Toru Kaneko

A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous γδ T cells,

OBJECTIVES Human gammadelta T lymphocytes can recognise and kill non-small-cell lung cancer cells by Vgamma9Vdelta2 T-cell receptor and/or NKG2D. We have established large-scale ex vivo expansion of gammadelta T cells by zoledronate and interleukin-2. This pilot feasibility study evaluates the safety and potential anti-tumour effects of activated autologous gammadelta T cells administered intravenously to patients. METHODS Patients who had measurable foci of recurrent non-small-cell lung cancer were registered to undergo gammadelta T-cell immunotherapy, designed as a one-way, open, clinical research, after their informed consent. Mononuclear cells collected from peripheral blood of the patient were cultured with zoledronic acid and interleukin-2. After 2-week incubation, the gammadelta T-cell fraction was proliferated and it was intravenously reinfused to the patient. RESULTS Ten patients had undergone the gammadelta T-cell immunotherapy. They were administered autologous gammadelta T cells 3-12 times (mean=6) every 2 weeks. No patient died during the study period. Adverse events, not directly related to the immunotherapy, were observed five times in four patients (grade 3 pneumonia in two and grade 1 coldness in three). According to the Response Evaluation Criteria in Solid Tumours, neither complete nor partial response was achieved in any patient; stable disease was observed in three; and progressive disease in five at 4 weeks after six consecutive injections of during immunotherapy. The Functional Assessment of Cancer Therapy-Biologic Response Modifier scores of the patients during immunotherapy were stable or improved, except for one patient who had suffered from pneumonia. The patients were followed up after immunotherapy for 240-850 days (median=401 days). At the end of the observation, six patients were alive. CONCLUSIONS We suggest that gammadelta T-cell immunotherapy might be safe and feasible for patients with recurrent non-small-cell lung cancer.

A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer.

Objectives: Dendritic cell (DC) therapy frequently induces a measurable immune response. However clinical responses are seen in a minority of patients, presumably due to insufficient expansion of antigen-specific cytotoxic T lymphocytes (CTLs) capable of eradicating tumor cells. To increase therapeutic efficacy of DC-based vaccination, we have undertaken the first clinical trial involving a combination therapy of gemcitabine (GEM) with immunotherapy for patients with inoperable locally advanced pancreatic cancer. Methods: Patients (n = 5) received the treatment course, which consisted of intravenous GEM administration at 1000 mg/m2 (day 1) and the endoscopic ultrasound-guided fine-needle injection of OK432-pulsed DCs into a tumor, followed by intravenous infusion of lymphokine-activated killer cells stimulated with anti-CD3 monoclonal antibody (CD3-LAKs) (day 4), at 2-week intervals. Results: No serious treatment-related adverse events were observed during the study period. Three of the 5 patients demonstrated effective responses to this clinical trial; 1 had partial remission and 2 had long stable disease more than 6 months. In the patient with partial remission, it has been shown that DC-based vaccination combined with GEM administration induces tumor antigen-specific CTLs. Conclusion: This combined therapy was considered to be synergistically effective and may have a role in the therapy of pancreatic cancer for inducing tumor antigen-specific CTLs.

Zoledronate-activated Vγ9γδ T cell-based immunotherapy is feasible and restores the impairment of γδ T cells in patients with solid tumors

Gamma/delta (γδ) T cells play a role in innate immunity and exhibit cytotoxicity toward a large range of tumor types. Recent studies have shown that aminobisphosphonates may be applied to a culture in which a large number of γδ T cells are proliferated ex vivo. We carried out a clinical study of 25 patients with various solid tumors to determine further the safety, immunologic effect and feasibility of zoledronate-activated Vγ9γδ T cell-based immunotherapy. No severe toxicity was observed. In the cells used for the first treatment, the total cell number, frequency and number of CD3(+) Vγ9(+) γδ T cells were 409 ± 284 × 10(7) cells, 56 ± 33% and 255 ± 242 × 10(7) cells, respectively. Aminobisphosphonate therapy or chemotherapy resulted in the suppression of CD3(+) Vγ9(+) γδ T-cell proliferation. The numbers of CD3(+) T cells, CD3(+) Vγ9(+) γδ T cells and CD27(-) CD45RA(-) Vγ9(+) subsets in peripheral blood were significantly lower in patients than in healthy subjects (P < 0.05). From such an impaired immunologic condition, the numbers and frequencies of CD3(+) Vγ9(+) γδ T cells and CD27(-) CD45RA(-) subsets significantly increased in patients treated with this immunotherapy. Zoledronate-activated Vγ9γδ T cell-based immunotherapy that restores the number of Vγ9γδ T cells in cancer patients may provide another mode of adoptive immunotherapy.

Impaired and imbalanced cellular immunological status assessed in advanced cancer patients and restoration of the T cell immune status by adoptive T-cell immunotherapy.

Recent progress has been made in understanding the mechanisms of antitumor immune responses, which may further clarify the immune status of cancer patients. In this study, we performed a detailed evaluation of the immunological status of 47 patients with advanced solid cancer, who had received no immunosuppressive treatment, and compared the results with 32 healthy subjects. Flow-cytometry data for peripheral blood were obtained using 19 monoclonal antibodies against various cell surface and intracellular molecules. Absolute numbers of T cells, several T cell subsets, B cells, and NK cells were significantly decreased in patients compared with healthy subjects. The percentage of CD27(+)CD45RA(+) T cells was lower and that of CD27(-)CD45RA(-) T cells was higher in patients compared with controls. Regulatory and type 2 helper T cells were elevated in patients relative to healthy subjects. The percentage of perforin(+) NK cells was significantly lower in patients than in controls. These results suggest a dysfunctional anti-tumor immune response in cancer patients. Furthermore, peripheral blood from 26 of 47 cancer patients was analyzed after adoptive T cell immunotherapy (ATI). ATI increased the number of T cell subsets, but not B and NK cells. The number and percentage of regulatory T cells decreased significantly. These results suggest that ATI can restore impaired and imbalanced T cell immune status.

Abstract 1926: Adoptive γδ T cell transfer therapy for non-small cell lung cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Human peripheral blood γδ T cells can recognize and kill tumor cells by their Vγ9Vδ2 T cell receptor and/or NKG2D. We have established large-scale ex vivo expansion of γδ T cells from peripheral blood mononuclear cells (PBMC) by zoledronate and IL-2. A clinical study of adoptive γδ T cell transfer therapy has been conducted for the treatment of non-small-cell lung cancer (NSCLC) patients to evaluate the safety profile and potential anti-tumor effects of γδ T cells. Prior to beginning the study, it was registered on a University hospital Medical Information Network Clinical Trials Registry (UMIN-CTR : http://www.umin.ac.jp/ctr/index.htm) on March 1, 2006 (UMIN ID: C00000036). PBMC were isolated from 67.5 ml of peripheral blood and stimulated with zoledronateand IL-2 for 14 days to obtain γδ T cells. The cultured cells on day 14 consisted mostly of γδ T cells. The predominant populations in fresh peripheral blood were cells with the CD45RA−CD27+ memory phenotype or a CD45RA+CD27− naive phenotype. In contrast, most γδ T cells were shown to have the CD45RA−CD27− effector memory phenotype after 14 days culture. They expressed NKG2D and up-regulated the expression of activation marker CD69, and had cytotoxicity against allogeneic tumor cell lines. Upon stimulation, γδ T cells produced IL-2, TNF-α and IFN-γ. Though certain patients were refractory to zoledronate stimulation, γδ T cells from patients demonstrated comparable cytotoxic effector activity once they were successfully expanded. Cultured cells were intravenously administered every 2 weeks for 6 injections. Fifteen patients who were resistant to current standard therapy were enrolled this study. The treatment was well tolerated and no severe adverse events related to γδ T cell therapy were observed. Two patients came off the study at the early stage of the treatment due to the aspiration pneumonia (grade 3) and radiation pneumonitis (grade3). The responses according to RECIST guideline were SD for 7 patients, PD for 6 patients and 2 inevaluable for response due to early drop off the study (The disease control rate was 46.7 %). Transferred γδ T cells gradually accumulated in peripheral blood in patients who received more than 1×109 γδ T cells per injection and accounted for 10 % of PBMC even 2 weeks after the final injection. When PBMC were stimulated with PMA/ionomycin or zoledronate-pulsed Daudi cells, IFN-γ production and surface expression of CD107 were detected on γδ T cells, suggesting that transferred γδ T cells sustained their effector function in vivo. These results indicated that γδ T cell therapy was well tolerated and feasible for the treatment of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1926.