Natural killer (NK) cells can be isolated from peripheral blood and expanded using cytokines in vitro in autologous and allogeneic settings as adoptive immunotherapy for hematologic malignancies and solid cancers. 1-5 We have reported an effective in vitro NK-cell expansion method using cytokines without feeder cells. 6-8 In addition, NK cells kill target cells by antibody-dependent cellular cytotoxicity (ADCC) in which an antibody such as an anti-CD20 monoclonal antibody (rituximab) crosslinks to the Fc receptor (CD16) on NK cells. 9, 10 Therefore, we have conducted a phase I study of therapy using ex vivo expanded auto-NK cells from patients’ peripheral blood mononuclear cells (PBMC) combined with rituximabcontaining chemotherapy for relapsed CD20-positive malignant lymphoma patients. Patients with relapsed or refractory CD20-positive malignant lymphoma in whom prior standard chemotherapy had failed were enrolled in this study. Eligibility criteria included age between 20 and 80 years, Eastern Cooperative Oncology Group (ECOG) performance status of not more than 3, expected life span of more than three months, and absence of severe active infection or severe organ dysfunction. This study was an open label, non-randomized phase I clinical trial with dose escalation of NK cells. The primary end point was evaluation of the safety of infusion of adoptive NK cells by our expansion method in vitro. The secondary end point was clinical response. This study was approved by the ethics committee of Tokyo Women’s Medical University and was registered in the University Hospital Medical Information Network (UMIN) Clinical Trail Registry as ID: UMIN000014072. This study was designed and conducted in accordance with the Declaration of Helsinki and the Ethical Guidelines for Clinical Research (Ministry of Health, Labor and Welfare, Japan), and was approved by the Certified Committee for Regenerative Medicine.

Peripheral blood mononuclear cells were obtained from the peripheral blood (20-50 mL) of each lymphoma patient. The PBMC (1x106/mL) with IL-15 (10 ng/mL)(PeproTech Inc., Rocky Hill, NJ, USA), IL-2 (5 ng/mL)(R & D Systems, Minneapolis, MN, USA), and anti-CD3 mAb (OKT3, 10-1,000 ng/mL, Janssen Pharmaceutical Company, Tokyo, Japan) with tacrolimus (0.1 ng/mL, Fujisawa, Osaka, Japan) and dalteparin sodium (Fragmin, 5-10 U/mL, Pfizer Japan, Tokyo, Japan) in culture medium SCGM (CeeGenix, Freiburg, Germany) that was produced under good manufacturing practice (GMP) were cultured with 2.5% auto-serum in a culture bag without feeder cells for three weeks. 7, 8 Cell cultures were split into approximately half and a quarter after 3-4 days of culture, and fresh medium, cytokines and reagents were