Abstract 1434: The Costimulatory Signal Domains 4-1BB and CD3ζ Do Not Improve the Function of CD16A Chimeric Antigen Receptor Transduced NK Cells
Authors: Chen Long , Li Vicky , Allan David , Reger Robert , Cherkasova Elena , Pierre Stephanie , Barisic Stefan , Granadier David , Levy Emily , Waller Giacomo , Doh Susan , Chakraborty Mala , Stringaris Kate , Childs Richard 
Published: August 2019

Abstract
Chimeric Antigen Receptors (CAR)-T cells are genetically modified to express an extracellular binding domain, transmembrane domain and intracellular signaling domain. The extracellular binding domain is usually derived from antibodies; however, it can be replaced with other ligand-receptor binding systems, such as with an Fc receptor. CD16A has been tested in second-generation 4-1BB-CD3ζ CAR T cells and NK cell lines (NK-92) and enhances tumor killing; however, it is not clear whether these cells provide superior tumor killing compared to cells modified to express CD16A alone.
In this study, we designed three CAR constructs containing the extracellular binding domain of high affinity mutant CD16A (CD16A-V158) and intracellular signal through 4-1 BB-CD3ζ that differed in leader sequences and transmembrane domains. CAR1 contained the CD16A leader and CD16A transmembrane domain; CAR2 had the CD16A leader and CD8α transmembrane domain; CAR3 contained the CD8α leader and CD8α transmembrane domain. When transduced into Jurkat cells via lentivirus, CD16A V158 and all three CARs showed stable high surface expression of CD16A (&gt; 70%). In contrast, NK-92 cells were only successfully transduced to express CD16 by CD16A V158 (29.8%), CAR2 (42.3%), and CAR3 (42.5%) with no expression of CD16A by CAR1. Transduced cells were sorted by FACs based on CD16A expression. All sorted cells showed &gt;90% stable expression of the constructs for up to 42 days post sorting. We observed a much higher CD16 MFI in cells transduced with CAR2 and CAR3 compared with CD16A V158. Western blotting with anti-CD3ζ identified the full assembly of CARs.
We then tested the function of the transduced cells by a Rituximab binding assay. Although there was no difference in binding to Rituximab at 0.01 and 0.1 mg/mL; CAR2 and CAR3 showed better binding at the 1 and 10 mg/mL. In a 4-hour coculture toxicity assay with 721.221 LCLs, CD16A V158 and CAR3 increased the target lysis by two-fold while CAR2 only showed a 50% increase. When cocultured with 721.221 HLA-E+ LCLs, a 721.221 subclone more resistant to killing by NK-92 cells, we observed similar tumor killing with CD16A V158 and CAR3 (killing doubled) in contrast to CAR2(killing increased by only 50%). We next tested against the B-cell line Raji and found that there was only a mild increase of target lysis with transduced NK-92 cells; however, the CD16A V158 and CAR3 still showed higher toxicity (around a 40% increase).
In summary, CAR2 and CAR3 expressed a higher CD16 MFI in NK cells and demonstrated better binding to Rituximab at1 and 10 mg/mL compared to CD16A V158; however, CAR3 and CD16A V158 had similar killing of target cells that was higher than was observed with CAR2. Our data indicate that NK cells transduced to have a CAR structure combining CD16A with 4-1BB-CD3ζ have augmented ADCC, although this augmentation is not superior to NK cells that are simply transduced to express CD16A alone.
Citation Format: Long Chen, Vicky Li, David Allan, Robert Reger, Elena Cherkasova, Stephanie Pierre, Stefan Barisic, David Granadier, Emily Levy, Giacomo Waller, Susan Doh, Mala Chakraborty, Kate Stringaris, Richard Childs. The Costimulatory Signal Domains 4-1BB and CD3ζ Do Not Improve the Function of CD16A Chimeric Antigen Receptor Transduced NK Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1434.