Abstract:
Despite the tremendous clinical success of chimeric antigen receptor (CAR)-expressing T cells, severe side effects have been associated with CAR T cell therapy. Moreover, production for individual patients still requires an extensive and time-consuming manufacturing process, which presses the need for clinical alternatives. The continuously expandable and well-established natural killer (NK) cell line NK-92 might represent such alternative. Irradiated NK-92 cells provide a safe and consistent way to produce NK effector cells in a GMP-compliant and cost-effective way. Furthermore, NK-92 can be redirected by CARs to mediate antigen specific tumor lysis.
We recently developed the universal adapter CAR (AdCAR) system. Utilizing adapter molecules (AM), antigen recognition and CAR-driven immune cell activation were split into a two-step process. The system allows precise quantitative (on-/off-switch) as well as qualitative (change and combination of target antigens) regulation of immune cell function. AdCARs are based on the unique properties of a novel scFv targeting a “neo”-epitope-like structure derived from the endogenous vitamin biotin. Biotinylation of antibodies, which are already approved by the American Food and Drug administration (FDA) and European Medicines Agency (EMA), such as cetuximab or trastuzumab, generates functional AM with a known safety profile for AdCAR NK-92 therapy and facilitates translation into clinical settings.
The primary goal of this thesis was the development of a highly flexible CAR system for universal tumor targeting to overcome the existing limitations of CAR therapy, especially for solid tumors. We used lentiviral vectors for the transduction process and single-cell sorted successfully transduced AdCAR NK-92 cells using flow cytometry. The clone with the highest CAR surface expression and highest viability was chosen for further experiments. We thoroughly characterized AdCAR NK-92 cells for their NK receptor expression as well as for the expression of immune checkpoints and chemokine receptors. For functional assessment we utilized standard calcein release cytotoxicity assays as well as impedance-based real-time live cell analysis technology. AdCAR NK-92 cells successfully induced significant lysis of various hematological malignancies such as lymphoma and leukemia as well as solid tumors such as melanoma and various carcinomas in vitro but only in the presence of biotinylated antibodies (bAb) targeting antigens that are sufficiently expressed on the tumor cell surface. Therefore, classic tumor antigen evasion mechanisms such as antigen downregulation can be counteracted by simultaneous or successive addition of different bAb. Specific AdCAR-mediated cytotoxicity could be demonstrated regardless of tumor cell expression of inhibitory ligands. To bridge the gap between in vitro and in vivo assessment of AdCAR NK-92 cell function, we generated three-dimensional tumor cell models that are physiologically more relevant and predictive than standard two-dimensional assays. AdCAR NK-92 cells were capable of successful tumor spheroid infiltration and specific long-term tumor cell lysis.
Utilizing CAR-modified NK-92 cells targeting the immune checkpoint molecule B7-H3 (CD276), a tumor-associated antigen severely overexpressed on a variety of solid cancers, we assessed the ability of CAR NK cell-based immunotherapy to overcome a variety of the obstacles exerted by the immunosuppressive tumor microenvironment (TME). Moreover, we used CRISPR/Cas9 technology to generate CAR NK-92 cells with a NKG2A (CD159a) knock-out (KO) and assessed whether we could boost CAR-mediated effector function by eliminating a highly expressed inhibitory NK receptor. The CRISPR mediated NKG2A KO as well as blocking the NKG2A receptor with an inhibitory antibody did not significantly boost CAR-mediated cytotoxic potential of CD276 CAR NK 92 cells suggesting that CAR-induced tumor lysis is independent of the expression of inhibitory receptors on NK-92 cells.
In conclusion, we generated a universal CAR-engineered and an antigen-specific CAR NK-92 cell line. These cells can be manufactured as an “off-the-shelf, on-demand” standardized product whose powerful effector function can be tightly regulated for tunable, patient-individualized targeting of hematological and solid cancers.
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