Cancer Therapy: Preclinical 2B4 (CD244) Signaling by Recombinant Antigen-specific Chimeric Receptors Costimulates Natural Killer Cell Activation to Leukemia and Neuroblastoma Cells

Purpose: Novel natural killer (NK) cell–directed strategies in cancer immunotherapy aim at specifically modulating the balance between NK cell receptor signals toward tumor-specific activation. The signaling lymphocyte activation molecule–related receptor 2B4 (CD244) is an important regulator of NK cell activation. We investigated whether 2B4-enhanced activation signals can redirect the cytolytic function of human NK cells to NK cell–resistant and autologous leukemia and tumor targets. Experimental Design: In vitro–stimulated NK cells from healthy donors and pediatric leukemia patients were gene modified with CD19 or GD2-specific chimeric receptors containing either the T-cell receptor ζ or 2B4 endodomain alone or combined. Results: Chimeric 2B4 signaling alone failed to induce interleukin-2 receptor up-regulation and cytokine secretion but triggered a specific degranulation response. Integration of the 2B4 endodomain into T-cell receptor ζ chimeric receptors significantly enhanced all aspects of the NK cell activation response to antigen-expressing leukemia or neuroblastoma cells, including CD25 up-regulation, secretion of IFN-γ and tumor necrosis factor-α, release of cytolytic granules, and growth inhibition, and overcame NK cell resistance of autologous leukemia cells while maintaining antigen specificity. Conclusion: These data indicate that the 2B4 receptor has a potent costimulatory effect in NK cells. Antigen-specific 2B4ζ-expressing NK cells may be a powerful new tool for adoptive immunotherapy of leukemia and other malignancies. Besides antigen-specific CTLs, cellular components of the innate immune system can contribute to immune surveillance of malignant cell growth. In particular, natural killer (NK) cells can eliminate abnormal cells without priming or sensitization (1). Their activity is determined by the balance of signals from inhibitory and activating NK cell receptors. Inhibitory receptors [e.g., killer immunoglobulin receptors (KIR)] interact with selfMHC class I antigens and protect normal cells from NK cell attack. Many malignant cells express MHC class I antigens and are thus naturally resistant to lysis by autologous NK cells. Accordingly, the first clinical trials using adoptive transfer of autologous NK cells have failed to produce significant therapeutic effects (2, 3). For these reasons, NK cell–based immunotherapies have mostly focused on the haploidentical hematopoietic stem cell transplantation setting, where KIR:MHC class I mismatches between the donor and recipient were found by many investigators to contribute to the control of residual myeloid leukemia cells (4, 5). Increasing awareness of the role of activating receptors in the recruitment of NK effector functions has motivated new efforts to target autologous malignancies. Indeed, engagement of activating NK cell receptors by ligands expressed on tumor cells can overcome inhibitory signals and stimulate NK cell responses even in the presence of autologous MHC class I (6, 7). This mechanism is efficiently counteracted in many human tumors, where cells evade NK cell–mediated killing by shedding or intracellular retention of ligands for activating receptors (8). Furthermore, inhibitory cytokines secreted within the malignant microenvironment result in systemic downmodulation of either NK cell receptors or their ligands in cancer patients, further contributing to NK cell resistance (9). Attempts at increasing the susceptibility of malignant cells to NK cell–mediated lysis have focused on modulating the balance between inhibitory and activating NK cell receptor signals using agonistic cytokines or drugs (6, 10). An alternative approach is the genetic modification of NK cells with chimeric receptors (CARs) that retarget cellular activation Authors' Affiliations: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster, Muenster, Germany; Department of Haematology, University College London, London, United Kingdom; and Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee Received 10/28/08; revised 3/17/09; accepted 4/6/09; published OnlineFirst