Expanded Natural Killer (NK) Cells Transfected with Anti-CD20 Chimeric Antigen Receptor (CAR) mRNA Have Significant Cytotoxicity Against Poor Risk B-Cell (CD20⁺) Leukemia/Lymphoma (B-L/L).

The outcome for children and adolescents with B-L/L has improved significantly but for patients who relapse or progress, the prognosis is dismal due to chemo-immuno-radiotherapy resistance (Cairo, et al, J Clin Oncol, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Ruggeri L et al., Science 2002) but cell number and tumor specific recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Genetically engineered and expanded NK cells with an anti-CD19 CAR have been previously reported by Campana et al (Li L et al, Cancer Gene Ther. 2010). Anti-CD20 CAR transduced primary NK cells by retrovirus were reported by our group (Chu & Cairo, et al, ASH, 2011).

To generate large-scale, efficiently modified NK cells with low cost, clinical applicable and a non-virus method, we investigated the functional activities of anti-CD20 chimeric antigen receptor (CAR⁺) modified PBNK cells following mRNA nucleofection against CD20⁺ B-L/L.

PBMC were expanded with mitomycin C treated K562-mbIL15–41BBL cells for 7 or 14 days. CD56⁺CD3⁻ expanded PBNK (exPBNK) cells were isolated using Miltenyi NK cell isolation kit. CD56, CD3 and receptor expression were evaluated by flow cytometry. Anti-CD20-4-1BB-CD3ζ was subcloned into a pcDNA3 vector. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit from T7 promoter. CAR mRNA was nucleofected into exPBNK using Amaxa nucleofector. CAR expression was detected using FITC-conjugated goat anti-mouse IgG, F(ab')₂ fragment-specific antibody. exPBNK cytotoxicity was assessed by europium release assay at different E:T ratios against CD20⁺ B-L/L. CD107a degranulation and intracellular IFNgƒnproduction in exPBNK were measured by flow cytometry after stimulation with medium, K562-mbIL15–41BBL, CD20⁺ Ramos, CD20⁺Daudi, or CD20⁻ RS4;11 for 4–6 hrs.

CD56⁺CD3⁻ exPBNK cells were significantly expanded by mitomycin C treated K562-mbIL15–41BBL cells at day7. exPBNK cells were selected with more than 96% purity of CD56⁺CD3⁻. 50 to 95% exPBNK cells were detected to express CAR at 16 hrs after CAR mRNA nucleofection. CAR mRNA nucleofection did not affect the expression of exPBNK activating receptors (CD16, CD69, NKG2D, CD244, NKp30, NKp44, NKp46) or inhibitory receptors (NKG2A, KIR2DS4, CD94, CD158a, CD158b, CD158e). exPBNK in vitro cytotoxicity was significantly enhanced by CAR⁺ exPBNK compared to CAR⁻ exPBNK against CD20⁺ B-L/L at 10:1 (n>3): Ramos (97.25+ 2.61% vs 82.5+ 4.058%, p<0.05), Daudi (71.5+ 3.26% vs 36.34+ 6.31%, p<0.001), Raji (21.45+ 1.98% vs 6.94+ 5.64%, p<0.05), Raji-2R (a Rituximab resistant cell line) (96.39+ 1.03% vs 86.3+ 1.52%, p<0.01), and U-698-M (82.84+ 1.17% vs 26.2+ 0.776%, p<0.001). However, there was no significant difference against CD20⁻ RS4;11 or Jurkat cells. Consistently, CD107a degranulation was enhanced in CAR⁺ exPBNK compared to CAR⁻ exPBNK in response to CD20⁺ Ramos (31.47+ 1.74% vs 15.2+ 0.26%, p<0.001, n=3) and Daudi (38.9+ 2.7% vs 19.73+ 0.58%, p<0.001, n=3) stimulation, however, there was no significant difference in response to RS4;11 or medium. Intracellular IFNγ production was also enhanced in CAR⁺ exPBNK compared to CAR⁻ exPBNK in response to CD20⁺ Ramos and Daudi specific stimulation. We also observed that the expression of exPBNK activating receptors (CD69, NKp44 and NKG2D) were enhanced similarly and inhibitory receptors (CD94 and CD158b) were unchanged in mock exPBNK and CAR⁺ exPBNK cells after incubation with U-698-M compared to medium, implying CAR⁺PBNK directed enhanced cytotoxicity is mainly mediated by engineered CAR but not by endogenous NK receptors.

Anti-CD20 CAR expression in exPBNK cells by mRNA nucleofection was associated with a significant increase in CD107 degradulation and INF-g production after stimulated with CD20⁺ BL/L compared to mock exPBNK cells. Consequently, Anti-CD20 CAR expression in exPBNK cells results in significant and specific exPBNK in vitro cytotoxicity against CD20⁺ B-L/L. Future directions include examining CAR⁺ exPBNK cytotoxic activity against CD20⁺ primary B-L/L tumor cells isolated from patients and testing the anti-tumor activity of CAR⁺ exPBNK against B-L/L and animal survival in xenograft mice.

No relevant conflicts of interest to declare.