Generation of an NFκB-Driven Alpharetroviral “All-in-One” Vector Construct as a Potent Tool for CAR NK Cell Therapy

Abstract

Immune cell therapeutics are increasingly applied in oncology. Especially chimeric antigen receptor (CAR) T cells are successfully used to treat several B cell malignancies. Efforts to engineer CAR T cells for improved activity against solid tumors include co-delivery of pro-inflammatory cytokines in addition to CARs, via either constitutive cytokine expression or inducible cytokine expression triggered by CAR recognition of its target antigen—so-called “T cells redirected for universal cytokine-mediated killing” (TRUCKs) or fourth-generation CARs. Here, we tested the hypothesis that TRUCK principles could be expanded to improve anticancer functions of NK cells. A comparison of the functionality of inducible promoters responsive to NFAT or NFκB in NK cells showed that, in contrast to T cells, the inclusion of NFκB-responsive elements within the inducible promoter construct was essential for CAR-inducible expression of the transgene. We demonstrated that GD2CAR-specific activation induced a tight NFκB-promoter-driven cytokine release in NK-92 and primary NK cells together with an enhanced cytotoxic capacity against GD2+ target cells, also shown by increased secretion of cytolytic cytokines. The data demonstrate biologically relevant differences between T and NK cells that are important when clinically translating the TRUCK concept to NK cells for the treatment of solid malignancies.

The prospect of genetically engineering natural killer cells for cancer immunotherapy

“…A more optimal control strategy is the use of an inducible promoter to regulate cytokine expression. A recent ‘proof-of-principle’ work focused on transducing NK-92 and primary NK cells with a vector containing constitutively expressed disialoganglioside GD2-specific CAR and inducible human IL-12 (hIL-12) regulated by NFκB (iIL-12/GD2.CAR-NK cells) ( Rudek et al, 2021 ). NFκB is a signaling molecule expressed in activated NK cells ( Goodier et al, 2016 ).…”

“…The ideal gene delivery method has to be safe and efficient. Retroviral transduction is often used to introduce CAR constructs into expanding primary NK cells ( Christodoulou et al, 2021 ; Liu et al, 2018 ; Rudek et al, 2021 ) and NK-92 cells ( Klopotowska et al, 2021 ; Rudek et al, 2021 ). Being capable to infect both non-dividing and dividing cells ( Naldini et al, 1996 ), lentivirus can also be employed to deliver PD-L1 CAR constructs into NK92 cells ( Klopotowska et al, 2021 ), and high-affinity, cleavage-resistant CD16 sequences into iPSC-derived NK cells and primary NK cells ( Zhu et al, 2020a , b ).…”

“…However, the efficiency to transduce primary NK cells is generally low with retroviruses ( Xie et al, 2020 ), i.e. the transduction efficiency with alpharetroviral vector into primary NK cells is around 30% ( Rudek et al, 2021 ), and the random insertion of transgene potentiates harmful mutations ( Xie et al, 2020 ). Therefore, non-viral alternatives are appealing to deliver the foreign construct.…”

Determinants of Retroviral Integration and Implications for Gene Therapeutic MLV—Based Vectors and for a Cure for HIV-1 Infection

“…Viral vectors can also be derived from other genera of retroviruses, such as alphaviruses. Due to the more random integration profile of alpharetroviruses [ 5 , 6 , 7 , 8 , 102 ], a safer profile for alpharetrovirus–based vector delivery systems has been predicted, which has resulted in the development of SIN alpharetrovirus–derived vectors [ 6 , 184 , 185 , 186 , 187 , 188 , 189 , 190 ]. These vectors have no preferential targeting for TSSs, transcribed genes, CpG islands, conserved non–coding regions, elements enriched in transcription factor binding sites, promotor regions, enhancer regions, repetitive elements and, most importantly, oncogene bodies [ 6 ].…”

Gene-edited and CAR-NK cells: Opportunities and challenges with engineering of NK cells for immunotherapy

“… 58 , 59 Engineering of CAR-NK cells with alpharetroviral vectors has been successful, with high efficiency of transduction. 60 …”