Randomized Study of Autologous Cytokine-Induced Killer Cell Immunotherapy in Metastatic Renal Carcinoma

Metastatic renal cell carcinoma (RCC) has a poor prognosis with a median overall survival (OS) of 12 months and a 5-year survival rate of less than 10% (1). Metastatic RCC seems to be resistant to conventional therapy regimens such as chemotherapy, hormone therapy, and radiotherapy with an objective response rate of less than 10% (1). The most successful therapy for metastatic RCC has been single-agent, high-dose interleukin (IL)-2 with durable complete remissions in a small percentage of patients. This is at the expense of considerable toxicity and thus careful patient selection by clinical and other criteria is appropriate (2). Prior attempts to improve IL-2 clinical outcome with the addition of other agents have shown a very limited success (3–5). New targeted agents, such as sunitinib, sorafenib, temsirolimus, and bevacizumab, have been developed and are used as a first-line therapy in many centers. Although these agents represent a major advance in the treatment of this disease, they are palliative treatments and rarely produce durable complete remissions (6–9). These limited successes indicate that further efforts are needed to improve the current therapeutic modalities and to explore novel therapies for RCCs to improve patient care and increase survival (10, 11).

Immunotherapy has recently become the fourth important treatment modality for malignant tumors, ranked after surgery, radiotherapy, and chemotherapy (12–14). A number of adoptive immunotherapies using various killer cells have been reported, including lymphokine-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), and anti-CD3 monoclonal antibody–induced killer cells (15–17). However, the therapeutic efficacy is limited because of their low antitumor activities (10). At present, cytokine-induced killer (CIK) cells have been recognized as a new type of antitumor effector cells, which can proliferate rapidly in vitro, with stronger antitumor activity and broader spectrum of targeted tumor than other reported antitumor effector cells (13). Moreover, CIK cells can regulate and generally enhance the immune functions in patients with cancer (18). Our previous phase I study showed the feasibility and low toxicity of CIK cell immunotherapy on patients with cancer (19). However, current data from phase I studies on the anti-RCC effects of CIK cells are highly limited (19–21), and the therapeutic benefits of CIK cells are unknown in metastatic RCCs. The purpose of this prospectively randomized study was to evaluate the clinical efficacy of CIK cell immunotherapy in patients with metastatic clear cell RCCs.

Between June 2005 and June 2008, 148 patients with metastatic clear cell RCCs were treated in this randomized clinical trial (Table 1). Criteria for entry onto this study were histologically confirmed progressive and irresectable metastatic clear cell RCCs, expected survival duration of more than 3 months, Karnofsky performance status (KPS) more than 40%, age between 18 and 80 years, serum bilirubin and creatinine less than 1.25 of the upper normal limit, and free of congestive heart failure, severe coronary artery disease, cardiac arrhythmias, HIV infection, chronic active hepatitis, and concomitant corticosteroid therapy. In all patients treated, no chemotherapy or immunomodulatory treatment had been conducted during the previous 4 weeks. Pregnant and lactating women were excluded.

This study was approved by the State Food and Drug Administration, China (2006L01023), and by the Ethical Committee of Cancer Hospital of Tianjin Medical University, Tianjin, China, according to the guidelines of the Declaration of Helsinki. Informed consent was obtained from all subjects before their entry onto the study. Follow-ups of all patients were started from June 1, 2005, and ended on June 1, 2011. Median follow-up of all patients was 27 months (range, 3–72 months).

Patients were randomly assigned to receive intravenous infusion autologous CIK cells (iv-CIKs; arm 1) or subcutaneous injection human IL-2 (sc-IL-2) in combination with subcutaneous injection human IFN-α-2a (sc-IFN-α-2a; arm 2). The major endpoint was OS and secondary endpoint was progression-free survival (PFS) in this trial. The sample size calculation assumed a 20% improvement in OS at 3 years with a one-sided α-risk of 5% and a β-risk of 20%. OS rates at 3 years were estimated to be 45% and 25% in arms 1 and 2, respectively. Under these assumptions, 140 patients would be required to detect a significant difference between groups.

Randomization was stratified by using a block method. Treatment allocation was done through a specific web site. An independent review committee evaluated the quality of the study, especially with respect to independent radiographic assessment, data monitoring, and statistical analysis.

As shown in Fig. 1, patients received iv-CIKs at days 15 and 16 per cycle in arm 1 and received cycle treatment once every month. Patients without disease progression were eligible for maintenance treatment. Patients received a median of 97 × 10⁸ (range, 76 × 10⁸–114 × 10⁸) autologous CIK cells every cycle.

In arm 2, sc-IL-2 (Proleukin, Chiron) and sc-IFN-α-2a (Roferon, Hoffmann-La Roche) were administered as previously reported in the CRECY study (22). IL-2 was administered to patients at 10 × 10⁶ IU/m²/d on days 1, 3, and 5, weeks 1 to 4. IFN-α-2a was administered at 3 × 10⁶ IU/m²/d on days 2, 4, and 6, weeks 1 to 4. After 2 weeks of rest, an identical treatment with IL-2 combination with IFN-α-2a was administered. Six-week treatment cycles were repeated for up to 1 year unless progression of disease occurred.

Detailed recommendations for dose modifications were given to the investigators. In summary, all toxic events judged as grade III for intensity and prolonged over 2 weeks, or events of grade IV intensity, could justify permanent treatment discontinuation. Concomitant medication was given as needed to control adverse effects. Patients with progressive disease in arms 1 or 2 left study to receive subsequent individual care. Crossover between treatment arms was not allowed. Reevaluation of the patient's tumor status was conducted every 2 months. In arm 1, one (1.4%) patient was delayed treatment because of grade III fatigue. There was no patient who received dose modification and no patient failing to fulfill the CIK cell immunotherapy in arm 1. The median cycle of CIK cell immunotherapy was 10 cycles (range, 2–35 cycles). In arm 2, 14 (19%) patients did not complete cycle 1 because of early progression before the first response evaluation (6%), intolerance (10%), or both early disease progression and intolerance (3%). Beside these, 4 (5.4%) patients delayed treatment and 6 (8.1%) patients received dose modification. Patients in arm 2 received a median of 2.5 six-week cycles (range, 1–8 cycles). All patients were seen at regular biweekly intervals by oncologic specialists; additional care was provided whenever needed.

Risk category of patients was preformed according to the Memorial Sloan-Kettering Cancer Center (MSKCC, New York) criteria (23), Tumor response was classified in accordance with the National Cancer Institute (Bethesda, MD) Response Evaluation Criteria in Solid Tumors (RECIST; ref. 24). Patients were assessed serially using computed tomography of chest, abdomen, pelvis, and brain, and technetium bone scan. Tumor evaluation was done 2 months after treatment start. Adverse events were evaluated according to World Health Organization (WHO) criteria.

CIK cells were prepared as described in our previous studies (19, 25). Briefly, peripheral blood mononuclear cells (PBMC) were collected from the patients with RCCs using a Cobe Spectra Apheresis System (CaridianBCT), and cultured in AIM-V medium (Invitrogen) containing 50 ng/mL anti-CD3 antibody (e-Bioscience), 100 U/mL recombinant human IL-1α (e-Bioscience), and 1,000 U/mL IFN-γ (PeproTech), at 37°C with 5% CO₂ for 24 hours. Then, 300 U/mL recombinant human IL-2 (rhIL-2; Proleukin) was added to the media. The medium was replaced by the fresh IL-2 and IFN-γ–containing medium was replaced every 5 days. At day 14, CIK cells were harvested and analyzed for phenotype and cytotoxicity. All products were free of bacterial, mycoplasma, or fungal contamination. The endotoxin was less than 5 EU.

Phenotypes of untreated PBMCs and autologous CIK cells from 29 patients selected randomly from the 74 patients in arm 1 were detected by two-color fluorescence as described in our previous studies (19, 25). Briefly, according to the routine method, 5 × 10⁵ CIK cells were resuspended in 20 μL of PBS containing 2% newborn calf serum and 1% sodium azide and then labeled with anti-CD3-FITC/anti-CD56-RPE (Dako), CD3-FITC (fluorescein isothiocyanate), CD4-RPE, CD8-RPE, CD45RO, and CD4-FITC/CD25-PE (BD Bioscience). The cell population was analyzed using flow cytometry (BD Aria).

The cytotoxicity of CIK cells from 29 patients selected randomly from the 74 patients in arm 1 was detected as described in our previous studies (19, 25). Briefly, the target cells used for this assay included K562, CALU-6, 823, MCF-7, 786-O, and SK-RC-42. These cell lines were obtained from the American Type Culture Collection. Target cells (1 × 10⁵ cells/mL) were incubated for 4 hours in triplicate sets with effector cells (CIK cells) at an effector-to-target cell ratio of 50:1. At the end of incubation, 50 μL culture supernatant was transferred to a new, flat 96-well plate, and incubated with 50 μL lactate dehydrogenase (LDH) substrate mixture (for detection of LDH released upon cell lysis) at room temperature for 30 minutes in dark. Then, 50 μL stop solution was added to each well. Absorbance was measured at 490 nm using a 96-well plate reader. Killing efficiency was calculated as: % killing efficiency = [(experimental counts − effector spontaneous counts − target spontaneous counts)/(target maximal counts − target spontaneous counts)] × 100.

OS was calculated from initiation of treatment to death, and patients alive were censored at the time of last contact. PFS was calculated from initiation of treatment until first progression, and patients alive in stable state were censored at the time of last contact. The χ² test and Fisher exact test were used for binary variable comparisons. The Mann–Whitney U test was used for median comparisons. Distributions of survival time and rate were estimated by using the Kaplan–Meier method; median survival time and 3-year survival rates along with 95% confidence intervals (CI) were reported. Associations between survival and potential prognostic factors were assessed using the log-rank test in univariable analyses. The Cox proportional hazards model was undertaken in multivariable analyses by using the forward-LR (likelihood ratio) method with a significance level of 0.15 for entering and removing variables. In univariate evaluations of the prognostic impact of continuous variable (the cycle count of CIK cell treatment), the optimal cutoff point was determined using the receiver operating characteristic curve method. A P value less than 0.05 using two-sided tests indicates statistical significance. All calculations were conducted using the SPSS 16.0 Software.

Seventy-four patients were randomized to the CIK cell therapy (arm 1) and 74 patients to the IL-2 plus IFN-α-2a treatment (arm 2). The characteristics of patients in the 2 groups are detailed in Table 1; there were no significant differences between the 2 groups.

The median count of untreated PBMCs of all patients in arm 1 was 21 × 10⁸ (range, 7 × 10⁸–32 × 10⁸) per cycle. The median count of autologous CIK cells after 14 days of amplification could reach 97 × 10⁸ (range, 76 × 10⁸–114 × 10⁸) per cycle. On the basis of trypan blue staining, the cellular vitality reached 95% or even higher.

Phenotypic analysis of autologous CIK cells in 29 patients of arm 1 before culture and after 14 days of culture showed that percentages of CD3⁺, CD3⁺ CD4⁺, CD3⁺ CD8⁺, CD3⁺ CD56⁺, and CD25⁺ cell subsets increased from 48.41% ± 6.19%, 28.07% ± 4.76%, 19.00% ± 5.54%, 3.65% ± 1.41%, and 13.11% ± 3.11% to 81.06% ± 9.22%, 42.70% ± 6.18%, 36.41% ± 5.19%, 18.21% ± 4.73%, and 33.13% ± 6.87%, respectively, with P values <0.01. But percentages of CD3⁻/16⁺/56⁺, CD14⁺, and CD20⁺ cell subsets decreased from 14.23% ± 3.34%, 17.44% ± 5.06%, and 12.11% ± 4.14% to 7.18% ± 2.01%, 6.17% ± 1.89%, and 8.14% ± 2.42%, respectively, with P values <0.05.

Autologous CIK cells from the 29 patients in arm 1 were mixed with various types of tumor cell lines at an effector-to-target ratio of 50:1. Mean values of cytotoxicity of CIK cells against K562, CALU-6, 823, and MCF-7 were 45.30% ± 7.95%, 28.51% ± 6.22%, 34.11% ± 8.27%, and 25.06% ± 6.61%, respectively. In addition, the cytotoxic activity of CIK cells from patients with metastatic RCCs was investigated against 2 human renal carcinoma cell lines 786-O and SK-RC-42. At an effector-to-target ratio of 50:1, CIK cells killed 786-O and SK-RC-42 cells with mean percentage lysis of 35.41% ± 6.11% and 32.17% ± 5.30%, respectively.

Thirteen patients (18%) in arm 1 achieved a complete response and 26 patients (35%) had a partial remission. The overall objective response rate was 53% (95% CI, 45%–61%). Twenty-five patients (34%) showed disease stabilization and 10 patients (14%) exhibited continuous disease progression despite therapy. In arm 2, there were 5 complete responders (7%) and 15 partial responders (20%), with an overall objective response rate of 27% (95% CI, 17%–37%). Twenty-five patients (34%) had disease stabilization, and in 29 patients (39%), a continuous disease progression was observed.

The 3-year PFS rate in arm 1 (18%; 95% CI, 10%–26%) was significantly higher than that in arm 2 (12%; 95% CI, 6%–18%; P = 0.031). The 3-year OS rate in arm 1 (61%; 95% CI, 52%–70%) was also significantly higher than that in arm 2 (23%; 95% CI, 16%–30%; P < 0.001). The median PFS in arm 1 (12 months; 95% CI, 10–14 months) was significantly longer than that in arm 2 (8 months; 95% CI, 4–12 months; P = 0.024; Fig. 2A). The median OS in arm 1 (46 months; 95% CI, 36–56 months) was also significantly longer than that in arm 2 (19 months; 95% CI, 13–25 months; P < 0.001; Fig. 2B).

The cycle count of CIK cell treatment significantly improved the PFS and OS of patients when analyzed as a continuous variable in the univariate analysis (P < 0.001 and <0.001, respectively) and in the multivariate analysis after adjustment for sex, age, KPS, hemoglobin, neutrophils, platelets, LDH, serum corrected calcium, time from initial diagnosis to current therapy, number of metastatic disease, nephrectomy, prior and poststudy therapies (HR = 0.88, 95% CI, 0.84–0.93, P < 0.001; and HR = 0.58, 95% CI, 0.48–0.69, P < 0.001; respectively). The optimal cutoff point of the cycle count was 7 cycles. All 14 potential predictive covariates with their univariate analyses are presented in Table 2. KPS less than 80, no prior nephrectomy, time from initial diagnosis to current treatment of less than 1 year, number of metastases greater than 1, anemia, hypercalcemia, neutrophils, and elevated LDH were significantly associated with poor prognosis. The cycle count of CIK treatments ≥7 cycles was significantly associated with good prognosis. In the multivariate analysis, KPS less than 80, time from initial diagnosis to current treatment of less than 1 year, number of metastases greater than 1, anemia, hypercalcemia, and elevated LDH were independent poor prognostic factors. The cycle count of CIK treatments ≥7 cycles was an independent good prognostic factor (Table 3).

The distributions of side effects in the 2 groups are shown in Table 4. No toxic death was observed. The toxicities in arm 2 were more frequent and more serious than that in arm 1. No patient in arm 1 failed to fulfill the CIK immunotherapy. There were no grade III–IV cell-related toxicities, and common grade I–II toxicities consisted of transient fever, chills, fatigue, headache, and anemia in arm 1.

Treatment of RCCs, especially metastatic RCCs, confronts a great dilemma in clinical practice. Although there are a number of therapeutic options available such as the immunoregulating cytokines and the new antiangiogenic targeted agents at present, these are commonly toxic and rarely produce durable complete remissions. The recent considerable success of cell immunotherapy in melanoma warrants further efforts to apply this treatment to other cancers including RCCs. Two conventional adoptive cell immunotherapy approaches are LAK cells and TILs (10). LAK cell in combination with IL-2 has been extensively studied and was shown to be heterogeneous and capable of killing both allogeneic and autologous tumors (26). The activity of LAK cells was mainly mediated by natural killer (NK) cells and also indirectly by MHC unrestricted T cells (26). TILs represent a part of the host immune response to human malignancy and contain an enriched population of cells with both cytotoxic and helper functions that are reactive to the autologous tumor (27). The majority of TILs expanded by IL-2 are composed of both CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells (27). In addition, TILs have been shown to contain antigen-specific as well as nonspecific cytotoxic lymphocytes (28). These cell immunotherapies get considerable success on the treatment of melanoma which is regarded as an immunosensitive tumor. Although RCC is an immunosensitive cancer, similar attempts in metastatic RCCs have shown a very limited success (10). CIK cells are a novel population of immune effector cells and are activated T cells with NK cell properties that can be expanded in vitro in presence of rhIL-2, starting from PBMCs stimulated by IFN-γ and anti-CD3 antibody (18). CIK cells express CD3 and CD56 as well as the NKG2D antigen and show MHC-unrestricted cytotoxicity toward neoplastic but not normal targets (18, 29, 30). CIK cells express several chemokine receptors and are shown to migrate to the tumor site after intravenous administration in in vivo models (31, 32). At the tumor site, CIK cells can exert their cytotoxic activity and control tumor growth. CIK cells can proliferate rapidly in vitro, with stronger antitumor activity, broader target tumor spectrum, and lower adverse effect than other reported antitumor effector cells (13, 18). Moreover, CIK cells can regulate and enhance the immune function in patients with cancer (18). Their ease of production in vitro and antitumor potential have made them suitable candidates for cell therapy regimens in solid and hematopoietic tumor treatments. Indeed both autologous and allogeneic CIK cells have been used in phase I/II clinical trials for the treatment of various tumor types. In these trials, they have shown limited toxicity in vivo and evidence of antitumor activity (19–21, 25, 33). However, current knowledge on the anti-RCC effects of CIK cells in phase I studies is highly limited (19–21), and the therapeutic benefit of CIK cells is unknown in metastatic RCCs. To our knowledge, the present report is the largest prognostic study in metastatic RCCs treated with CIK cell immunotherapy. We have shown for the first time that CIK cell treatment significantly improves the prognosis of metastatic RCCs. Heng and colleagues reported that the median OS of metastatic RCCs treated with VEGF-targeted therapy was 28 months (34). In this study, the median OS in patients with metastatic clear cell RCCs treated with CIK cell immunotherapy was 46 months. These data suggest that CIK cell immunotherapy could improve the prognosis of patients with metastatic RCCs.

In multivariate analysis, the cycle count of CIK cell treatment is significantly associated with prognosis in the arm 1, when viewed as a continuous variable. The optimal cutoff point of the cycle count was determined to be 7 cycles. The prognosis of patients who received CIK cell treatments for ≥7 cycles was significant better than that of patients who received <7 cycles of the treatments. The median time from the first to seventh treatment was 9 months (range, 8–12 months). Previous studies have shown that the minimal time for immunotherapy to display an effect in patients with cancer is about 8 months (35, 36). These data indicate that the time CIK cell immunotherapy starts to display an effect in metastatic RCCs is approximately 9 months, and the maintenance treatments are required after the initial effect is observed for maximal benefits. However, the preferred length for maintenance treatments is still unclear and needs further investigations.

In conclusion, we have revealed, for the first time, the relationship between the CIK cell immunotherapy and the prognosis of metastatic RCCs. Our study has indicated that CIK cell treatment could improve the prognosis of metastatic clear cell RCCs, and increased frequency of CIK cell immunotherapy could result in additional benefits.

No potential conflicts of interest were disclosed.

L. Liu, H. Li, J. Yu, and S. Wei contributed to the conception and design of the study. L. Liu and H. Li contributed to the development of methodology and writing, review, and/or revision of the manuscript. L. Liu, W. Zhang, and H. Li contributed to the acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.). L. Liu and X. Qi contributed to the analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis) and fulfilled the statistical analysis. L. Liu provided administrative, technical, or material support (i.e., reporting or organizing data, constructing databases) and conducted study supervision. X. Hao and X. Ren were the principal investigators and took the primary responsibility for the manuscript. W. Zhang, X. Ren, and X. Hao recruited the patients. L. Liu, H. Li, and J. Yu conducted the laboratory work for this study. L. Liu, X. Ren, and S. Wei wrote the manuscript.

This work was supported partially by the National Natural Science Funds (No. 30872986 and No. 30901754) and by the Tianjin Key Natural Science Funds (No. 09JCZDJC20400).

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