Entinostat, a histone deacetylase inhibitor, enhances CAR-NK cell anti-tumor activity by sustaining CAR expression

Abstract

Allogeneic natural killer (NK) cell therapy has demonstrated significant potential in cancer immunotherapy by harnessing NK cells to target malignancies. CD138-targeting chimeric antigen receptor (CAR)-engineered NK cells offer a promising therapeutic option for multiple myeloma (MM). However, sustaining CAR expression on CAR-NK cells during ex vivo expansion poses a challenge to developing effective immunotherapies. In this study, primary NK cells were isolated, cryopreserved, and modified to express anti-CD138 CARs through retroviral transduction. Histone deacetylase inhibitors (HDACi), particularly entinostat (ENT), were applied to enhance CAR expression stability in CAR-NK cells. Our findings indicate that ENT treatment significantly improves and maintains CAR expression, thereby enhancing the cytotoxic activity of CAR-NK cells against CD138-positive multiple myeloma cells. ENT-treated CAR-NK cells exhibited prolonged persistence and more significant tumor reduction in an MM tumor-bearing mouse model, highlighting the therapeutic potential of HDACi-treated CAR-NK cells. This study provides the first evidence that HDAC inhibitors can sustain CAR expression in CAR-NK cells in a promoter-dependent manner, potentially enhancing anti-tumor efficacy in multiple myeloma and underscoring the possible need for further clinical evaluation.

Keywords: CD138; chimeric antigen receptor; cryopreservation; entinostat; genetic engineering; histone deacetylase inhibitors; multiple myeloma; natural killer cells.

Figure 1

Cryopreserved master stock NK cells can be expanded and engineered for CAR therapy. (A) The schematic diagram for expansion of cryopreserved NK cells. (B) NK cell expansion folds at two-day intervals (n=3 donors’ NK cells) (C) NK cell purity analysis from expanded NK cells (n=3 donors’ NK cells). (D) The timeline for NK cell engineering and expansion. (E) Expansion folds in UT and chimeric antigen receptor (CAR) engineered NK cells. (n=2 donors’ NK cells with triplicate transduction) (F) CAR expression on days 5 and 12 after the transduction (TD + 5 and TD + 12). (n=2 donors’ NK cells with triplicate transduction); UT, Untransduced; Cryo, Cryopreserved NK cells.

Figure 2

Functionality and CAR expression of anti-CD138-CAR engineered NK cells. (A) Design of the anti-CD138 CAR construct. (B) GFP and CAR expressions in engineered NK92 cells. (C) The CD107a and IFN-γ-expression patterns in modified NK92 cells co-cultured with CD138-positive target MM1.R and CD138-negative target MDA-MB-231 cells. (D) CD107a and IFN-γ expressions in NK92 cells with or without target cells. (E) NK92 cell killing assay with CD138 positive and negative target cells (F) Killing assay using engineered pNK cells with MM1.S (CD138-positive, CD138^Pos) and Raji (CD138-negative, CD138^Neg) cells. (n=2 donors’ NK cells) (G) Injected NK cell GFP and CAR expressions and timeline of the performed in vivo experiment. (H) In vivo imaging. Signals indicate firefly luciferase-expressing MM1.S cells. (I) CAR expressions in NK cells on TD + 5 and TD + 12 (n=3 cryopreserved donors’ NK cells).

Figure 3

Enhanced CAR expression in NK cells with the treatment of histone deacetylase inhibitors. (A) Effects of different concentrations of Histone deacetylase inhibitor (HDACi) in GFP+ CAR+ population. (n=2 donors’ NK cells) (B) CAR and GFP MFIs in HDACi-treated pNK cells. (n=2 donors’ NK cells) (C) ENT 300nM and 500nM-treated MSCV, EFS, and EFS-CAR-engineered pNK cells. (n=1 donor’s NK cells with triplicate transduction) (D) Puromycin-based NK cell translation capacity confirmation. Puromycin staining was performed with or without metabolic inhibitors, 2DG, and oligomycin. (n=1 donor’s NK cells) (E) GFP expressions in the cells tested for the puromycin staining; ENT, Entinostat; VPA, Valproic acid; RGFP, RGFP966; MSCV, Murine stem cell virus promoter; EFS, EF1-α small promoter; CMV, Cytomegalovirus promoter; 2DG, 2-Deoxy-D-glucose.

Figure 4

CAR maintenance, degranulation, and survival of ENT-treated NK cells in vitro. (A) GFP+ CAR+ population and CAR MFI analysis after overnight resting. (n=2 donors’ ENT 500nM-treated NK cells) (B) GFP+ CAR+ population maintenance analysis for five days post-ENT removal. (n=2 donors’ ENT 500nM-treated NK cells) (C) CD107a assay using ENT 500nM-treated NK cells. (n=2 donors’ NK cells) (D) NK cell survival without IL-2. NK cells were treated with IL-2 and with or without ENT 500nM, then both IL-2 and ENT were withdrawn to confirm their survival. (n=2 donors’ NK cells).

Figure 5

The CAR and NK cell phenotypes after long-term ENT treatment (A) CAR expression analysis during expansion ex vivo following CAR transduction in the presence of ENT (n=2 donors’ NK cells). (B) NK cell expansion for seven days with ENT. (n=4, 2 donors’ NK cells with or without engineering). NK cells were transduced, stimulated, and expanded for five days (TD + 5). The modified NK cells were then cultured with or without ENT. (C) Comparison of NK cell cytotoxicity between ENT 300nM-treated CAR NK cells and ENT-treated untransduced (UT) NK cells in vitro. (n=2 donors’ NK cells) (D) Examine cytokine production in DMSO or ENT 300nM-treated UT-NK cells. (n=2 donors’ NK cells) (E) Target cell killing activity of the UT-NK cells. (n=2 donors’ NK cells) (F) DMSO-treated TD + 12 NK cells were treated with 500nM ENT for two days (brief stimulation, bENT). NK cells showed improved GFP+ CAR+ populations (n=2 donors’ NK cells).

Figure 6

Anti-tumor activity of CAR-NK cells treated with ENT in vitro and in vivo. (A) GFP and CAR expressions in NK cells used for in vivo injection. (n=1 donor’s NK cells) (B) In vitro cytotoxicity of the expanded NK cells. Cytotoxicity was assessed on the day of the NK cell injection. (C) Schematic diagram of the in vivo experiment. (D) Newton imaging. The brighter color (blue to red) indicates the higher luminescence signals from MM1.S-FLUC cells. Marked mice were exposed for 30 seconds and not included in the statistical analysis. Other mice received 2 minutes of exposure. Red numbers indicate the analysis scale for the 2-minute exposed mice. (n=5 mice for NK cell treatments and n=2 mice for PBS control). (E) Analysis of total flux from each mouse. (F) Percentages of human CD45 positive populations in the blood on days 10, 16, and 22 post-MM1.S-FLUC cell injection. (G) GFP MFIs from the hCD45 positive cells. (H) Multiple myeloma tumor burden on day 29 in bone marrow (BM) and spleen (SP).; FLUC, Firefly luciferase; bENT 500nM, three days ENT 500nM treated NK cells; BM, Bone marrow. The days in (D–H) indicate the timeline post-MM1.S-FLUC cell injection.