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Clinical Cancer Research Vol. 12, 6079-6086, October 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Clinical

A Phase I Study of In vitro Expanded Natural Killer T Cells in Patients with Advanced and Recurrent Non–Small Cell Lung Cancer

Shinichiro Motohashi1,2, Aki Ishikawa1,2, Eiichi Ishikawa1, Mizuto Otsuji2, Toshihiko Iizasa2, Hideki Hanaoka4, Naomi Shimizu5, Shigetoshi Horiguchi3, Yoshitaka Okamoto3, Shin-ichiro Fujii6, Masaru Taniguchi6, Takehiko Fujisawa2 and Toshinori Nakayama1

Authors' Affiliations: Departments of 1 Immunology, 2 Thoracic Surgery, and 3 Otorhinolaryngology, Graduate School of Medicine, Chiba University; 4 Clinical Research Center; and 5 Division of Blood Transfusion, Chiba University Hospital, Chiba, Japan; and 6 Laboratory of Immune Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan

Requests for reprints: Toshinori Nakayama, Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 Japan. Phone: 81-43-226-2186; Fax: 81-43-227-1498; E-mail: tnakayama{at}faculty.chiba-u.jp.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: Human V{alpha}24 natural killer T (V{alpha}24 NKT) cells bearing an invariant V{alpha}24J{alpha}Q antigen receptor are activated by a glicolipid ligand {alpha}-galactosylceramide ({alpha}GalCer; KRN7000) in a CD1d-dependent manner. The human V{alpha}24 NKT cells activated with {alpha}GalCer and interleukin-2 have been shown to produce large amounts of cytokines, such as IFN-{gamma}, and also exerting a potent killing activity against various tumor cell lines. We did a phase I study with autologous activated V{alpha}24 NKT cell therapy.

Experimental Design: Patients with advanced or recurrent non–small cell lung cancer received i.v. injections of activated V{alpha}24 NKT cells (level 1: 1 x 107/m2 and level 2: 5 x 107/m2) to test the safety, feasibility, and clinical response of this therapeutic strategy. Immunomonitoring was also done in all cases.

Results: Six patients were enrolled in this study. No severe adverse events were observed during this study in any patients. After the first and second injection of activated V{alpha}24 NKT cells, an increased number of peripheral blood V{alpha}24 NKT cells was observed in two of three cases receiving a level 2 dose of activated V{alpha}24 NKT cells. The number of IFN-{gamma}-producing cells in peripheral blood mononuclear cells increased after the administration of activated V{alpha}24 NKT cells in all three cases receiving the level 2 dose. No patient was found to meet the criteria for either a partial or a complete response.

Conclusions: The clinical trial with activated V{alpha}24 NKT cell administration was well tolerated and carried out safely with minor adverse events even in patients with advanced diseases.

A unique lymphocyte subpopulation, consisting of invariant natural killer T (iNKT) cells, is characterized by the coexpression of an invariant antigen receptor and natural killer (NK) receptors (1–4). Human iNKT cells express the invariant V{alpha}24J{alpha}Q paired with the Vß11 antigen receptor and are activated by a specific glycolipid antigen {alpha}-galactosylceramide ({alpha}GalCer) in a CD1d-dependent manner. CD1d is an HLA class Ib antigen-presenting molecule, which is well conserved through mammalian evolution with a lack of allelic polymorphism (5, 6). After activation, human V{alpha}24 NKT cells show a strong antitumor activity against various malignant tumors both in vitro and in vivo (2, 7, 8) and produce high levels of cytokines, such as IFN-{gamma} and interleukin-4 (IL-4), thereby activating other antitumor effector cells (9–12). Decreased numbers of V{alpha}24 NKT cells in human peripheral blood mononuclear cells (PBMC) have been shown in patients with malignant diseases (13–15). At the same time, functional alterations of V{alpha}24 NKT cells have also been reported after in vitro stimulation with {alpha}GalCer in patients with some malignant diseases (13, 16–18). For the patients possessing severely decreased or functionally deficient V{alpha}24 NKT cells, the expansion and activation of these cells in vitro and the subsequent adoptive transfer may be therapeutically beneficial. The in vitro expansion of V{alpha}24 NKT cells has been reported to be successful in the presence of {alpha}GalCer and IL-2 in both healthy volunteers and cancer-bearing patients (19–21).

Based on theses findings, we carried out a phase I study using in vitro expanded V{alpha}24 NKT cells in patients with recurrent or advanced non–small cell lung cancer. The goal of this study was to confirm the safety profile of activated V{alpha}24 NKT cell immunotherapy, and no severe adverse events were observed. We detected an increased number of V{alpha}24 NKT cells and also an increased number of IFN-{gamma}-producing cells in the PBMCs of the patients receiving 5 x 107/m2 in vitro expanded V{alpha}24 NKT cells.


   Patients and Methods
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 Abstract
 Patients and Methods
Results
 Discussion
 References
 
Patient eligibility criteria. Patients between 20 and 80 years of age, with a histologic or cytologic diagnosis of non–small cell lung cancer for which no standard treatment was available, were eligible for the study. Further inclusion criteria were a performance status of 0, 1, or 2; an expected survival of ≥6 months; normal or near normal renal, hepatic, and hematopoietic function; and no chemotherapy or radiotherapy received for at least 4 weeks before enrollment. In enrolled patients, V{alpha}24+Vß11+ NKT cells were detected at a level of >100 cells in 1 mL peripheral blood by flow cytometry. The exclusion criteria were a positive response to HIV, hepatitis C virus, or human T-cell lymphotrophic virus antibodies; positive for hepatitis B antigen; the presence of active inflammatory disease or active autoimmune disease; a history of hepatitis; pregnancy or lactation; concurrent corticosteroid therapy; and evidence for another active malignant neoplasm. The histologic type, tumor-node-metastasis classification, and the antitumor effect of treatment were classified according to the general rules for clinical and pathologic recording of lung cancer as described by the Japan Lung Cancer Society.

Clinical protocol and study design. The study was carried out in the Department of Thoracic Surgery, Chiba University Hospital, Japan, according to the standards of Good Clinical Practice for Trials on Medicinal Products in Japan. The protocol was approved by the Institutional Ethics Committee (no. 1972). In addition, this trial underwent ad hoc reviews by the Chiba University Quality Assurance Committee on Cell Therapy.

The study design is illustrated in Fig. 1 . Written informed consent was obtained from all of the patients before undergoing a screening evaluation to determine eligibility. Clinical and laboratory assessments were conducted once a week, consisting of a complete physical examination and standard laboratory values. Any adverse events and changes in laboratory values were graded according to the National Cancer Institute Common Toxicity Criteria version 2.0. All patients underwent an assessment of the tumor status at baseline and 4 weeks after the second NKT cell administration (7 weeks after study entry). Disease progression was defined as >25% increase in target lesions and/or the appearance of new lesions.


Figure 1
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  		Fig. 1. Experimental design of activated V{alpha}24 NKT cell administration. The patients received in vitro expanded activated V{alpha}24 NKT cells (NKT#1 and NKT#2). Timing for both apheresis and activated V{alpha}24 NKT cell administration.

 
Preparation of activated V{alpha}24NKT cells from peripheral blood. All procedures were done according to the Good Manufacturing Practice standards. Eligible patients underwent peripheral blood leukapheresis (Spectra, COBE). PBMCs were collected and then further separated by density gradient centrifugation (OptiPrep, Axis-Shield, Oslo, Norway). After washing three times, the cells were resuspended in AIM-V (Invitrogen Corp., Carlsbad, CA) with 100 JRU/mL of recombinant human IL-2 (Imunace, Shionogi, Japan) and 100 ng/mL of {alpha}GalCer (KRN7000; Kirin Brewery Co., Gunma, Japan). Restimulation with {alpha}GalCer-pulsed autologous PBMCs was done on days 7 and 14. After 14 or 21 days of cultivation, the cells were harvested, washed thrice, and then resuspended in 100 mL of 2.5% albumin in saline. The patients received the cultured cells i.v. The criteria for activated V{alpha}24 NKT cell administration included a negative bacterial culture 48 hours before V{alpha}24NKT cell injection, cell viability >70%, and an endotoxin test 48 hours before cell injection with a result <0.7 EU/mL. The activated V{alpha}24 NKT cells were given in a dose escalation design at a dose level per cohort of 1 x 107 and 5 x 107 cells/m2 per injection. The cell dose represents the V{alpha}24+Vß11+ NKT cell number but not the bulk population of the cells.

Activated V{alpha}24NKT cell phenotype evaluation. The phenotypes of peripheral blood lymphocytes and activated V{alpha}24NKT cells were determined by a flow cytometry analysis. The monoclonal antibodies (mAb) used were FITC-labeled anti-TCR V{alpha}24 mAb (C15; Immunotech, Marseilles, France), anti-CD8 mAb (HIT8a; BD Biosciences PharMingen, San Diego, CA), phycoerythrin-labeled anti-TCR Vß11 mAb (C21; Immunotech), anti-CD56 mAb (B159; BD Biosciences PharMingen), and cychrome-labeled anti-CD3{varepsilon} mAb (UCTH1; BD Biosciences PharMingen), anti-CD4mAb (RPA-T4; BD Biosciences PharMingen). Phycoerythrin-labeled {alpha}GalCer-loaded CD1d tetramer was prepared in our laboratory using a baculovirus expression system kindly provided by Dr. M. Kronenberg (La Jolla Institute, La Jolla, CA; ref. 22). Isotype-matched control mAbs were used as negative controls. To determine the percentages of V{alpha}24NKT cells in the in vitro {alpha}GalCer/IL-2–cultured PBMCs, we presented the values of V{alpha}24+Vß11+CD3+ cells because the percentages of {alpha}GalCer/CD1d tetramer-positive cells and V{alpha}24+Vß11+CD3+ cells are basically identical (Supplementary Fig. S1).

51Cr release cytotoxic assay. Target Daudi cells (B lymphoma) and PC-13 cells (lung large cell carcinoma; 5 x 106) were labeled with 100 µCi sodium chromate (Amersham LIFE SCIENCE, Little Chalfont, Buckinghamshire, England) for 1 hour. Activated NKT cells were seeded into 96-well round-bottomed plates at the indicated effector/target ratios on 51Cr-labeled Daudi cells or PC-13 cells (1 x 104). Radioactivity released from lysed target cells was counted on a {gamma}-counter after 4 hours of incubation at 37°C in 5% CO2 incubator. The percentage of 51Cr release was calculated by the following formula: % specific lysis = (sample cpm – spontaneous cpm) x 100 / (maximum cpm – spontaneous cpm) as described (19). Spontaneous cpm was calculated from the supernatant of target cells alone, and the maximum release was obtained by adding 1 N HCl to the target cells. The data are expressed as the mean value of triplicate cultures with SDs.

Cytokine measurement. The amount of cytokines in the culture supernatant was measured by ELISA. Cultured V{alpha}24 NKT cells were plated at 2 x 105 per well with 1 x 106 irradiated PBMCs that had been previously pulsed for 2 to 3 hours with 100 ng/mL {alpha}GalCer. The supernatants were collected after 24 to 48 hours of activation and stored –80°C. IFN-{gamma} and IL-4 were measured using ELISA kits (BD Biosciences, San Jose, CA) according to the manufacturer's instructions.

Flow cytometric analysis of the peripheral blood NKT cells and NK cells. PBMC samples were obtained from the patients twice before V{alpha}24 NKT cell administration and weekly until 4 weeks after the final injection. The frequencies of V{alpha}24 NKT cells (V{alpha}24+Vß11+CD3+), NK cells (CD3–CD56+), T cells (CD3+ cell), and other cells, including B cells (CD3–CD56– cells) in PBMCs, were assessed by flow cytometry using automated full blood counts. The frequency of monocyte was calculated from the cytogram findings. The absolute numbers of these cells were also calculated.

Single-cell enzyme-linked immunospot assay. The PBMCs were washed thrice with PBS and then were stored in liquid nitrogen until use. For detecting IFN-{gamma}-secreting cells, 96-well filtration plates (Millipore, Bedford, MA) were coated with mouse anti-human IFN-{gamma} (10 µg/mL; Mabtech, Nacka Strand, Sweden). PBMCs (5 x 105 per well) were incubated for 16 hours with or without {alpha}GalCer (100 ng/mL) in 10%FCS containing RPMI. Phorbol 12-myristate 13-acetate (10 µg/mL) plus ionomycin (10 nmol/L) was used as a positive control. After culture, the plates were washed and incubated with biotinylated anti-IFN-{gamma} (1 µg/mL; Mabtech). Spot-forming cells were quantified by a microscopy. In our enzyme-linked immunospot assay protocol, the majority of the IFN-{gamma}-producing cells detected after {alpha}GalCer stimulation for 16 hours was CD56+ NK and NKT cells (Supplementary Table S1).


   Results
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 Abstract
 Patients and Methods
 Results
Discussion
 References
 
Patient characteristics. In accordance with the protocol, a total of six patients were enrolled in the study from July 2003 to March 2004. The patient characteristics are summarized in Table 1 . All patients showed recurrent lung cancer after surgical treatment. The study included four patients with adenocarcinoma and two patients with squamous cell carcinoma. All patients received previous treatments, including two cases who had undergone an incomplete surgical resection of the recurrent legions, one who had received radiation therapy, and four who had received chemotherapy >4 weeks before enrollment into this study.


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  		Table 1. Profiles of each patient and any adverse events observed

 
Phenotypes of activated V{alpha}24NKT cells cultured with {alpha}GalCer and IL-2. The surface phenotypes of activated V{alpha}24 NKT cells were analyzed by flow cytometry for each administration. Representative profiles (V{alpha}24/Vß11 and CD3/CD56) of a patient in the level 2 (case 005) are shown in Fig. 2A . Freshly isolated PBMCs contained very small percentages of V{alpha}24+Vß11+ NKT cells (0.06%; Fig. 2A, top left). After cultivation in the presence of {alpha}GalCer and IL-2, this population efficiently expanded (25%, 2 weeks and 21.5%, 3 weeks; Fig. 2A, top center and right). The CD3-negative population decreased after cultivation (Fig. 2A, bottom). The percentages of NKT cells, CD3+ cells, and CD3–CD56+ cells in the cultured cells of six patients (before, 2 weeks, and 3 weeks after cultivation) are summarized in Table 2 . The CD4/CD8 expression of {alpha}GalCer/CD1d tetramer-positive cells was also investigated. CD8+ and CD4–CD8– phenotype were dominant in one patient (case 005), and CD4+, CD8+, and CD4–CD8– phenotypes were all observed in other two patients in the levels 2 (Fig. 2B). It is interesting to note that we detected a substantial number of CD8+V{alpha}24+Vß11+ NKT cells, which correlated with the findings of a previous report (23). Next, functional evaluations of the levels of cytotoxic activities and cytokine production were done. Activated V{alpha}24 NKT cells simulated with {alpha}GalCer and IL-2 showed efficient cytotoxic activities against human tumor cell lines, including Daudi cells and PC-13 cells (Fig. 2C). In addition, they also produced a large amount of IFN-{gamma} and a small amount of IL-4 in response to a rechallenge with {alpha}GalCer-pulsed PBMCs (Fig. 2D). The number of V{alpha}24+Vß11+ NKT cells in the PBMC cultures for 1 to 3 weeks increased efficiently with some variations (Fig. 2E). The expansion rate of V{alpha}24 NKT cells in six donors averaged 1,290-fold (range: 50- to 3,460-fold) for the 14-day culture period and 2,380-fold (range: 280- to 5,250-fold) for the 21-day culture period, and these results are consistent with those shown in our previous report (19).


Figure 2
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  		Fig. 2. Phenotypic and functional evaluation of activated V{alpha}24 NKT cells. PBMCs from a patient were cultured with {alpha}GalCer and IL-2 for 2 or 3 weeks, and whole cultured cells were used for analysis. A, flow cytometric analysis of precultured (Before) or cultured cells (2 wks or 3 wks). Top, percentages of V{alpha}24+Vß11+ NKT cells. Bottom, NK cells (CD56+CD3–), NKT cells (CD56+CD3+), and T cells (CD3+CD56–). Representative results of a patient (case 005) in the level 2 group. B, CD4/CD8 expression on {alpha}GalCer/CD1d tetramer-positive cells. Cultured cells were stained with phycoerythrin-labeled {alpha}GalCer/CD1d tetramer followed by FITC-labeled anti-CD8 mAb and cychrome-labeled anti-CD4 mAb. CD4/CD8 expression was determined by a flow cytometry analysis at the end of the 2- or 3-week culture period. C, cytotoxic activity of in vitro expanded V{alpha}24 NKT cells. Whole cultured cells for 2 or 3 weeks were incubated with two target cell lines (1 x 104 per well) for 4 hours at various effector/target ratios (E/T ratio) in triplicate. Percentages of specific 51Cr release with SD. D, cytokine production from activated V{alpha}24 NKT cells. After 2- or 3-week cultivation with {alpha}GalCer and IL-2, whole cells were collected and seeded onto a 96-well plate (2 x 105 per well) in triplicate. Restimulation was done with {alpha}GalCer-pulsed, irradiated, autologous PBMCs for 24 hours, and the amounts of cytokines (IFN-{gamma} and IL-4) in the culture supernatant were determined by ELISA. E, expansion of V{alpha}24 NKT cells after cultivation with {alpha}GalCer and IL-2. The PBMCs were cultured with {alpha}GalCer (100 ng/mL) and IL-2 (100 JRU/mL). On days 7, 14, and 21, all cultured cells were harvested, and the total cell number and the percentage of V{alpha}24+Vß11+NKT cells were evaluated by a flow cytometry analysis. The fold increase in V{alpha}24+Vß11+ NKT cells was determined as follows: [(total number of live cells recovered on days 7, 14, and 21) x (%V{alpha}24+Vß11+CD3+ cells)] / [(total number of live cells before culture) x (%V{alpha}24+Vß11+CD3+ cells before culture)]. Number (001-006) represent each enrolled patient.

 

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  		Table 2. Profiles of {alpha}GalCer/IL-2–cultured PBMCs

 
Adverse events. No major (grade >2) toxicity or severe side effects were observed in any patients (Table 1). One patient in the level 1 group (case 001) experienced a transient flush and headache soon after the V{alpha}24 NKT cell injection, but no additional treatment was required. One patient in the level 1 group (case 003) experienced transient arrhythmia a few hours after the V{alpha}24 NKT cell injection, and the symptom disappeared in several minutes without any medication. One patient in the level 2 group (case 004) experienced a mild facial paralysis (numbness) on day 42 (21 days after the final injection). A zygomatic bone metastasis was found after precise medical check-up. The numbness was improved by a radiation therapy on the metastatic legion in the zygomatic bone. One patient in the level 2 group (case 006) experienced an increased fever (grade 1) a few hours after the V{alpha}24 NKT cell administration, which abated 6 hours after the use of loxoprofen sodium. Regarding the laboratory data, an elevation in the serum glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase (day 42), and total bilirubin level (day 22) was observed in one patient in the level 1 group (case 001); a lactate dehydrogenase elevation (day 28) was observed in one patient in the level 1 group (case 003); and an elevation in the {gamma}-GTP level (day 22) was observed in one patient in the level 2 group (case 006). The Common Terminology Criteria for Adverse Events grade of these abnormal data was categorized as grade 1, and no cases needed any additional treatment.

Immunologic monitoring. Immunologic assays were done for the six patients who completed the study. The frequency of peripheral blood V{alpha}24+Vß11+ NKT cells in all patients was measured by a flow cytometry analysis (Fig. 3A-F ). A level 1 patient (case 003) showed increased circulating V{alpha}24 NKT cells on days 35 to 49 (Fig. 3C). Two patients in the level 2 group showed increases in the V{alpha}24 NKT cells after the administration on day 28 (Fig. 3D) or on days 21 and 28 (Fig. 3E) compared with the initial NKT cell numbers detected (before or on day 14: before 1st injection). The absolute numbers of V{alpha}24 NKT cells decreased transiently to a nadir around 1 day after the V{alpha}24 NKT cell injection in these three cases. The number of NK cells slightly increased in one patient on day 28 (Fig. 3D), but no obvious increase was detected in the other four patients. In addition, we monitored the absolute numbers of peripheral blood CD3+ cells, CD3–CD56– cells, and monocytes in patients 005 and 006 (Fig. 3G and H). We observed a slight increase in the number of peripheral blood CD3+ cells, but no obvious changes in the number of CD3–CD56– cells or monocytes.


Figure 3
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  		Fig. 3. Immunologic monitoring of PBMCs of patients with V{alpha}24 NKT cell administration. Absolute number of peripheral blood NKT cells (V{alpha}24+Vß11+ cells) and NK cells (CD56+CD3– cells; A-F) and that of T cells (CD3+ cell), other cells including B cell (CD3–CD56– cells), and monocytes (G and H) during the course of treatment in each patient. With the use of the results of a flow cytometry analysis and automated full blood counts (Chiba University Hospital), the absolute number of NKT cells, NK cells, T cells, and other cells was calculated. The absolute number of monocytes was calculated from the cytogram. 1st inj., first in vitro expanded NKT cell injection; 2nd inj., second in vitro expanded NKT cell injection.

 
Concurrently, we assessed the ability to produce IFN-{gamma} after restimulation with {alpha}GalCer. The patient PBMCs were stimulated with {alpha}GalCer for 16 hours, and the IFN-{gamma}-producing cells in the culture were assessed by an enzyme-linked immunospot assay. The number of cells with IFN-{gamma} production increased 1 and 2 weeks after the second injection in a patient in level 1 (case 001, Fig. 4A ) and a level 2 patient (case 004, Fig. 4C), immediately after the first injection in a patient in level 2 (case 005, Fig. 4D), and 2 weeks after the second injection in case 006 (Fig. 4E). Regarding the production of IL-4, we did not observe a detectable number of {alpha}GalCer-dependent IL-4-producing cells in any cases in our assay system (data not shown).


Figure 4
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  		Fig. 4. Monitoring of IFN-{gamma}-producing cells in PBMCs of patients with V{alpha}24 NKT cell administration. Detection of {alpha}GalCer-reactive IFN-{gamma}-producing cells by enzyme-linked immunospot assay. Cryopreserved PBMCs were thawed and cultured overnight with either {alpha}GalCer or vehicle. The presence of IFN-{gamma}-producing cells was quantified by an enzyme-linked immunospot assay. Spot number of IFN-{gamma} with SD for triplicate cultures. N.D., not done; 1st inj., first in vitro expanded NKT cell injection; 2nd inj., second in vitro expanded NKT cell injection; SFC, spot-forming cells.

 
Clinical outcome. All six cases were evaluated at the end of the clinical trial period. From the chest X-ray and computed tomography findings, there were no cases of complete response or partial response, four cases of stable disease (cases 001, 003, 005, and 006), and two cases of progressive disease (cases 002 and 004). Two patients receiving the level 2 dose were followed up for 9 and 12 months after the clinical trial period, and both of them were classified as stable disease.


   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
The primary aim of this study was to assess the feasibility and toxicity of adoptive immunotherapy using activated V{alpha}24 NKT cells in patients with advanced or recurrent non–small cell lung cancer. Because this is the first clinical trial for the administration of V{alpha}24 NKT cells activated in vitro with {alpha}GalCer and IL-2, we did the trial very carefully. Our results indicate that activated V{alpha}24 NKT cell therapy has no major side effects and is well tolerated with minor adverse events, even in patients with advanced stages of lung cancer. There were no clinical symptoms suggesting the development of an autoimmune disease during the observation period. Furthermore, the therapy was safe with minor adverse events, and it can be easily done on an outpatient basis. Although activated iNKT cells in the mouse liver induced severe hepatitis (24, 25), only slight liver dysfunction was detected in two patients in the level 1 group and one patient in the level 2 group. These patients recovered without any additional treatments. This could be due to the limited number of activated V{alpha}24 NKT cells that migrated into the liver of the patients receiving the V{alpha}24 NKT cell administration.

We chose PBMCs as a source of V{alpha}24 NKT cells for administration. An effective method to obtain a large number of purified, functional V{alpha}24 NKT cells from human peripheral blood has been reported (21). In this report, autologous whole PBMCs were used as antigen-presenting cells instead of the additional preparation of monocyte-derived dendritic cells for stimulation. Our culture system was similar to that described in this report. The expansion of V{alpha}24 NKT cells was sufficient (Fig. 2E); thus, this simple procedure seemed to be the most appropriate for the clinical use. The fold increase in the V{alpha}24 NKT cell number varied among the patients (Fig. 2E), and it seems to be partially depended on the initial frequency of V{alpha}24 NKT cells (see Table 2). We previously reported that the number of V{alpha}24 NKT cells in the peripheral blood significantly decreased in patients with lung cancer (14). We, therefore, set relatively tight entry criteria (>100 mL) regarding the number of peripheral blood V{alpha}24 NKT cells. The number of V{alpha}24+Vß11+CD3+ NKT cells and {alpha}GalCer/CD1d tetramer-positive cells were almost identical in the cultures with {alpha}GalCer and IL-2 (Supplementary Fig. S1); therefore, the expansion of V{alpha}24-negative {alpha}GalCer/CD1d tetramer-reactive T cells reported by Gadola et al. seemed to be negligible in our culture system (26).

Deficiencies in IFN-{gamma} production or the proliferative potential of V{alpha}24 NKT cells have recently been reported in some patients with advanced malignancies (13, 15, 17). In prostate cancer patients, ex vivo expansion of {alpha}GalCer-activated V{alpha}24 NKT cells as well as their IFN-{gamma} production was decreased (13). The number of peripheral blood V{alpha}24 NKT cells was decreased in the cancer patients regardless of the tumor type or tumor load, but the ability to produce IFN-{gamma} at a per cell basis did not decrease (15). V{alpha}24 NKT cells from different types of solid cancer patients failed to proliferate even with {alpha}GalCer stimulation, thus producing reduced levels of cytokines compared with those from healthy individuals (17). In lung cancer patients, a reduced proliferative response of V{alpha}24 NKT cells to {alpha}GalCer was detected, and the reduction was partially recovered by granulocyte-colony stimulating factor (27). These studies indicate that V{alpha}24 NKT cells in cancer patients have some numerical and functional defects. In contrast, the number and the function of V{alpha}24 NKT cells has been reported not to be suppressed in glioma patients (28). We previously reported the ability to produce IFN-{gamma} in peripheral blood V{alpha}24 NKT cells to be preserved in non–small cell lung cancer patients (14). The reason for the discrepancy between the NKT cell function in the lung cancer patients in our report (14) and that in the report by Konishi et al. (27) is still unclear at this time. However, the levels of expansion and the ability to produce IFN-{gamma} in our prepared activated V{alpha}24 NKT cells were sufficient for the use of clinical trials (Table 2; Fig. 2). Although we still do not know whether there is functional alteration in the V{alpha}24 NKT cells in the lung, we detected an accumulation of V{alpha}24 NKT cells in the cancerous lesion in the lung, thus suggesting the occurrence of antitumor reactions against tumor cells (14).

In patients with either decreased or functionally altered V{alpha}24 NKT cells, the expansion and activation of these cells in vitro and subsequent adoptive transfer should be therapeutically beneficial, if these deficiencies can be rectified through in vitro cultivation. From the results shown in this report, in vitro expanded V{alpha}24 NKT cells seemed to be functional regarding IFN-{gamma} production (Fig. 2). More importantly, we detected an increased number of IFN-{gamma}-producing cells in the peripheral blood after the injection of activated V{alpha}24 NKT cells. The timing of the increase detected was from a day to 2 weeks (Fig. 4). Because the number of injected activated V{alpha}24 NKT cells was within the order of 1 x 108, it is very likely that some important change occurred in patients after the administration of activated V{alpha}24 NKT cells, such as a robust expansion of endogenous V{alpha}24 NKT cells and the activation of NK cells. In fact, the specific activation of iNKT cells has been reported to lead to a rapid induction of extensive NK cell proliferation and cytotoxicity, partially depending on the IFN-{gamma} production by iNKT cells (9, 10, 29, 30). Regarding the transient decrease in the number of V{alpha}24 NKT cells observed after NKT cell administration (Fig. 3C, D, and E), such a decrease was also observed in our previous clinical trials with {alpha}GalCer-pulsed dendritic cells (31). We do not know the reason at this time, but a possibility for the down-modulation of V{alpha}24 NKT cell receptors from the cell surface has been suggested (32–34).

Immunotherapy methods with {alpha}GalCer or {alpha}GalCer-pulsed dendritic cells in patients with malignant diseases have been recently described (31, 35–37). In these trials, it is expected that the given {alpha}GalCer or {alpha}GalCer-pulsed dendritic cells induces the activation of V{alpha}24 NKT cells in situ. We detected an increase in the number of V{alpha}24 NKT cells in the peripheral blood of patients receiving {alpha}GalCer-pulsed dendritic cells (31). The most characteristic difference in the present study from these studies is that the activation process of V{alpha}24 NKT cells with {alpha}GalCer presented on dendritic cells is done in vitro. We detected notable immune responses in vivo after the administration of activated V{alpha}24 NKT cells (Figs. 3 and 4). Although we need to more precisely assess the nature of immune responses induced by the administration of activated V{alpha}24 NKT cells with an increased number of patients, the results shown in this report provide an alternative procedure of cancer immunotherapy aimed at the activation of V{alpha}24 NKT cells and the subsequent activation of NK cells.

No clear antitumor effect was observed in the present clinical trials. However, these trials are still small scale; thus, we need to await the findings of relatively large-scale studies to evaluate the clinical efficacy of this immunotherapy with activated V{alpha}24 NKT cells. A study with a relatively longer monitoring period (at least a few years) may help to answer the question whether this immunotherapy can induce a long stable disease status in advanced cancer patients. In addition, a combined immunotherapy with the administration of {alpha}GalCer or {alpha}GalCer-pulsed dendritic cells is expected to result in a more effective clinical effect.

Primary lung cancer is hard to cure, although the primary tumor lesions tend to be small enough to be diagnosed at an early stage. Approximately half or more of patients with lung carcinomas who undergo complete resection had clinically undetectable local or distant micrometastases (38–40). These findings point to the importance of preoperative or postoperative immunotherapy to suppress the growth of micrometastasis. For this purpose, among the lymphocytes possessing antitumor activity, cells for tumor surveillance, such as NK and NKT cells, are considered to be the most appropriate. From this point of view, non–small cell lung cancer patients undergoing radical surgery may thus be the optimal candidates for immunotherapy aimed at V{alpha}24 NKT cell activation.

In summary, immunotherapy of activated V{alpha}24 NKT cell administration is well tolerated, and it can be carried out safely with minor adverse events even in patients with advanced disease. In vivo immunologic responses, including the elevation of IFN-{gamma}-producing cells in the peripheral blood, were detected after the administration of activated V{alpha}24 NKT cells. With a greater number of treatments, we should eventually obtain more conclusive evidence regarding evaluation of the antitumor effect of this therapy. Furthermore, a combination of this therapy with a potentially additive or synergistic therapeutic strategy may also result in a more prominent antitumor effect.


   Acknowledgments
 
We thank the Kirin Brewery for providing clinical-grade {alpha}GalCer (KRN7000) for these studies and the nurses and staff surgeons in the Department of Thoracic Surgery, Chiba University Hospital for their excellent help with the patient care and continuous support.


   Footnotes
 
Grant support: Ministry of Education, Culture, Sports, Science and Technology (Japan) grants-in-aid for Scientific Research in Priority Areas #17016010 and #17047007, Scientific Research B #17390139, and Scientific Research C #18590466; grant-in-aid for Young Scientists #17790317; and Special Coordination Funds for Promoting Science and Technology; Ministry of Health, Labor and Welfare (Japan); Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (Japan); Cancer Translational Research Project; The Japan Health Science Foundation; Kanae Foundation; and Uehara Memorial Foundation and Mochida Foundation.

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 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Received 1/17/06; revised 5/10/06; accepted 5/11/06.


   References
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 Patients and Methods
 Results
 Discussion
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Commentary

Natural Killer T Cell–Based Cancer Immunotherapy
Hans J.J. van der Vliet, Steven P. Balk, and Mark A. Exley
Clin. Cancer Res. 2006 12: 5921-5923. [Full Text] [PDF]



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