Therapeutics, Targets, and Chemical Biology Ex Vivo Expansion of Highly Cytotoxic Human NK Cells by Cocultivation with Irradiated Tumor Cells for Adoptive Immunotherapy Seon Ah Lim 1 , Tae-Jin Kim 1,5 , Jung Eun Lee 1,4 , Chung Hee Sonn 1 , Kwanghee Kim 1 , Jiyoung Kim 1 , Jong Gwon Choi 2 , Il-Kyu Choi 3 , Chae-Ok Yun 3 , Jae-Hong Kim 4 , Cassian Yee 6,7 , Vinay Kumar 5 , and Kyung-Mi Lee 1 Abstract Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I–negative or –mismatched tumor cell lines we identified a Jurkat T- lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotox- icity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1–mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcg receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1–lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation. Cancer Res; 73(8); 2598–607. Ó2012 AACR. Introduction Natural killer (NK) cells have been characterized as effector cells of the innate immune system with potent cytotoxic activity. As the first line of defense against tumor cells and viruses, NK cells are poised to mediate killing, without the requirement for prolonged preactivation, through patterned recognition of target ligands (1, 2). Selective engagement of activating receptors (e.g., NKp46, NKp44 and NKp30, NKG2D, DNAM-1, 2B4) in the absence of target inhibitory ligands leads to NK cell activation, expansion, and cytotoxicity (3). Clinical evidence of antitumor responses attributable to NK cells can be found in the setting of haploidentical allogeneic stem cell transplantation for leukemia where improved sur- vival following transplant was noted among patients receiv- ing donor NK cells which were KIR-ligand mismatched (4, 5). A more refined approach using autologous or allogeneic NK cells for adoptive transfer has been used for treating patients with ovarian, breast, renal cell cancer, glioblastoma, and other solid tumors with occasional clinical responses (6–10). In contrast to vaccine therapy or antigen-specific adoptive T-cell therapy, identification of target tumor anti- gens is not required for NK cell therapy, which can be more universally applied and particularly effective for treating solid tumor malignancies that have lost expression of self- MHC as a mechanism of immune escape from T cells (11, 12). However, a major challenge to the broader application of adoptive NK cell therapy has been to develop a method for enriching and expanding the low-frequency NK cells to numbers sufficient for adoptive transfer from the peripheral blood mononuclear cell (PBMC) population (13, 14). For T cells, in vitro expansions to more than 1,000-fold can be routinely achieved by T-cell receptor (TCR) triggering using anti-CD3 monoclonal antibody (mAb) in combination with Authors' Affiliations: 1 Global Research Laboratory, Department of Bio- chemistry and Molecular Biology, Korea University College of Medicine; 2 Oncology-Hematology, Internal Medicine, Korea University Guro Hospi- tal; 3 Department of Bioengineering, College of Engineering, Hanyang University; 4 School of Life Sciences and Biotechnology, Korea University, Seoul, Korea; 5 Department of Pathology, University of Chicago, Chicago, Illinois; 6 University of Washington; and 7 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S.A. Lim, T-J. Kim, and J.E. Lee contributed equally to this work. Corresponding Author: Kyung-Mi Lee, Global Research Laboratory, Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul 136-713, Korea. Phone: 82-2-920-6251; Fax: 82-2-920-6252; E-mail: kyunglee@korea.ac.kr doi: 10.1158/0008-5472.CAN-12-2893 Ó2012 American Association for Cancer Research. Cancer Research Cancer Res; 73(8) April 15, 2013 2598 Downloaded from http://aacrjournals.org/cancerres/article-pdf/73/8/2598/2697948/2598.pdf by guest on 21 June 2022