Despite the recent success of CAR T cells targeting CD19 and CD22 in hematological malignancies, the production of CAR T cells still requires an extensive manufacturing process. The well-established NK-92 cell line provides a promising alternative to produce CAR-modified effector cells in a GMP-compliant, costeffective way. NK-92 can be redirected against a variety of surface antigens by our adapter CAR (AdCAR) system utilizing biotinylated antibodies (bAb) as adapter molecules. Selected bAb were capable of inducing significant AdCAR NK-92-mediated lysis of non-Hodgkin lymphoma (NHL) and mantle-cell lymphoma (MCL) cell lines as well as primary MCL and chronic lymphocytic leukemia (CLL) cells. AdCAR specificity was proven using a JeKo-1 CD19/CD20 knockout antigen-loss model. Moreover, through combinations of bAb, AdCAR NK-92 cells are capable of combatting tumor antigen evasion mechanisms. In conclusion, we successfully generated the AdCAR NK-92 cell line which can be manufactured as an "offthe-shelf, on-demand" product allowing universal and tunable tumor targeting.

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Background: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). Methods: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. Results: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. Conclusions: The strong cytotoxic effect of a CD276-specific CAR in combination with an “off-the-shelf” NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor in adults. The current standard-of-care treatment, which includes surgical resection, radiation, and chemotherapy, extends the survival of patients to merely 14.6 months. GBM is virtually incurable due to its heterogeneity, which is represented by tumor cells with different patterns of gene expression (proneural, neural, classical, and mesenchymal GBM), the presence of GBM cancer stem (like) cells (CSC), and immune evasion. Thus, new therapeutic strategies directed at eliminating GBM CSC in particular are urgently needed. Utilizing fresh patient material from surgical resections, GBM CSC were successfully enriched and cultivated in serum-free medium and subsequently differentiated by switching to medium containing FBS. The gene expression pattern of both, GBM CSC and differentiated cells, was characterized. Furthermore, a screening platform was tailored for flow-cytometric characterization of patient-specific antigen expression of all newly established glioblastoma cell lines. In this work we present a promising immunotherapeutic approach towards eradication of GBM CSC by combining the universal targeting and controllability of our recently developed adapter chimeric antigen receptor (AdCAR) system with the “off-the-shelf” properties of the continuously expandable and well-characterized NK cell line NK-92. The AdCAR system is based on the unique properties of a novel scFv targeting a “neo”-epitope-like structure derived from the endogenous vitamin biotin, which can be linked to monoclonal antibodies (mAb). Utilizing these biotinylated mAb as adapter molecules, the system allows precise quantitative (on-/off switch) as well as qualitative (change and combination of target antigen) regulation of immune cell function. AdCAR-transduced NK-92 cells demonstrated significant target cell lysis, which, importantly, is largely independent from activation of endogenously expressed NK receptors and the presence of their ligands on target cells but mainly mediated by AdCAR activation through binding to the respective tumor antigen. In the presence of the respective antigen, first and foremost biotinylated antibodies directed at CD9, CD146, CD276, and EGFR were capable of inducing significant AdCAR NK-92-mediated lysis of GBM cells after 2 hours. Interestingly, due to overexpression of various target antigens on GBM cancer stem cells, CSC lysis was noticeably higher as compared to AdCAR-mediated lysis of differentiated glioblastoma cells. In conclusion, we have successfully generated a CAR-modified NK cell line, AdCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens, allowing universal and tunable targeting of glioblastoma cells. Most importantly, AdCAR NK-92 cells provide a promising strategy to eradicate GBM cancer stem (like) cells. Citation Format: Stefan Grote, Chun-Ho Chan, Caroline Baden, Stephan M. Huber, Franziska Eckert, Joerg Mittelstaet, Andrew Kaiser, Christian Seitz, Patrick Schlegel, Rupert Handgretinger, Sabine Schleicher. Universal adapter CAR-engineered NK-92 cells target patient-derived glioblastoma cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B70.

Chimeric antigen receptor (CAR) expressing T cells (CAR-Ts) have demonstrated tremendous clinical success, especially when targeted against the B-phenotypic antigens CD19 and CD22 in ALL, CLL as well as NHL. Despite the recent success, production of CAR-Ts still requires an extensive and time consuming manufacturing process. Moreover, CAR-Ts have to be prepared individually for each patient. Especially in heavily pretreated and rapidly progressing patients, which often lack sufficient numbers of healthy T cells for CAR-T production, alternatives are a significant clinical need. NK cells might represent a promising alternative effector cell source. The continuously expandable and well established NK cell line NK-92 can provide a safe and consistent way to produce NK effector cells in a GMP-compliant and cost-effective way. Irradiated NK-92 CARs as an "off-the-shelf on-demand" cell therapeutic are currently tested in pre-clinical and early-phase clinical trials. Furthermore, NK-92 can be redirected by CARs to mediate direct antigen specific lysis. We have recently developed a universal adapter CAR (aCAR) system. By splitting antigen recognition and CAR-immune cell activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combination of target antigens) regulation of immune cell function. aCARs are based on the unique properties of a novel scFv targeting a "neo"-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugated on novel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner. In the present study, we intended to combine the universal and flexible targeting as well as controllability of the aCAR with the "off-the-shelf" properties of NK-92 cells. NK-92 was obtained from ATCC and transduced with aCARs containing either CD28 or 4-1BB co-stimulatory plus CD3-ζ signaling domains. Importantly, only CD28 containing aCARs sufficiently mediated specific target cell lysis in the presence of biotinylated antibodies (LLE-AMs). Using single cell sorting, aCAR NK-92 clones with the highest CAR expression were selected and demonstrated significantly improved target cell lysis, in a LLE-AMs dependent manner. Both, LLE-AMs against CD19 and CD20, were capable of inducing significant NK-92-mediated lysis against the NHL cell lines Raji, Daudi and JeKo-1. Cytotoxicity experiments using aCAR NK-92 cells and primary lymphoma cells are ongoing. Specificity of the LLE-AM directed effector cell elimination was further proven using a JeKo-1 CD19 and/or CD20 knock out (KO) antigen-loss model. aCAR NK-92 mediated antigen specific lysis only in the presence of the target antigen and the specific LLE-AM. Moreover, combinations of anti-CD19 and anti-CD20 LLE-AMs are capable of avoiding antigen evasion. To test the universal applicability of aCAR NK-92, specific target cell lysis against a variety of different tumor entities was demonstrated using LLE-conjugated therapeutic antibodies. Importantly, irradiation of effector cells, as required in all active clinical trials using NK-92, prior to testing had no observable effect on target cell lysis. Finally, the investigation of potential on-target off-tumor reactivity against healthy B cells showed no cytotoxic effects of aCAR NK-92 cells in combination with LLE-AMs against CD19 or CD20. In conclusion, we have generated an NK cell line, aCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens allowing tunable and universal targeting. Moreover, aCAR NK-92 cells can be manufactured as an "off-the-shelf on-demand" standardized product improving the practicality of NK CAR therapy combined with the possibility of tailoring a specific LLE-AM platform for patient-individualized treatment. Disclosures Seitz: Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding.