Vol. 4, 2859-2868, November 1998 Clinical Cancer Research 2859 Antileukemia Activity of a Natural Killer Cell Line against Human Leukemias1 Ying Yan,2 Peter Steinherz, Hans-Georg Klingemann, Dieter Dennig, Barrett H. Childs, Joseph McGuirk, and Richard J. O’Reilly Bone Marrow Transplantation Service [Y. Y., B. C., J. M., R. J. 0.] and Department of Pediatrics [P. S., D. D., R. J. 0.], Memorial Sloan- Kettering Cancer Center, New York, New York 1002 1 , and The Terry Fox Laboratory and University of British Columbia, Vancouver, British Columbia V5Z 1L3. Canada IH-G. K.] ABSTRACT We describe here the in vitro and in vivo antileukemia activity of a recently described natural killer (NK) cell line (NK-92), which has features of human activated NK cells. The cytotoxic activity of rhIL2-dependent cultured NK-92 cells against primary patient-derived leukemic target cells [12 acute myelogenous leukemias (AMLs), 7 T acute lym- phoblastic leukemias (T-ALLs), 14 B-lineage-ALLs, and 13 chronic myelogenous leukemias (CMLs)], human leukemic cell lines (K562, KG!, HL6O, Raji, NALM6, TALL-!04, CEM-S, and CEM-T) and normal bone marrow cells was measured in 5tCr-release assay (CRA). The patient-derived leukemias could be subdivided into three groups based on their sensitivity to NK-92 cells: insensitive (!9% lysis), sensitive (20-49% lysis), and highly sensitive (50% lysis) at an E:T ratio of 9:!. Of 46 patient-derived samples, 24 (52.2%) were sensitive or highly sensitive to NK-92-medi- ated in vitro cytotoxicity (6 of 12 AMLs, 7 of 7 T-ALLs, 5 of 14 B-lineage-ALLs, and 6 of 13 CMLs). NK-92 cells were highly cytotoxic against all of the eight leukemic cell lines tested in a standard 4-h CRA. Normal human bone marrow hematopoietic cells derived from 18 normal donors were insensitive to NK-92-mediated cytolysis. In comparison with human lymphokine-activated killer cells, normal NK cells, and T cells, NK-92 cells displayed more powerful antileuke- mia activity against a patient-derived T-ALL as well as K562 and HL6O cells, both in in vitro CRA and in a xe- nografted human leukemia SCID mouse model. The NK-92 cells did not induce the development of leukemia in SCID Received 5/12/98; revised 8/17/98; accepted 8/26/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1 734 solely to indicate this fact. I Supported by NIH Grant CA23766 and grants from the Lisa Bilotti Foundation, the Andrew Gaffney Foundation, the Zelda Radow Wein- traub Cancer Foundation, and the Guy M. Stewart Foundation. 2 To whom requests for reprints should be addressed, at Cancer Institute! Hospital, Beijing Union Medical College, Chinese Academy of Medical Sciences, Beijing 100021, China. Phone: 86-10-67718927; Fax: 86-10- 67715058. mice after i.v., i.p., or s.c. inoculation. In adoptive transfer experiments, SCID mice receiving i.p. inoculations of human leukemias derived from a T-ALL (TA27) and an AML (MA26) that were highly sensitive to the cytolysis of NK-92 cells in vitro, as well as a pre-B-ALL (BA3I) that was insensitive to the in vitro cytohysis of NK-92 cells, were treated by administration of NK-92 cells with or without rhLL2 (2 x iO NK-92 cells i.p.; one dose or five doses). Survival times of SCID mice bearing the sensitive TA27 and MA26 leukemias were significantly prolonged by adoptive cell therapy with NK-92 cells. Some of the animals who received five doses of NK-92 cells with or without rhIL2 administration were still alive without any signs of leukemia development 6 months after leukemia inoculation. In con- trast, survival of mice bearing the insensitive BA31 leukemia were not affected by this treatment. This in vitro and in vivo antileukemia effect of NK-92 cells suggests that cytotoxic NK cells of this type may have potential as effectors of leukemia control. INTRODUCTION Cytotoxicity mediated by NK3 cells, has been hypothesized to play an important role in a host’s defenses against several forms of cancer (1, 2). Human NK cells are an immunopheno- typically distinguishable subset of lymphocytes that exhibit a striking and, as yet, poorly understood capacity to distinguish and destroy tumor cells or virally infected cells. This cytotoxic activity does not require prior sensitization and is not restricted by MHC antigens (1-4). Much research has been focused on ways to manipulate the functions of NK cells and other immune cells for therapeutic purposes (5-8). However, the inability to generate sufficient numbers of such cells in vitro, which main- tam their in vivo tumoricidal and tumor-targeting capabilities, remains a major obstacle to their use in clinical applications of adoptive cell immunotherapy (6, 9, 10). Studies of the mecha- nisms whereby NK cells exert their tumoricidal effects are also limited by both difficulties in enriching the NK cell fractions without compromising their biological functions and obtaining pure NK cells without contaminating T cells or other immune- response effector cells. To avoid these problems, many investi- gators have used established NK-like cell lines to explore the biological mechanisms of natural cytotoxicity to target cells (11-14). NK cells as well as CTL clones established from patients 3 The abbreviations used are: NK, nature killer; AML, acute myeloge- nous leukemia; ALL. acute lymphoblastic leukemia; CML. chronic myelogenous leukemia: CRA, 51Cr-release assay; MHC, major histo- compatibility; CTL. cytotoxic T cell; BM, bone marrow; FACS, flu- oroescence-activated cell sorting; LAK, hymphokine-activated killer; MST. median survival time; MTX, methotrexate. Association for Cancer Research. by guest on September 4, 2020. Copyright 1998 American https://bloodcancerdiscov.aacrjournals.org Downloaded from
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Vol. 4, 2859-2868, November 1998 Clinical Cancer Research 2859
Antileukemia Activity of a Natural Killer Cell Line against
Human Leukemias1
Ying Yan,2 Peter Steinherz,
Hans-Georg Klingemann, Dieter Dennig,
Barrett H. Childs, Joseph McGuirk, and
Richard J. O’Reilly
Bone Marrow Transplantation Service [Y. Y., B. C., J. M., R. J. 0.]and Department of Pediatrics [P. S., D. D., R. J. 0.], Memorial Sloan-
Kettering Cancer Center, New York, New York 1002 1 , and The
Terry Fox Laboratory and University of British Columbia,Vancouver, British Columbia V5Z 1L3. Canada IH-G. K.]
ABSTRACTWe describe here the in vitro and in vivo antileukemia
activity of a recently described natural killer (NK) cell line
(NK-92), which has features of human activated NK cells.
The cytotoxic activity of rhIL2-dependent cultured NK-92cells against primary patient-derived leukemic target cells
[12 acute myelogenous leukemias (AMLs), 7 T acute lym-
phoblastic leukemias (T-ALLs), 14 B-lineage-ALLs, and 13
chronic myelogenous leukemias (CMLs)], human leukemic
Fig. 1 In vitro and in vivo antileukemic efficacy of NK-92 cells against K562 and HL6O leukemias as compared with human LAK cells and other
effectors. 51CR-labeled K562 (A) and HL6O (B) cells were tested for susceptibility to cytolysis by NK-92 cells as compared with various other
effectors [including LAK, NK (CD56�CD3), and T cells (CD3�CD56)] at indicated E:T ratios in a 4-h CRA. Results are means ± SD of threeseparate tests for NK-92 cells. and two tests for each of the same two donor- derived effectors for LAK, CD56�, and CD3� cells. SCID mice receive
s.c. inoculations of K562 cells (C) or HL6O cells (D; 5 X 106 each mouse) alone or combined with NK-92, LAK, or NK cells at a 4: 1 E:T ratio. The
tumor sizes were measured once a week after inoculation (n = 5).
ison, with the exception of three samples (a CML in blast crisis,
a T-ALL, and a pre-B-ALL), the majority of leukemic samples
were resistant to the other NK-like cell line tested (YT; data not
shown).
Cytotoxicity of NK-92 toward Human Leukemic Cell
Lines. NK-92 cells were highly cytotoxic to all of the eight
leukemic cell lines tested in a 4-h standard CRA (Table 2). The
MTX-sensitive T-ALL cell line CEM-S, as well as its MTX-
transport resistant subline CEM-T, displayed a similar sensitiv-
ity to the NK-92 cells. TALL-l04 was cytotoxic to K562,
NALM6, and HL6O cells; however, Raji cells exhibited only
22.2% lysis at a 9: 1 E:T ratio, and KG1 cells (CEM-S as well as
CEM-T) were resistant. The YT cell line did not exhibit signif-
icant cytotoxic activity to most leukemia cell lines, with the
exception of K562 cells and Raji cells, which showed a 32% and
25% lysis at a 9: 1 E:T ratio, respectively.
Effect of NK-92 Cells on Normal Human BM Hemato-
poietic Cells. Hematopoietic cell-enriched fractions of normal
BMs from 18 normal donors were tested by standard CRA to
determine their susceptibility to lysis by NK-92 cells. All of the
normal BM samples were insensitive to the NK-92-mediated
cytolysis. The median values of the cytolysis mediated by
NK-92 at a 90:1 and 9:1 E:T ratio were 5.5% (range, 1.9-
13.5%) and 3.2% (range, 0.6-12.1%), respectively.
In Vivo Leukemogenesis of NK-92 cells in SCID Mice.
CB-l7 scid/scid mice as well as pfp-Rag-2 mice received inoc-
ulations of NK-92 cells by iv. (n = 3, each group), s.c. (n = 2,
each group), and i.p. (CB-l7, n = 8; Rag-2, n = 3) injection.
Survival of the animals was followed at least 6 months after
inoculation. All of the animals appeared healthy, and there was
no hepatosplenomegaly, lymphadenopathy, or leukemic nodular
growth, which would indicate leukemia development in these
animals during the period of follow up. Leukemic cellular
infiltration was not detected in any of the tissues of the sacri-
ficed animals by histopathology, and there were no cells of
human origin detectable in the tissues by FACS analysis.
Comparison of Antileukemic Effect of NK-92 Cells with
LAK, NK, and T Cells against Human Leukemic Cell Lines.The antileukemic effects of NK-92 cells, human LAK cells, NK
cells (CD56�CD3), and T cells (CD3�CDS6), were assessed
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by measuring in vitro cytolytic activity in standard CRA and by
measuring inhibition of heukemic cell xenograft growth in vivo
when the effector cells and targets were coinoculated s.c. into
SCID mice. NK-92 cells displayed high in vitro cytotoxicity
against K562 and HL6O with a mean specific lysis of 89% and
78% at a 9: 1 E:T ratio, respectively. This was superior to the
killing mediated by human LAK (52% and 1 1%), NK (72% and
28%), and T (12% and 1.2%) cells (Fig. 1, A and B). Correspond-
ingly, the NK-92 cells demonstrated a more intense in vivo inhibi-
rion of the growth of K562 and HL6O leukemic cells xenografts
than did the human LAK and NK cells (Fig. 1 , C and D).Comparison of Antileukemic Effect of NK-92 Cells with
Leukemia-reactive Allocytotoxic T Lymphocytes cells. To
compare the antileukemia effect of NK-92 cells with leukemia-
reactive allocytotoxic T lymphocytes, we generated allogeneic
leukemia-reactive T cells against leukemia derived from a pa-
tient with T-ALL (TA27). More than 98% of the leukemia-
reactive allocytotoxic T cells stained CD3�CD56 by FACS
analysis and cytolysis of the T cells against an NK-sensitive cell
line K562 at a 9: 1 E:T ratio was <3% in a CRA. Both NK-92
and the leukemia-reactive allocytotoxic T cells (TA27-T) dis-
played a significantly higher specific cytolysis (70% and 58% at
a 9: 1 E:T ratio, respectively) than the other effectors [LAK cells
(22%), NK cells (38%), and T cells (1.5% specific lysis),
respectively] against the TA27 leukemic cells (Fig. 14). Corre-
spondingly, the s.c. growth of TA27 leukemic cells was inhibited
by coinjection of either NK-92 cells or TA27-T cells (Fig. 2B).
Antileukemia Effect of NK-92 Cells in a Human Leu-
kemia Xenografted SCID Mouse Model. SCID mice who
received i.p. inoculations of human heukemic cells were treated
by i.p. injections of NK-92 cells with or without rhIL2. Leuke-
mic cells derived from a patient (TA27) with T-ALL and a
patient (MA26) with AML M4 leukemia were highly sensitive
to the NK-92 cells (73% and 66% specific killing at a 9:1 E:T
ratio in CRA, respectively), whereas cells from a patient with
pre-B-ALL (BA3 1 ) were insensitive to the NK-92 cells (4%
specific killing at a 9: 1 E:T ratio in CRA). All these human
leukemias grew aggressively in SCID mice. The survival of
mice bearing TA27 leukemia was significantly prolonged by the
NK-92 cell administration (Fig. 3). The MST of the animals
with no treatment or 1L2 alone was 72 days (n 5) and 63 days
(n 5; P >0.05), respectively. All of these animals died of
leukemia. In contract to the animals without treatment or 1L2
alone, treatment with NK-92 cells alone, or with addition of
rhIL2 significantly increased the MST to 102 days (n 6; P
<0.05) and 1 14 days (n = 6; P <0.05), respectively, for the
one-dose injection schedule (2 X l0� NK-92 cells, day 1). The
MST increased to 160 days (n = 6) and 129 days (n 6),
respectively, with five doses of NK-92 with or without rhIL2
injection (2 x l0� NK-92 cells, days 1, 3, 5, 7, and 9; Fig. 3).
Three animals who received five doses of NK-92 cell injections
with or without rhIL2 administration survived without any signs
of leukemia development 6 months after inoculation. Survival
was significantly prolonged for all of the groups that received
NK-92 cells (Fig. 3). There was no significant survival differ-
ence between the treatments with or without rhIL2 administra-
tion either in the one-dose group (P = 0.75), or the five-dose
group of NK-92 cell administration (P = 0.45) schedule. Com-
pared with the one dose of NK-92 cell injection with or without
Fig. 2 Antiheukemic effect of NK-92 cells, leukemia-reactive allocy-totoxic T cells, and other effectors against a patient-derived T-ALL(TA27). A, specific killing of TA27 target cells by NK-92, leukemia-
reactive allocytotoxic T cells (TA27-T), as well as other effectors, were
determined by a 4-h CRA using the indicated E:T ratios. Results aremeans ± SD of two or three separate tests. B, SCID mice received s.c.inoculations of TA27 cells (5 X 106 each mouse) alone or with NK-92,
TA27-T, or other effector cells at a 4: 1 E:T ratio. rhIL2 was adminis-tered to mice i.p. for 2 weeks at the dose of 5 X l0� IU every other day.
Leukemic tumor sizes were measured once a week after inoculation
(n 5).
rhIL2 treatment, survivals were significantly extended in ani-
mals that received five doses of NK-92 cells without rhIL2
treatment (P = 0.009 and P = 0.009, respectively).
In SCID mice who received inoculations of human pre-B-
ALL (BA3 1) leukemia with or without rhIL2 treatment, the
MST was 63 days (n 5) and 64 days (n 5), respectively. For
the animals who received 2 X l0� NK-92 cells for five doses
with or without rhIL2 administration, the MST was increased to
79 days (n = 5) and 76 days (n = 5), respectively. However,
these survival times were not significantly different from the
animals that were not treated by NK-92 cells (P >0.05; Fig. 4).
In animals bearing human AML (MA26) leukemia with or
without rhIL2 treatment, MST was 97 days (n 6) and 100
days (n = 6; Fig. 5). The MST was extended to 173 days in the
animals that received 2 X !0� NK-92 cells 5 times (P < 0.01;
n 6). Three of six animals who received NK-92 cells were
alive 6 months after leukemia inoculation. Two mice appeared
healthy without any signs of leukemia development. One mouse
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5 Y. Yan, B. H. Childs, and R. J. O’Reilly, unpublished observation.
human T-ALL as well as AMLs that were sensitive to in vitro
cytolysis by NK-92 cells, was not only significantly prolonged
by NK-92 cell treatment with or without rhIL2, but some of the
animals did not develop leukemia at all during a long-term
follow-up period (Figs. 3 and 5). During the same follow-up
interval, no animals were curable in this T-ALL SCID mouse
model when treated by maximally tolerated doses of either
MTX or idarubicin.5
Our results indicate that primary leukemias vary in their
susceptibility to NK-92 and other NK-like effector cells, but that
the in vitro susceptibility of a given primary leukemia to the
NK-92-cell-mediated cytolysis is predictive of its in vivo sus-
ceptibility. The sensitivity of such primary leukemias to the
cytolytic activity of NK-92 cells or other NK cells cannot be
predicted on the basis of the activity of these effectors against
established leukemia cell lines. Indeed, in our study, all of the
eight leukemic cell lines displayed marked susceptibility to
cytolysis by NK-92 cells at a 9: 1 E:T ratio (Table 2). Addition-
ally, in a previous report, NK-92 cells were found to be highly
cytotoxic against 14 established leukemia, lymphoma, and my-
eloma cell lines at a 10: 1 E:T ratio (20). Our studies, therefore,
suggest that the determination of the in vitro susceptibility of
patient-derived leukemia samples to the NK-92 cells might be
an informative predictor of activity if NK-92 cells were to be
used for in vitro marrow graft purging or for in vivo adoptive
immunotherapy.
In conclusion, this study represents the first data available
on the effect of NK-92 cells against primary patient-derived
leukemias. The in vitro and in vivo antileukemic activity of
NK-92 cells suggests that this type of cell and other well
characterized NK cells, may have potential as antileukemic
effectors in adoptive immunotherapy.
ACKNOWLEDGMENTS
We are grateful to Sharon Bleau for BM processing assistance, toTeresa Diaz-Barrientos for CU cells culture, to Thoma Delohery andMacthu Menon for flow cytometry technical assistance, and to ThomasWilliams for statistics consultation.
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