A Novel Platform Technology for the Development of NK Cell-Based Cellular Immunotherapies
Authors: Boje Ammelie Svea , Langner Anna , Gehlert Carina Lynn , Reitinger Carmen , Nimmerjahn Falk , Murga Penas Eva Maria , Bendig Sonja , Chitadze Guranda , Brüggemann Monika , Diemer Katharina , Jochimsen Alexander , Winterberg Dorothee , Baum Natalie , Krohn Steffen , Valerius Thomas , Stölzel Friedrich , Gramatzki Martin , Klausz Katja , Kellner Christian , Peipp Matthias 
Published: December 2024

Adoptive cell therapy based on natural killer (NK) cells holds great promise for the treatment of cancer. Recent early-phase clinical trials using genetically modified NK cells (CAR-NK cells) or unmodified memory-like NK cells demonstrated promising activity in cancer patients. Due to a different mode of target cell recognition and toxicity profile, NK cells differentiate from T cells in adoptive cell therapy. For all approaches aiming at utilizing NK cells in immunotherapy, efficient ex vivo expansion technologies for the generation of highly cytotoxic NK cells are a prerequisite for clinical translation.
Here, we developed a novel ex vivo NK cell expansion technology based on a multifunctional fusion protein allowing potent NK cell amplification using different sources of NK cells and obviating the requirement of genetically modified feeder cells. The novel fusion protein is designed to bind B cells and as a consequence, to provide interleukin (IL)-15 trans-presentation along with 4-1BB co-stimulation to NK cells in an autologous setting. The multifunctional fusion protein was produced in CHO-S cells and is active at low nanomolar concentrations to trigger strong NK cell proliferation when used in co-culture experiments with autologous B cells. Expansion rates between 100- to 6,000-fold (mean value: 1,200-fold) were achieved using NK cells from peripheral blood of healthy donors (n=22), cord blood (n=3), multiple myeloma patients (n=4) and acute myeloid leukemia (AML) patients (n=6). Interestingly, with our technology also significant expansion rates of NK cells that have been polarized to a memory-like phenotype by triple cytokine stimulation (IL-12, IL-15, IL-18) were achieved. Immunophenotyping of a set of surface receptors and single cell sequencing demonstrated an activated NK cell phenotype. The expanded NK cells showed increased expression levels of activating NK cell receptors (e.g. NKG2D, NKp30) and about 75-80 % of NK cells strongly expressed FcγRIIIa. Importantly, the expanded and activated NK cells did not lyse autologous non-malignant B cells, indicating that NK cells generated by this novel approach are still physiologically regulated. In contrast, the expanded NK cells showed high cytotoxic capacity towards tumor cells over a wide range of effector-to-target cell ratios. The extent of tumor cell lysis ranged between 20-80% against a panel of 13 tumor cell lines representative for various hematological and solid tumor entities. The cytotoxic activity of the expanded NK cells was further enhanced by combination with therapeutic antibodies (e.g., rituximab, daratumumab, elotuzumab) thereby triggering antibody-dependent cell-mediated cytotoxicity (ADCC) of antigen-positive tumor cells via engagement of FcγRIIIa. In addition, specifically designed bispecific NK cell engagers induced strong lysis of leukemia and lymphoma cells in combination with the expanded NK cells. Furthermore, we were able to show that NK cells amplified with our novel approach can be successfully modified by non-viral CRISPR/Cas9-based genome editing achieving 95% efficacy. These data further underline the broad applicability our novel technology. Expanded NK cells may well serve as the basis for the generation of CAR-NK cells or cellular products optimized by genomic remodelling of critical signalling pathways.
In conclusion, a novel platform technology is provided for the ex vivo expansion of NK cells by using a multifunctional fusion protein and obviating the need for genetically modified feeder cells. Our approach may be well-suited for the development of NK cell-based immunotherapies.