Two for one: targeting BCMA and CD19 in B-cell malignancies with off-the-shelf dual-CAR NK-92 cells

Abstract

Background Chimeric antigen receptor (CAR) T-cell therapy has proven to be a valuable new treatment option for patients with B-cell malignancies. However, by applying selective pressure, outgrowth of antigen-negative tumor cells can occur, eventually resulting in relapse. Subsequent rescue by administration of CAR-T cells with different antigen-specificity indicates that those tumor cells are still sensitive to CAR-T treatment and points towards a multi-target strategy. Due to their natural tumor sensitivity and highly cytotoxic nature, natural killer (NK) cells are a compelling alternative to T cells, especially considering the availability of an off-the-shelf unlimited supply in the form of the clinically validated NK-92 cell line. Methods Given our goal to develop a flexible system whereby the CAR expression repertoire of the effector cells can be rapidly adapted to the changing antigen expression profile of the target cells, electrotransfection with CD19-/BCMA-CAR mRNA was chosen as CAR loading method in this study. We evaluated the functionality of mRNA-engineered dual-CAR NK-92 against tumor B-cell lines and primary patient samples. In order to test the clinical applicability of the proposed cell therapy product, the effect of irradiation on the proliferative rate and functionality of dual-CAR NK-92 cells was investigated. Results Co-electroporation of CD19 and BMCA CAR mRNA was highly efficient, resulting in 88.1% dual-CAR NK-92 cells. In terms of CD107a degranulation, and secretion of interferon (IFN)-γ and granzyme B, dual-CAR NK-92 significantly outperformed single-CAR NK-92. More importantly, the killing capacity of dual-CAR NK-92 exceeded 60% of single and dual antigen-expressing cell lines, as well as primary tumor cells, in a 4h co-culture assay at low effector to target ratios, matching that of single-CAR counterparts. Furthermore, our results confirm that dual-CAR NK-92 irradiated with 10 Gy cease to proliferate and are gradually cleared while maintaining their killing capacity. Conclusions Here, using the clinically validated NK-92 cell line as a therapeutic cell source, we established a readily accessible and flexible platform for the generation of highly functional dual-targeted CAR-NK cells.

Clinical application and prospect of immune checkpoint inhibitors for CAR-NK cell in tumor immunotherapy

“…mRNA transfection is also considered to be a safe and practical transfection method for CAR-NK cells. In a xenograft tumor model, receptor expression exceeded 80% at 24h after electroporation of mRNA, and NK cells transfected with mRNA exerted marked cytotoxicity ( 92 ). Studies have recently shown that mRNA transfection avoids targeted non-tumor toxicity, a major limiting factor in the clinical application of CAR-modified cell immunotherapy ( 92 , 93 ).…”

“…In a xenograft tumor model, receptor expression exceeded 80% at 24h after electroporation of mRNA, and NK cells transfected with mRNA exerted marked cytotoxicity ( 92 ). Studies have recently shown that mRNA transfection avoids targeted non-tumor toxicity, a major limiting factor in the clinical application of CAR-modified cell immunotherapy ( 92 , 93 ). However, the anti-tumor effect of CAR-NK cells transfected with mRNA by electroporation is temporary because the expression of CARs does not exceed 3 days.…”

Anti-BCMA novel therapies for multiple myeloma

“…A good efficacy has been demonstrated in early-phase clinical trials with bispecific CAR-Ts that target BCMA, CD19, or CD38 [112] . Future alternative approaches could be represented by allogenic BCMA CAR-T cells or CAR-NK (CARnatural killer), which are now investigated in early clinical trials [113][114][115][116][117][118][119][120][121] .…”

Chimeric antigen receptor-engineered NK cells: new weapons of cancer immunotherapy with great potential

“…There are a series of activated receptors but almost no inhibitory killer receptors expressed on NK-92 cells, and NK-92 cells are unable to mediate ADCC because of shortage of the CD16 receptor [ 143 – 145 ]. CAR-engineered NK-92 cells have strong cytotoxicity and can obtain a large number of cells with the same phenotypes in a short time [ 86 , 146 ]. The disadvantage of CAR-NK92 cells is that they must be irradiated to avoid malignant proliferation before infused into patients [ 83 ].…”