Download Antitumor activity of cytokine-induced killer cells against human lung

International Immunopharmacology 7 (2007) 1802 – 1807
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Preliminary report
Antitumor activity of cytokine-induced killer cells against
human lung cancer
Hwan Mook Kim a,1 , Jaeseung Lim b,1 , Song-Kyu Park a , Jong Soon Kang a , Kiho Lee a ,
Chang Woo Lee a , Ki Hoon Lee a , Mi-Jung Yun b , Kyu-Hwan Yang c , Gyoonhee Han d ,
Soon Woo Kwon e , Youngsoo Kim e , Sang-Bae Han e,⁎
a
Bioevaluation center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Chungbuk 363-883, South Korea
b
Innocell Ltd., SJ technoville, 60-19 Gasandong, Geumcheon, Seoul 153-769, South Korea
c
Kyungwon University, Seongnam, Gyeonggi 461-701, South Korea
d
Department of Biotechnology, Yonsei University, Seodaemun, Seoul 120-740, South Korea
e
College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea
Received 23 June 2007; received in revised form 16 August 2007; accepted 16 August 2007
Abstract
Lung cancer is the leading cause of cancer-related death among men and women in the world. Despite the aggressive treatment
with surgery, radiation and chemotherapy, the long term survival for lung cancer patients remains low. In this study, the anti-tumor
activity of cytokine-induced killer (CIK) cells against human lung cancer was evaluated in vitro and in vivo. Although CD3+CD56+ CIK cells were rare in fresh human peripheral blood mononuclear cells, they could expand more than 1000-fold on day 14
in the presence of anti-CD3 antibody plus IL-2. At an effector-target cell ratio of 30:1, CIK cells destroyed 98% of NCI-H460
human lung cancer cells, which was determined by the 51Cr-release assay. In addition, CIK cells at doses of 3 and 30 million cells
per mouse inhibited 57% and 77% of NCI-H460 tumor growth in nude mouse xenograft assay, respectively. This study suggests
that CD3+CD56+ CIK cells may be used as an adoptive immunotherapy for patients with lung cancer.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Cytokine-induced killer cells; Lung cancer; Adoptive immunotherapy
1. Introduction
The development of adoptive cell immunotherapy for
the treatment of cancer has received considerable
attention. The goal of immune cell-based cancer therapy
is to eliminate cancer cells through the transfer of ex vivo
⁎ Corresponding author. Tel.: +82 43 261 2815; fax: +82 43 268
2732.
E-mail address: [email protected] (S.-B. Han).
1
These two authors equally contributed to this work.
1567-5769/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2007.08.016
expanded and activated immune cells. Immune cells such
as dendritic cells [1], lymphokine-activated killer cells [2],
natural killer cells [3], cytotoxic T cells [4], and cytokineinduced killer (CIK) cells [5] have been explored for the
active immunotherapy of cancer. Among them, CIK cells
were known to have several attractive advantages; 1) It is
very easy to generate large number of CIK cells ex vivo
and they are readily expandable from peripheral blood
mononuclear cells of cancer patients [6]; 2) CIK cells
have strong anti-tumor effects [7]; 3) Cytotoxicity is
MHC-unrestricted; 4) CIK cells are the final effector cells,
H.M. Kim et al. / International Immunopharmacology 7 (2007) 1802–1807
which are able to directly kill cancer cells; 5) More
importantly, the toxicity is minimal and no graft-versus
host reaction is observed [8]. Allogenic spleen cells with
bone marrow cells could induce graft-versus-host disease
(GVHD) in recipient mice by showing body weight loss
and death within 9 days after transplantation [9].
However, CIK cells did not induce GVHD, even when
injected with 10 times the amount of CIK cells compared
to spleen cells [8]. This reduction in GVHD was known to
be related with IFN-γ production of CIK cells. IFN-γ+/+
CIK cells produced no GVHD, but IFN-γ−/− CIK cells
caused acute lethal GVHD [8,10].
CIK cells showed promising antitumor effects on
various cancers, such as hepatoma [11], leukemia [12], and
liver [11], renal [4], and gastric cancer [13], in preclinical
and clinical studies. In this study, we examined the
antitumor activity of CIK cells against human non-small
cell lung cancer. We generated CIK cells from human
peripheral blood mononuclear cells, characterized their
phenotypes, and evaluated the anti-tumor activity of CIK
cells in vitro and in vivo nude mouse xenograft model.
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2. Materials and methods
2.1. Cells
Human lung cancer cells, such as NCI-H460, A549, NCIH556, NCI-H441, and NCI-H23, were obtained from the
American Type Culture Collection (Manassas, VA, USA). Cell
lines were grown in the RPMI-1640 medium supplemented
with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/
ml streptomycin.
Cytokine-induced killer (CIK) cells were generated from
peripheral mononuclear cells (PBMC) of healthy volunteers.
After informed consent, 10–20 ml of blood was drawn from
volunteers in evacuated tubes with heparin (Vacutainer, Becton
Dickinson). The PBMC were obtained from buffy coats of
PBMC by Ficoll-Hypaque density centrifugation and washed
three times with PBS [11]. All blood draws and PBMC
isolation experiments were finished within 1.5 h. Next, the
PBMC were re-suspended at 1 × 106 cells/ml in Lymphomedia
(Lymphotech, Japan) containing 10% fetal bovine serum
(Invitrogen, CA, USA) and cultured with immobilized antiCD3 antibody (5 μg/ml, BD Pharmingen, NJ, USA) as well as
recombinant human IL-2 (700 U/ml, R&D systems, MN, USA)
Fig. 1. The phenotypic characterization of CIK cells. CIK cells cultured for 14 days were stained with human monoclonal antibodies, such as antiCD3-FITC plus anti-CD56-PE (A), anti-CD4-FITC plus anti-CD8-PE (B), anti-CD4-PE plus anti-CD56-APC (C), and anti-CD8-PE plus anti-CD56APC (D).
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measured in a gamma counter. The percentage of specific lysis
was calculated according to the following equation:
%cytotoxicity ¼ ½ðsample spontaneousÞ=ðmaximum
spontaneousÞ 100
Spontaneous release was obtained by incubating target cells in
medium alone, whereas maximal release was obtained after
treatment with 2% Nonidet P-40 (Sigma, St Louis, MO, USA).
2.4. Nude mouse xenograft assay
Fig. 2. The cytotoxicity of CIK cells. Cytotoxicity of CIK cells
cultured for 14 days were assessed against NCI-H460, A549, NCIH596, NCI-H441, and NCI-H23 target cells. The target cells were
labeled with 51Cr and incubated for 4 h with CIK cells at effector-totarget ratios of 1–30:1.
for 5 days. Following this, the cell suspension was further
incubated in a new Lymphomedia containing recombinant
human IL-2 only (170 U/ml) for 9 days. Furthermore, fresh IL2 and medium were replenished every third day. The viability
of CIK cells was usually 85–95%.
2.2. Phenotype analysis
Cells were obtained from CIK cultures for phenotype
analysis with the appropriated monoclonal antibodies, including CD3-FITC, CD4-FITC, CD8-PE, CD56-APC, and CD56PE. One million CIK cells were washed once in PBS containing
1% bovine serum albumin (BSA) and re-suspended in 100 μl of
PBS/BSA buffer. The cells were incubated with various
conjugated monoclonal antibodies for 20 min at 4 °C, washed
twice in PBS, and re-suspended in 400 μl of PBS. A flow
cytometric analysis was performed on a FACSCalibur flow
cytometry (BD Biosciences), and the data were analyzed using
the WinMDI statistical software (Scripps, La Jolla, CA, USA).
Forward and side scatter parameters were used to gate on live
cells [14].
2.3.
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Nude mice (6–8 weeks old) were obtained from the Charles
River (Japan) and were housed under specific pathogen-free
conditions according to the guidelines of the Animal Care
Committee at the KRIBB. To eradicate NK cells, the study mice
received 200 cGy of whole body irradiation on day-1. On day 0,
six million NCI-H460 cells were injected subcutaneously into
irradiated nude mice. Following this, CIK cells were injected
intravenously twice in a week at dose ranges from 0.3 to 30
million cells per mouse. The tumor volumes were estimated by
the formula length (mm) × width (mm) × height (mm) / 2. On day
18, the mice were sacrificed and the tumor weights were
measured. To determine the animal toxicity, the body weights of
the animals were measured [16].
2.5. Statistics
The in vivo results represent samples from six mice per
experiment, and the in vitro results represent the mean values
of four samples. Standard deviations (SD's) and p-values were
calculated using the Student's t-test and ANOVA (Prisim,
GraphPad Software, USA) [17].
Cr release assay
The human cancer cell lysis by CIK cells was measured in a
4 h 51Cr release assay [15]. Briefly, two million target cells
were labeled with 100 μCi of sodium chromate (Dupont-NEN,
Boston, MA, USA) for 4 h at 37 °C. The labeled cells were
washed three times in PBS and re-suspended in 10 ml of RPMI
1640 medium supplemented with 10% fetal bovine serum. The
labeled cells were plated in round bottom 96-well plates at
1 × 104 cells/100 μl per well in triplicate. CIK cells were added
at specified E:T ratios (1:1, 3:1, 10:1, and 30:1) and incubated
for 4 h. The supernatant was removed and the radioactivity
Fig. 3. Inhibition of the NCI-H460 tumor growth by CIK cells in nude
mouse xenograft models. Irradiated nude mice (n = 6) were implanted
subcutaneously with six million NCI-H460 lung cancer cells. CIK cells at
doses from 0.3 to 30 × 106 cells/mouse were injected intravenously twice
in a week. Adriamycin (ADR) was injected intravenously at 2 mg/kg.
Tumor volumes were estimated by the formula length (mm)× width
(mm) × height (mm)/ 2. Statistical significance was determined using the
Student's t-test versus PBS-treated control group (⁎p b 0.01).
H.M. Kim et al. / International Immunopharmacology 7 (2007) 1802–1807
3. Results
CD3+CD56+ CIK cells are rare in fresh human PBMC, but
they will markedly expand and be generated from T cell
precursors. After two weeks of culturing ten million PBMCs,
the absolute number of total cells increased up to 2500 +/− 150
million CIK cells. During the generation period, cell number
was maintained at approximately one million cells per
milliliter. The viability of expanded cell populations on day
14 was 85–95 %. First, we examined the phenotypes of
cultured cell population with fluorescence-activated cell
sorting analyses. Cell population showed 92% CD3+CD56−,
7% CD3−CD56+, and 38% CD3+CD56+ (Fig. 1A). Fresh
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PBMC is known to include less than 5% of CD3+CD56+ cells.
In addition, most cells were CD8+, but not CD4+ (Fig. 1B–D).
Next, we examined the cytotoxicity of CIK cells in an in vitro
condition by 4 h-Cr51 release assay. CD3+CD56+ cells have been
demonstrated to have strong cytotoxicity in a non-MHC-restricted
manner. At an effector-target cell ratio of 30:1, CIK cells
destroyed 98%, 61%, 36%, 22%, and 61% of NCI-H460, NCIH441, A549, NCI-H596, and NCI-H23 human lung cancer cell
lines, respectively (Fig. 2). We also examined the cytotoxicity of
fresh PBMC to these target cells, and observed that fresh PBMC
destroyed less than 4 % of all human lung cancer cell lines.
Finally, we evaluate the antitumor activity of CIK cells in
nude mouse xenograft assay. Nude mice were irradiated to
Fig. 4. Inhibition of the NCI-H460 tumor growth by CIK cells in nude mouse xenograft models. Irradiated nude mice (n = 6) were implanted
subcutaneously with six million NCI-H460 lung cancer cells. CIK cells at doses from 0.3 to 30 × 106 cells/mouse were injected intravenously twice in
a week. Adriamycin (ADR) was injected intravenously at 2 mg/kg. On day 18, the mice were sacrificed and the tumor weights were measured (A).
Representative photographs are shown (B). Statistical significance was determined using the Student's t-test versus PBS-treated control group
(⁎p b 0.01).
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Fig. 5. Body weight changes of tumor-bearing nude mice. Irradiated
nude mice (n = 6) were implanted subcutaneously with six million
NCI-H460 lung cancer cells. CIK cells at doses from 0.3 to
30 × 106 cells/mouse were injected intravenously twice in a week.
Adriamycin (ADR) was injected intravenously at 2 mg/kg. The body
weights of the tumor bearing nude mice were measured to estimate
toxicity.
eradicate intrinsic NK cells. Preliminary experiments revealed
that one hundred million CIK cells did not show any
observable toxicity in irradiated-nude mice. Mice did not
show hair ruffling, lowered morbidity, and weight loss. Thus,
we injected CIK cells intravenously at dose levels under one
hundred million cells. Six million NCI-H460 cells were
injected subcutaneously and they grew to a tumor volume of
316 mm3 18 days after implantation (Fig. 3). CIK cells were
injected intravenously at doses of 0.3, 3 and 30 million cells
per mouse, and they inhibited in vivo tumor growth by 32%,
67%, and 77%, respectively. Adriamycin (ADR), used as a
reference drug, strongly inhibited the growth of NCI-H460
tumors. On day 18, all tumors were isolated from nude mice
and weighed, which demonstrated the strong anti-tumor effect
of CIK cells against NCI-H460 (Fig. 4A and B). The body
weights of tumor-bearing nude mice were examined to assess
the toxicity. Control and CIK cell-injected nude mice showed a
body weight gain of 121–125%; whereas, adriamycin-treated
mice showed a body weight gain of 110%, which shows the
typical characteristics of a cytotoxic anti-cancer drug (Fig. 5).
4. Discussion
Lung cancer is the leading cause of cancer-related
death among men and women in the world and in Korea
[18]. Despite the aggressive treatment with surgery,
radiation and chemotherapy, the long term survival for
lung cancer patients remains low [19]. The mean survival
for patients with advanced stage treated with platinumbased chemotherapy is a disappointing 8–10 months
[20,21]. As a result, new therapeutic approaches to
improve patient mortality and morbidity are urgently
needed.
Lung tumors appear to have fewer tumor-infiltrating
lymphocytes, suggesting that immune response of
cancer patients does not properly work to eradicate
cancer cells [22]. The development of immunotherapy
for the treatment of cancer has received considerable
attention. The goal of immunotherapy is to eliminate
cancer cells through the activation of host immunity by
using biological response modifying polysaccharides,
cytokines, monoclonal antibody, cytokines, and ex vivo
activated immune cells. Adjuvant immunotherapy with
IFN-α, IL-2, thymosin, and Bacille Calmette–Guerin
(BCG) showed promising effect on lung cancer
[23,24]. Anti-HER2 antibody therapy had been recently applied to human antitumor therapy of lung cancer,
although in vivo activity was disappointing [25]. Antiganglioside fucosyl GM-1 antibody appeared to
decrease formation of metastatic lung cancer via
antibody-dependent cell-mediated cytotoxicity [26].
Cell immunotherapy had been also attempted for the
treatment of lung cancer. Dendritic cells pulsed with
lung cancer cell lysates could induce tumor cellspecific immune response and showed promising
clinical outcomes in patients with lung cancer [27].
Dendritic cells pulsed with alpha-galactosylceramide
induced NKT cell expansion and exerted a strong
antitumor activity in patient with lung cancer [28].
Except from dendritic cells, there were a few, or no,
reports on cell immunotherapy with lymphokineactivated killer cells, natural killer cells, cytotoxic T cells,
and CIK cells against lung cancer.
Here, we provide good preclinical evidence that CIK
cell immunotherapy can be used in patient with lung
cancer. CIK cells from human PBMC could expand
more than 1000 times in the presence of anti-CD3
antibodies and IL-2 for 14 days. CIK cells could break
various lung cancer cells down in a non-MHC-restricted
manner, and the adoptive transfer of CIK cells was
highly effective in preventing NCI-H460 lung tumor
growth in the nude mouse xenograft model. This study
suggested that CIK cells could be good candidates for
cell immunotherapy in patients with lung cancer.
Acknowledgements
This research was partially supported by a grant from
the KRIBB Research Initiative Program and by the
Korea Research Foundation Grant funded by the Korean
Government (MOEHRD) (The Regional Research
Universities Program/Chungbuk BIT Research-Oriented University Consortium).
H.M. Kim et al. / International Immunopharmacology 7 (2007) 1802–1807
References
[1] Kalinski P, Nakamura Y, Watchmaker P, Giermasz A, Muthuswamy R, Mailliard RB. Helper roles of NK and CD8+ T cells in
the induction of tumor immunity. Polarized dendritic cells as
cancer vaccines. Immunol Res 2006;36:137–46.
[2] Takashima K, Fujiwara H, Inada S, Atsuji K, Araki Y, Kubota T,
et al. Tracking of green fluorescent protein (GFP)-labeled LAK
cells in mice carrying B16 melanoma metastases. Anticancer Res
2006;26:3327–32.
[3] Raja Gabaglia C, Diaz de Durana Y, Graham FL, Gauldie J,
Sercarz EE, Braciak TA. Attenuation of the glucocorticoid
response during Ad5IL-12 adenovirus vector treatment enhances
natural killer cell-mediated killing of MHC class I-negative LNCaP
prostate tumors. Cancer Res 2007;67:2290–7.
[4] Wang W, Epler J, Salazar LG, Riddell SR. Recognition of breast
cancer cells by CD8+ cytotoxic T-cell clones specific for NY-BR-1.
Cancer Res 2006;66:6826–33.
[5] Thorne SH, Negrin RS, Contag CH. Synergistic antitumor effects
of immune cell-viral biotherapy. Science 2006;311:1780–4.
[6] Alvarnas JC, Linn YC, Hope EG, Negrin RS. Expansion of
cytotoxic CD3+CD56+ cells from peripheral blood progenitor cells
of patients undergoing autologous hematopoietic cell transplantation. Biol Blood Marrow Transplant 2001;7:216–22.
[7] Lu PH, Negrin RS. A novel population of expanded human
CD3+CD56+ cells derived from T cells with potent in vivo
antitumor activity in mice with severe combined immunodeficiency.
J Immunol 1994;153:1687–96.
[8] Baker J, Verneris MR, Ito M, Shizuru JA, Negrin RS. Expansion
of cytolytic CD8(+) natural killer T cells with limited capacity for
graft-versus-host disease induction due to interferon gamma
production. Blood 2001;97:2923–31.
[9] Han SB, Lee CW, Yoon YD, Kang JS, Lee KH, Yoon WK, et al.
Effective prevention of lethal acute graft-versus-host disease by
combined immunosuppressive therapy with prodigiosin and
cyclosporine A. Biochem Pharmacol 2005;70:1518–26.
[10] Leemhuis T, Wells S, Scheffold C, Edinger M, Negrin RS. A
phase I trial of autologous cytokine-induced killer cells for the
treatment of relapsed Hodgkin disease and non-Hodgkin
lymphoma. Biol Blood Marrow Transplant 2005;11:181–7.
[11] Takayama T, Sekine T, Makuuchi M, Yamasaki S, Kosuge T,
Yamamoto J, et al. Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised
trial. Lancet 2000;356:802–7.
[12] Kornacker M, Moldenhauer G, Herbst M, Weilguni E, Tita-Nwa F,
Harter C, et al. Cytokine-induced killer cells against autologous
CLL: direct cytotoxic effects and induction of immune accessory
molecules by interferon-gamma. Int J Cancer 2006;119:1377–82.
[13] Sun S, Li XM, Li XD, Yang WS. Studies on inducing apoptosis
effects and mechanism of CIK cells for MGC-803 gastric cancer
cell lines. Cancer Biother Radiopharm 2005;20:173–80.
[14] Han SB, Moratz C, Huang NN, Kelsall B, Cho H, Shi CS, et al.
Rgs1 and Gnai2 regulate the entrance of B lymphocytes into
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
1807
lymph nodes and B cell motility within lymph node follicles.
Immunity 2005;22:343–54.
Verneris MR, Arshi A, Edinger M, Kornacker M, Natkunam Y,
Karami M, et al. Low levels of Her2/neu expressed by Ewing's
family tumor cell lines can redirect cytokine-induced killer cells.
Clin Cancer Res 2005;11:4561–70.
Han SB, Lee CW, Jeon YJ, Hong ND, Yoo ID, Yang KH, et al.
The inhibitory effect of polysaccharides isolated from Phellinus
linteus on tumor growth and metastasis. Immunopharmacology
1999;41:157–64.
Kim JY, Yoon YD, Ahn JM, Kang JS, Park SK, Lee K, et al.
Angelan isolated from Angelica gigas Nakai induces dendritic
cell maturation through toll-like receptor 4. Int Immunopharmacol 2007;7:78–87.
Park MS, Chang YS, Shin JH, Kim DJ, Chung KY, Shin DH, et al.
The prevalence of human papillomavirus infection in Korean nonsmall cell lung cancer patients. Yonsei Med J 2007;48:69–77.
Tsai JR, Chong IW, Chen YH, Yang MJ, Sheu CC, Chang HC, et al.
Differential expression profile of MAGE family in non-small-cell
lung cancer. Lung Cancer 2007;56:185–92.
Ruttinger D, Winter H, van den Engel NK, Hatz RA, Schlemmer
M, Pohla H, et al. Immunotherapy of lung cancer: an update.
Onkologie 2006;29:33–8.
Stinchcombe TE, Lee CB, Socinski MA. Current approaches to
advanced-stage non-small-cell lung cancer: first-line therapy
in patients with a good functional status. Clin Lung Cancer
2006;7(Suppl 4):S111–7.
Wakabayashi O, Yamazaki K, Oizumi S, Hommura F, Kinoshita I,
Ogura S, et al. CD4+ T cells in cancer stroma, not CD8+ T cells in
cancer cell nests, are associated with favorable prognosis in human
non-small cell lung cancers. Cancer Sci 2003;94:1003–9.
Lipson SD, Chretien PB, Makuch R, Kenady DE, Cohen MH.
Thymosin immunotherapy in patients with small cell carcinoma
of the lung: correlation of in vitro studies with clinical course.
Cancer 1979;43:863–70.
Raez LE, Fein S, Podack ER. Lung cancer immunotherapy. Clin
Med Res 2005;3:221–8.
Clamon G, Herndon J, Kern J, Govindan R, Garst J, Watson D, et al.
Lack of trastuzumab activity in nonsmall cell lung carcinoma with
overexpression of erb-B2: 39810: a phase II trial of Cancer and
Leukemia Group B. Cancer 2005;103:1670–5.
Hanibuchi M, Yano S, Nishioka Y, Yanagawa H, Kawano T,
Sone S. Therapeutic efficacy of mouse-human chimeric antiganglioside GM2 monoclonal antibody against multiple organ
micrometastases of human lung cancer in NK cell-depleted SCID
mice. Int J Cancer 1998;78:480–5.
Hirschowitz EA, Foody T, Kryscio R, Dickson L, Sturgill J,
Yannelli J. Autologous dendritic cell vaccines for non-small-cell
lung cancer. J Clin Oncol 2004;22:2808–15.
Motohashi S. Translational research in patients with lung cancerclinical application of NKT cell immunotherapy. Gan To Kagaku
Ryoho 2007;34:550–3.