Adapter Chimeric Antigen Receptor (aCAR)-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Universal Tumor Targeting
Authors: Grote Stefan , Seitz Christian M. , Diepold Simon , Buchner Markus , Baden Caroline , Malenke Elke , Dieckmann Sophie Marie , Schwaemmle Hanna , Mittelstaet Joerg , Kaiser Andrew , Schlegel Patrick , Handgretinger Rupert , Schleicher Sabine 
Published: October 2019

Abstract

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.