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bioRxiv · 10.1101/2021.09.23.461509

Engineering CAR-NK cells to secrete IL15 sustains their anti-AML functionality, but is associated with systemic toxicities

Christodoulou, I.·Ho, W. J.·Marple, A.·Ravich, J. W.·Tam, A.·Rahnama, R.·Fearnow, A.·Rietberg, C.·Yanik, S.·Solomou, E. E.·Varadhan, R.·Koldobskiy, M. A.·Bonifant, C. L.
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Abstract

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. NK cells can elicit an anti-leukemic response via a repertoire of activating receptors that bind AML surface ligands. NK cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity, and interleukin-15 (IL15) to enhance their persistence. MethodsWe characterized in detail NK cell populations expressing a panel of AML (CD123)-specific CARs and/or IL15 in vitro and in AML xenograft models. ResultsCARs with 2B4.{zeta} or 4-1BB.{zeta} signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.{zeta} CAR-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.{zeta} CAR-NK cells. However, the benefit was transient due to limited CAR-NK cell persistence. Transgenic expression of secretory (s)IL15 in 2B4.{zeta} CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK cell subsets. 2B4.{zeta}/sIL15 CAR and sIL15 NK cells maintained an overall activated NK cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK cell groups. In vivo, 2B4.{zeta}/sIL15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.{zeta}/sIL15 CAR and sIL15 NK cells induced lethal toxicity in a second model. ConclusionTransgenic expression of CD123-CARs and sIL15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early phase clinical testing. O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/461509v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@4d09eaorg.highwire.dtl.DTLVardef@1c61411org.highwire.dtl.DTLVardef@14f8097org.highwire.dtl.DTLVardef@8375ac_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefSecretion of IL15 from anti-CD123.2B4.CD3{zeta} CAR-NK cells enhances their activation and killing potential against Acute Myelogenous Leukemia, but carries the risk of severe systemic toxicity.

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Christodoulou, I., Ho, W. J., Marple, A., Ravich, J. W., Tam, A., Rahnama, R., Fearnow, A., Rietberg, C., Yanik, S., Solomou, E. E., Varadhan, R., Koldobskiy, M. A., Bonifant, C. L.. 2021-09-24. Engineering CAR-NK cells to secrete IL15 sustains their anti-AML functionality, but is associated with systemic toxicities. https://doi.org/10.1101/2021.09.23.461509

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