NK92 Expressing Anti-BCMA CAR and Secreted TRAIL for the Treatment of Multiple Myeloma: Preliminary In Vitro Assessment

Abstract

Multiple myeloma (MM) has witnessed improved patient outcomes through advancements in therapeutic approaches. Notably, allogeneic stem cell transplantation, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies have contributed to enhanced quality of life. Recently, a promising avenue has emerged with chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA), expressed widely on MM cells. To mitigate risks associated with allogenic T cells, we investigated the potential of BCMA CAR expression in natural killer cells (NKs), known for potent cytotoxicity and minimal side effects. Using the NK-92 cell line, we co-expressed BCMA CAR and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) employing the piggyBac transposon system. Engineered NK cells (CAR-NK-92-TRAIL) demonstrated robust cytotoxicity against a panel of MM cell lines and primary patient samples, outperforming unmodified NK-92 cells with a mean difference in viability of 45.1% (±26.1%, depending on the target cell line). Combination therapy was explored with the proteasome inhibitor bortezomib (BZ) and γ-secretase inhibitors (GSIs), leading to a significant synergistic effect in combination with CAR-NK-92-TRAIL cells. This synergy was evident in cytotoxicity assays where a notable decrease in MM cell viability was observed in combinatorial therapy compared to single treatment. In summary, our study demonstrates the therapeutic potential of the CAR-NK-92-TRAIL cells for the treatment of MM. The synergistic impact of combining these engineered NK cells with BZ and GSI supports further development of allogeneic CAR-based products for effective MM therapy.

Keywords: allogenic; cancer; chimeric antigen receptor; immunotherapy; multiple myeloma; natural killer.

Figure A1

Overview of the Synergistic Combination Therapy: In our proposed approach, we have genetically modified the NK-92 cell line to express both the anti-BCMA CAR receptor and soluble TRAIL to induce apoptosis in multiple myeloma cells by targeting BCMA and the TRAIL receptor, respectively. Combination therapy with γ-secretase inhibitor and bortezomib improved the expression of the therapeutic targets on myeloma cells, significantly enhancing the killing potential of the proposed NK cell model.

Figure 1

Validation of the CAR-NK92-TRAIL construct. (A) The expression of the anti-BCMA CAR, sTRAIL, and Nluc-GFP is driven by the CMV promoter. The coding sequences are spaced by 2A self-cleaving peptides to permit the whole expression under a unique promoter. The piggyBac LTR flanking sequences ensure the integration of the transgene in the host cell genome under the action of the piggyBac transposase. (B) Fluorescence microscopy image of the CAR-NK92-TRAIL cells (GFP—expressed from the transgene; RFP—conjugated to the BCMA-APC protein bound to the anti-BCMA CAR expressed at the surface of NK92 cells). Scale bar—25 μm. (C) Flow cytometry analysis of the GFP and anti-BCMA CAR expression by CAR-NK92-TRAIL.

Figure 2

Characterization and cytotoxic effect of CAR-NK92-TRAIL against different cancer cell lines. (A) The expression of phenotypic markers (CD56, CD16, NKG2A, NKG2C, NKG2D) in both wt-NK92 and CAR-NK92-TRAIL cells. (B) Viability of the MM (RPMI-8226, MM1.S, U266, KMS-12-PE) after 24 h co-culture with wt-NK92/CAR-NK92-TRAIL cells (1:2 E:T ratio). (C–E) The expression of activation markers after 24 h co-culture with the panel of targets at (1:2 E:T ratio). Control: no target cells (F) Quantification of IFN-γ produced by NK cells after 24 h co-culture with the panel of targets (1:2 E:T ratio). Control: no target cells. Data are presented as means ± SD from three technical replicates. ns—not significant; * = p < 0.01; ** = p < 0.05; *** = p < 0.001; one-way ANOVA, repeated measures test and Student’s t-test.

Figure 3

MM sensitization to TRAIL by BZ (A,B) DR5 (TRAIL-receptor) expression by MM cells after 24 h incubation with BZ (10 nM (MM1.S, U266) or 15 nM (RPMI-8226, KMS-12-PE) concentration). Mean fluorescence intensity (A) and positive populations (B) are indicated. (C–F) TRAIL-mediated lysis of MM cell lines after 24 h sensitization with BZ. Cells were exposed to recombinant TRAIL protein at variable concentrations and after 24 h, cell viability was assayed. Data are presented as means ± SD from three technical replicates. ns—not significant; * = p < 0.01; ** = p < 0.05; *** = p < 0.001; one-way ANOVA, repeated measures test and Student’s t-test.

Figure 4

Improvement of BCMA exposure by the action of GSI and combination treatment assay (A,B) BCMA expression by MM cell lines, after 24 h incubation with γ-secretase inhibitor (GSI; 1 mM). Mean fluorescence intensity (A) and positive populations (B) are indicated. (C) Concentration of BCMA shed by MM cells in 24 h culture (1.5 × 10⁵ cells, 500 μL of medium) with different concentrations of GSI (0; 1 mM; 10 mM). Duplicates are represented with dots, with bars as means. (D) Specific lysis of MM cells in 4 h co-culture with CAR-NK92-TRAIL cells (1:1 E:T ratio). Prior assay, MM cell lines were incubated for 24 h with BZ, GSI, or BZ and GSI together. Data are presented as means ± SD from three (A,B,D) resp. two (C) technical replicates. ns—not significant; * = p < 0.01; ** = p < 0.05; *** = p < 0.001; one-way ANOVA, repeated measures test and Student’s t-test.

Figure 5

Efficiency of the engineered CAR-NK92-TRAIL cells against primary cells (A) Diagram depicting the obtention of primary MM cells from newly diagnosed MM patients. (B) Data of patients (n = 5) from whom primary MM cells were obtained. The percentage of aberrant plasmatic cells within the FACS-sorted CD138+ population is numbered in the “aPCs [%]” column. (C) Percentage of DR5+ (TRAIL-R) and BCMA+ cells within the aPC population (n = 5). (D) DR5 and BCMA mean expression by positive cells (MFI) (n = 5). (E) Cytotoxic effect of the wt-NK92 and CAR-NK92-TRAIL cells against primary MM cells (4 h assay; 1:1 and 5:1 E:T ratios). Data are presented as means ± SD from three technical replicates. ns—not significant *** = p < 0.001; one-way ANOVA, repeated measures test and Student’s t-test.