Anti-PSMA CAR-engineered NK-92 Cells: An Off-the-shelf Cell Therapy for Prostate Cancer

Abstract

Prostate cancer (PCa) has become the most common cancer among males in Europe and the USA. Adoptive immunotherapy appears a promising strategy to control the advanced stages of the disease by specifically targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the treatment of hematological malignancies, solid tumors still represent a challenge. To overcome current limits, other cellular effectors than T lymphocytes are under study as possible candidates for CAR-engineered cancer immunotherapy. A novel approach involves the NK-92 cell line, which mediates strong cytotoxic responses against a variety of tumor cells but has no effect on non-malignant healthy counterparts. Here, we report a novel therapeutic approach against PCa based on engineering of NK-92 cells with a CAR recognizing the human prostate-specific membrane antigen (PSMA), which is overexpressed in prostatic neoplastic cells. More importantly, the potential utility of NK-92/CAR cells to treat PCa has not yet been explored. Upon CAR transduction, NK-92/CAR cells acquired high and specific lytic activity against PSMA-expressing prostate cancer cells in vitro, and also underwent degranulation and produced high levels of IFN-γ in response to antigen recognition. Lethal irradiation of the effectors, a safety measure requested for the clinical application of retargeted NK-92 cells, fully abrogated replication but did not impact on phenotype and short-term functionality. PSMA-specific recognition and antitumor activity were retained in vivo, as adoptive transfer of irradiated NK-92/CAR cells in prostate cancer-bearing mice restrained tumor growth and improved survival. Anti-PSMA CAR-modified NK-92 cells represent a universal, off-the-shelf, renewable, and cost-effective product endowed with relevant potentialities as a therapeutic approach for PCa immunotherapy.

Keywords: CAR; NK-92 cell line; PSMA; cancer immunotherapy; prostate cancer.

Figure 1

Anti-PSMA (prostate-specific membrane antigen) CAR (chimeric antigen receptors)-engineered NK-92 cells acquire high and specific cytotoxicity to antigen-expressing cancer cells. (A) CAR surface expression on a NK-92/CAR eGFP-based sorted and enriched population was determined by flow cytometry with an antibody to a Myc tag present in the CAR moiety [19]. Left and central panels, eGFP and c-myc expression in the sorted population, respectively (open areas); parental NK-92 cells for eGFP expression and an isotype antibody for c-myc analysis served as controls (shaded areas). Right dot plot reports the events gated on total viable cells. (B) Cytotoxicity by NK-92/CAR (open squares) was investigated in ⁵¹Cr-release assays using as targets PSMA-negative K562 and PC3 cells (upper panels), and PSMA-expressing LNCaP and PC3 (bottom panels) cells. Parental NK-92 cells were included for comparison (full circles). Results are reported as mean values ± SD (n = 3) at different effector to target (E/T) ratios.

Figure 2

The irradiation of CAR-transduced or parental NK-92 cells fully blocks the proliferation but not impinges on phenotype. (A) Viability of NK-92 (left panel) and NK-92/CAR (right panel) cells undergoing or not γ-irradiation at 5 or 10 Gy. Proliferation was analyzed by counting viable cells at the indicated time points using trypan blue exclusion. Results are reported as mean values ± SD of 3 independent experiments. (B) Surface expression of different NK (natural killer) cells’ activation/inhibition receptors (CD85j, NKG2D, CD158d, and CD27) after γ-irradiation was analyzed by flow cytometry staining with appropriate antibodies. Solid lines refer to NK-92/CAR cells, while dotted lines represent parental counterparts.

Figure 3

Degranulation and IFN-γ secretion by irradiated NK-92/CAR cells upon engagement with PSMA-expressing prostate cancer cells. (A) Degranulation of NK-92/CAR cells (solid lines) was analyzed by flow cytometry assessment of CD107a surface expression after 5h of co-culture with PC3 or PC3-PSMA cells. Parental NK-92 cells (dotted lines) were included for comparison. Effector cells kept alone (shaded areas) or stimulated with PMA/ionomycin served as basal and positive controls, respectively. (B) IFN-γ release was analyzed in supernatants of NK-92/CAR cells stimulated with PSMA-negative or -positive cancer cell lines. NK-92/CAR cells treated with PMA/ionomycin served as positive controls. Parental NK-92 cells were included for comparison. Results are reported as mean values ± SD of 3 independent experiments. * p < 0.05.

Figure 4

Lytic activity of irradiated NK-92/CAR cells before and after NKG2D blocking. (A) Cytotoxicity of 10 Gy-irradiated NK-92/CAR (open squares) and NK-92 (filled circles) cells was investigated against K562, PC3, LNCaP, and PC3-PSMA cells, at different E/T ratios. (B) To evaluate the effects of NKG2D blocking, irradiated NK-92/CAR (open squares) and parental NK-92 (filled squares) cells were pre-treated with anti-NKG2D antibody for 1 h before incubation with K562, PC3, LNCaP, and PC3-PSMA targets. Irradiated non-blocked CAR-transduced (open circles) and wild-type (filled circles) effectors were included for comparison. Lysis data are shown as mean values ± SD of 3 independent experiments.

Figure 5

Assessment of NK-92/CAR cells’ in vivo therapeutic efficacy against locally implanted prostate cancers. (A) Winn assay. PC3-PSMA (left panel) and PC3 (right panel) cancer cells were inoculated s.c. in NSG (NOD/SCID common γ chain knockout) mice alone (filled triangles), or mixed with NK-92/CAR (filled squares) or parental NK-92 (filled circles) cells. Tumor growth was monitored over time by caliper measurement (6 mice/group). (B) Therapeutic activity against established tumors. NK-92/CAR cells or untransduced counterparts were administered i.v. in NSG mice for 3 times at alternate days, starting 4 days after s.c. injection of PC3-PSMA (left panel) or PC3 (right panel) tumor cells. Untreated animals served as control group. Symbols and groups as in a. Data are reported as mean values ± SD of tumor volumes at different time points.

Figure 6

Antitumor activity of NK-92/CAR cells against orthotopic PC3-PSMA xenografts. (A) Representative schedule of the experiment. Luciferase-expressing PC3-PSMA cells were injected into the anterior prostatic lobe of NSG mice. Starting from two days later, mice received i.v. injections of NK-92/CAR or parental NK-92 cells thrice per week for 4 weeks. Control mice were treated with PBS, and tumor growth was monitored by BLI. (B) Representative images of mice from experimental groups (one mouse/group) at day 28. (C) Graph reports cumulative results of the regions of interest (ROI) in total body. Tumor growth was monitored as total flux (ph/sec). Graphs show mean ± SD of three independent experiments. (D) Cumulative Kaplan–Meier survival curves of NSG mice (untreated mice, black line; NK-92 treated mice, red line; NK-92/CAR treated mice, blue line). * p < 0.05, ** p < 0.01 and *** p < 0.001.