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Copyright (c) 2018 Ruth Esser, Ulrike Koehl, Wolfgang Glienke, Michael Morgan, Andrew Kaiser, Hinrich Abken, Michael Heuser, Krasimira Aleksandrova, Jana Leise, Olaf Oberschmidt, Christoph Priesner, Lubomir Arseniev, Axel Schambach, Stephan Kloess
This work is licensed under a Creative Commons Attribution 4.0 International License.
Adaptive immunotherapy using redirected chimeric antigen receptor (CAR) T cells against leukemia has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products leads to a manufacturing challenge regarding automation, process robustness, and cell storage. In this respect our results from a study for relapsed Melanoma regarding manufacturing of CAR T cells in a closed and automated system gives rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies. In contrast to T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease, which makes them a promising source for third-party-donor immunotherapy. However, tumor cells can escape NK cell immunosurveillance by tumor immune escape mechanisms (TIEMs). In order to overcome TIEMs and to make NK cell-based therapies more specific, we engineered primary human NK cells to express a CAR designed to recognize CD19 or CD123, which are highly expressed on the surface of primary acute lymphoblastic or myeloid leukemia, respectively. NK cells were transduced with state-of-the-art alpharetroviral self-inactivating (SIN) vectors encoding EGFP alone as control or a second or third generation CAR engineered with an anti-CD19 or anti-CD123 single chain variable fragment (scFv) and containing the CD28 transmembrane domain, the 4-1BB costimulatory domain, the CD3? signaling domain and an internal ribosomal entry site (IRES) element for EGFP expression. CAR-modified NK cells showed a strongly improved cytotoxicity against leukemic cells compared to activated NK cells with a nearly complete elimination of leukemic cells after 48 h. Moreover, our side-effect studies demonstrated minimal or no cytotoxicity of CAR NK cells against PBMNCs and lung epithelia cells, respectively.
Since autologous T cells cannot consistently be expanded ex vivo for all patients, third-party allogeneic CAR NK cells as an “off the shelf product” may serve as an alternative strategy. Since NK cells have a significantly shorter lifespan than T cells, off-tumor toxicity might be reduced. However, for durable anti-tumor effects immature CAR NK cells or repeated cell infusions of mature CAR NK cells might be necessary.