ABSTRACT1. Grouper are of considerable economic value in tropical and subtropical regions and most particularly in south-east Asia. Moreover, it is in Asia, where this fish is highly prized, that the grouper farming began in the 1980s.2. The organoleptic qualities of the groupers are not the only characteristics to be appreciated; in culture, their robustness in heavily populated conditions, as well as their rapid growth at elevated temperatures makes them a good species for aquaculture. However, it is, above all, market demand that is outstripping the supply of fish, which is motivating the expansion of grouper aquaculture.3. At the present time, aquaculture is unable to satisfy demand. The chronic shortage of broodstock means that the alevins, destined for aquaculture, are taken mainly from natural habitats with the aid of fish traps, nets and cyanide, thus contributing to the destruction of south-east Asian reefs. Nevertheless, research is progressing and the number of species whose reproduction is totally controlled is increasing. It is in Taiwan that grouper aquaculture is at its most advanced with a total of five species being raised. 4. Aquaculture is divided into four stages, carried out by different farmers, allowing a more rapid turnover for each farmer and therefore a better financial return. On the other hand, in the Mediterranean at present time, there are no completely controlled grouper captive-breeding programmes. The first attempts at controlled breeding have been carried out since 1995 in Italy, Croatia and Greece. However, farming groupers in captivity, and in particular the control of their reproduction, poses numerous problems.
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 (> 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 >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.
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