autologous A-NK cells. Simultaneously, re-
combinant human interleukin 2 (rhIL-2) was initiated as a
4-day continuous i.v. infusion at 2 x 106 IU/m2/day, referredto as high-dose rhIL-2. Therapy with high-dose rhIL-2 wasfollowed by a 90-day continuous i.v. infusion at 3 x i05IU/m2/day, referred to as low-dose rhIL-2. All patients en-grafted and nine completed treatment. Posttransplant daysto a neutrophil count of 500/pJ and to a platelet count of50,000/p.l were similar to comparable patients treated withHDC-PBSCT alone. Generation of A-NK cells for therapywas feasible in all patients except the three patients with
Hodgkin’s disease, whose cells did not proliferate in culture.Overall toxicity associated with early posttransplant trans-fer of A-NK cells and interleukin 2 did not differ from thatobserved with peripheral blood stem cell transplantationalone in comparable patients. There was early amplificationof natural killer cell activity in the peripheral blood of fourpatients that appeared to result from the transfused A-NKcells. Adoptive transfer of A-NK cells and rhIL-2 during the
pancytopenic phase after HDC-PBSCT was feasible and well
tolerated, did not adversely affect engraftment, and resultedin amplified natural killer activity in the peripheral bloodduring the immediate posttransplantation period.
INTRODUCTION
The use of HDC3 and hemopoietic stem cell support has
become widely employed in the treatment of relapsed lym-
phoma and metastatic breast cancer (1-7). In chemotherapy-
sensitive lymphoma and metastatic breast cancer, HDC can
provide disease-free survival at S years in 40% and 15% of
remitting patients, respectively. Nevertheless, the majority of
completely remitting patients will relapse and die of their dis-
ease. Dose escalation of chemotherapy and radiation therapy
above conventional transplant doses has not improved disease-
free survival.
AlT with in vitro-activated autologous effector cells, either
LAK cells or tumor-infiltrating lymphocytes, in combination
with IL-2 has been shown to induce long-term responses in a
small proportion of patients with metastatic melanoma or renal
cell carcinoma (8, 9). These two types of cancer are generally
chemoresistant but seem to be among the most responsive to
biological therapy. Experience with the therapeutic use of cel-
lular effectors and IL-2 in lymphoma or breast cancer has been
limited, and only patients with advanced disease have been
treated (10). Nevertheless, a few responses have been seen (1 1),
indicating that AlT might be effective in these diseases. The
treatment protocol described here is an attempt to evaluate the
feasibility of AlT with A-NK cells and IL-2 in the setting of
HDC-PBSCT in patients with lymphoma or breast cancer at the
time of maximum chemotherapy effect.
A-NK cells are a subset of CD3�CD56�m peripheral blood
NK cells selected by adherence to plastic in the presence of
22 nM of IL-2 (12). A-NK cells can be expanded in culture with
IL-2 for 2 to 3 weeks (12). This subset of NK cells has been
shown recently to preferentially enter human tumor spheroids or
established human tumor xenografts in nude mice and to kill
NK-resistant tumor cells (13). In murine models of tumor me-
tastasis, systemically delivered A-NK cells have been shown to
preferentially localize to metastases (14). Our hypothesis has
Received 11/i 1/94; accepted 3/9/95.
1 This study was supported in part by the Alcoa Foundation (Pittsburgh,
PA), NIH/National Cancer Institute Grant 5U01-CA58271-02, and
Pathology Education and Research Foundation.
2 To whom requests for reprints should be addressed, at Division ofHematology/Bone Marrow Transplantation, University of Pittsburgh
Medical Center, 200 Lothrop Street, Pittsburgh, PA 15213-2582.
3 The abbreviations used are: HDC, high-dose chemotherapy; AlT,
adoptive immunotherapy; LAK. lymphokine-activated killer cells; IL-2,
Table 3 NK activity and percentage of MNC with NK cell phenotype in the blood of patients treated with HDC-PBSCT and AlT
UPN (diagnosis)”Before therapy”
LU’
Days +3 to �8d Day +90�
LUC
% NKcellse LU�
% NKcellse
380 (BRC)
385 (DSCCL)
386 (FML)
475 (DML)
319
33
325
415
772
2641673
287
32
2
41
n/a
833
440883
1290
25
3
48
32
a Only the patients cited exhibited increased NK activity in the blood on days +3 to +8, the other patients had values between 0 and 33 LU (see
Table 1 for diagnosis codes).6 This measurement (LU) was obtained before any protocol therapy had started and at least 4 weeks past any prior therapy.
C NK activity was measured in the PBMC of patients using 4-h 51Cr release assays with K562 as targets and is expressed as LU2(,/107 effector
cells (LU). The normal range in our laboratory is 50-300 LU, with a median of 132 LU.d The absolute leukocyte count was <200/jil at the time of this measurement. No lytic activity is detectable in the absence of A-NK cell infusion.
e % NK cells was determined by flow cytometry in two (CD356�) or three-color (CD356� 16k) analysis. The normal range for our laboratoryis 6-21%, with a median of 12%.
1The absolute leukocyte count was normal at the time of this measurement.
400
0
350 B�C’
0�
C’
0
zC.)
C’
.�
0-I
0�
00
0.0
-20 0 20 40 60 80 100
Day relative to transplant
Fig. 3 Changes in NK activity (LU7�1/107 cells) and in the absolute number of NK cells/pA in the peripheral blood of UPN 386 between days -20and + 100 of therapy relative to transplantation (day 0).
612 Stem Cell Transplantation and Immunotherapy
1800
. 1600
� 1400
U
� 1200
� 1000
I: 800
.� 600
� 400
200
0
CDrCD56(�mCD16_ or dim NK cells has potent antitumor
activity in vitro and in vivo (12, 19) and is particularly effective
in targeting metastases (14, 20). A-NK cells have been shown to
be able to produce a variety of cytokines (20, 21), localize to
metastases (20), and rapidly eliminate established metastases in
animal tumor models (13, 14, 19). Activities of A-NK cells are
strictly dependent on the presence of IL-2 (10, 22). At 2 X 106
IU/m2/day (HD-rhIL-2), the serum concentration of IL-2 was
estimated to be sufficient to bind high affinity IL-2 receptor
(22). Thus, we reasoned that the infused A-NK cells, a propor-
tion of which expresses the high affinity IL-2 receptor (12),
were likely to be stimulated in vivo, at least during the initial 4
days after adoptive transfer.
We hypothesized that the timing of A-NK cell transfusion
might be critical for cure. Animal models of combined chemo-
therapy and AlT have shown that the smaller the time interval
between the two therapies, the greater the cure rate (23, 24). On
300
250
200
150
100
50
0
the basis of earlier observations, we planned to give the A-NK
cells and IL-2 as close to the chemotherapy as possible.
Our main concerns were the possible adverse effect of AlT
on engraftment and the added toxicity of AlT in the pancyto-
penic phase after HDC-PBSCT. Our preclinical studies showed
no suppression of colony formation in vitro by human A-NK
cells and that at the higher A-NK to progenitor ratios, there may
actually be enhancement of growth (25). Murphy et a!. (26, 27)
and Siefer et a!. (28) have shown accelerated platelet production
after marrow transplantation, in a murine model, that is depen-
dent upon infusion of murine NK cells. We believe that we may
have observed an analogous phenomenon in humans.
The administration of IL-2 at doses sufficiently high to
support the antitumor activity of A-NK cells was an essential
component of the therapy. To reduce IL-2-related toxicity, two
design principles were used in this trial. First, the doses of
rhIL-2 used were one to two orders of magnitude less than those
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