NK cells engineered with a IL-15/IL-15Ra complex and CD19-targeted CAR show in vitro expansion and tumor regression in a murine xenograft model of B-cell leukemia in vivo

Natural killer (NK) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers and unlike T cells, offer their use in allogeneic settings. The translation of NK cells into the clinic is still hampered by the inability of CAR-NK cells to expand in vitro and in vivo. Adoptive transfer of CAR-NK cells holds the promise of therapeutic benefit with a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, adoptive transfer of CAR-NK cells has not yet achieved breakthrough clinical results, and further improvement of CAR-NK cells is necessary. Here, we demonstrated that a fourth-generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Ra (CAR.19-IL-15/IL-15Ra) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model. Surprisingly, CAR construct comprising the IL-15/IL-15Ra was superior to CAR co-expressing the soluble form of IL-15 in terms of sustained proliferation, viability, but importantly also in their ability to control CD19+ B-cell lymphoma in a murine xenograft model. Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Ra comprising fourth-generation CARs may overcome the limitations of NK cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals Overall design: Natural Killer 92 (NK-92) cells were maintained in serum free X-VIVO 10 media (Lonza, Cologne, Germany) containing 5% human heat inactivated AB plasma (Brazilian Blood Donation Service of Hemocentro de Ribeirao Preto), supplemented with 500 IU/ml IL-2 (Clinigen), as previously described (17,18) . Human leukemia cell lines K562, Raji and NALM-6 were cultured in RPMI medium (ThermoFisher Scientific) supplemented with 10% Fetal Bovine Serum (FBS). All cell lines were tested negative for mycoplasma using the MycoAlertPLUS Mycoplasma Detection Kit (Lonza) (reference value < 1.0).NK cells were cocultured with Raji cell 10:1 ratio (E:T) for 24h at 37°C, enough time to eliminate target cells. Total RNA from NK cells was isolated from two biological replicates. In short, RNA was isolated using RNeasy kit (Qiagen) according to manufacturer's protocol. Poly(A) RNA sequencing library was prepared following Illumina's TruSeq-stranded-mRNA protocol and conducted by LC Sciences (Houston, TX, USA). Poly(A) tail-containing mRNAs were purified using oligo-dT Briefly, magnetic beads with two rounds of purification, and fragmented using divalent cation buffer at elevated temperature. Quality control analysis and quantification of the sequencing library were performed using an Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing was performed on Illumina's NovaSeq 6000. For transcript assembly and estimating transcript expression levels, software is described by Figueiredo et al. (2020) (21). For differential expression analysis of mRNAs, StringTie was used by calculating fragments per kilobase million (FPKM). The differentially expressed mRNAs were selected with log2 (fold change) > 1.5 or log2 (fold change) < -1.5 and with statistical significance (p < 0.05) by edgeR.