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. 2018 Aug 2;23(2):181-192.e5.
doi: 10.1016/j.stem.2018.06.002. Epub 2018 Jun 28.

Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity

Ye Li  1 , David L Hermanson  2 , Branden S Moriarity  3 , Dan S Kaufman  4
Affiliations

Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity

Ye Li et al. Cell Stem Cell. .

Abstract

Chimeric antigen receptors (CARs) significantly enhance the anti-tumor activity of immune effector cells. Although most studies have evaluated CAR expression in T cells, here we evaluate different CAR constructs that improve natural killer (NK) cell-mediated killing. We identified a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling. NK cells derived from human iPSCs that express this CAR (NK-CAR-iPSC-NK cells) have a typical NK cell phenotype and demonstrate improved anti-tumor activity compared with T-CAR-expressing iPSC-derived NK cells (T-CAR-iPSC-NK cells) and non-CAR-expressing cells. In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells significantly inhibited tumor growth and prolonged survival compared with PB-NK cells, iPSC-NK cells, or T-CAR-iPSC-NK cells. Additionally, NK-CAR-iPSC-NK cells demonstrate in vivo activity similar to that of T-CAR-expressing T cells, although with less toxicity. These NK-CAR-iPSC-NK cells now provide standardized, targeted "off-the-shelf" lymphocytes for anti-cancer immunotherapy.

Keywords: chimeric antigen receptors; iPSCs; immunotherapy; natural killer cells; ovarian cancer.

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Conflict of interest statement

Competing Financial Interests

DSK has research funding and serves as a consultant for Fate Therapeutics. DSK, DLH, and BSM have filed a patent related to these studies.

Figures

Figure 1
Figure 1. CAR mediated anti-tumor activity in NK92 cells
(A) Surface expression of mesothelin (meso) in leukemia cell lines K562 and K562meso, ovarian cancer cell lines MA148 and A1847. (B–E) NK92 cells that express the indicated CARs were co-cultured with (B) mesoneg target cells of K562, (C) mesohigh target cells of K562meso, (D) mesolow target cells of MA148 cells, and (E) mesohigh target cells of A1847 at indicated effector to target ratios. The mean of % specific tumor cell lysis ± S.D are shown. (F and G) CD107a expression was determined in anti-CD56 labeled effector cells after stimulation by (F) K562 and K562meso cells, and (G) MA148 and A1847. Data were normalized to the percentage of media-treated effector cells, shown as mean ± S.D. Statistical analysis by two-tailed Student t-test, * P<0.05, ** P<0.001. See also Figure S1.
Figure 2
Figure 2. Domain-deficient CAR4-mediated NK cell activation response and cytolysis in NK92 cells
(A) Expression of GFP and surface expression of CAR4 in NK92 cells assessed by flow cytometry. (B) Schematic representation of the transposon vector encoding the domain-deficient CAR4 mutation TM: NKG2D (NKG2DΔ), CD: 2B4 (2B4Δ), SD: CD3ζ (CD3zΔ), and all domain-deficient mutated (ALLΔ). (C–F) NK92 cells were co-cultured with europium-loaded target cells of (C) mesoneg K562, (D) mesohigh K562meso, (E) mesolow MA148, and (F) mesohigh A1847 at indicated effector to target ratios. The mean of % specific tumor cell lysis ± S.D are shown. (G and H) CD107a expression, and IFN-γ production was analyzed in anti-CD56 labeled effector cells after co-culture with target cells. (I) Protein analysis of total protein and phospho-protein molecular of Syk, PLC-γ2 and Erk1/2 in cell lysate of NK92 cells by immunoblots in different conditions. Left: unstimulated; middle: 1-hour mesolow MA148 cell stimulation; right: 1-hour mesohigh A1847 cell stimulation. Anti-CD3ζ antibody was used to determine CAR expression with estimated molecular size. GAPDH and P-CK were used as loading control. (J and K) Quantification of phosphorylated level over total protein level in NK effector cells co-cultured with mesohigh A1847 target cells were performed in protein lysates stained for of (J) PLC-γ2, (K) Erk1/2. Data were shown as mean ± S.D. Statistics by two-tailed Students t-test: NS: not significant. * P<0.05, ** P<0.01,
Figure 3
Figure 3. Phenotype and anti-tumor activities of CAR-expressing iPSC derived NK cells
(A) Flow cytometric analysis of GFP, and NK cell surface receptors in the gate of CD56+ NK cell populations. (B) iPSC-NK cells derived from pooled or clonal CAR4(meso)-iPSC (#1 and #4), CAR4(-)-iPSC (#2 and #3) were co-cultured with europium-loaded K562meso cells (left), or A1847 cells (right) as different effector to target ratios. The mean of % specific tumor cell lysis ± S.D are shown. (C and D) CD107a expression (C), and IFN-γ production (D) was accessed by flow cytometery in anti-CD56 labeled iPSC-NK populations after the stimulation of K562meso or A1847. Data were plotted and shown as mean ± S.D. (E) Membrane protein analysis in cell lysate of iPSC-NK populations by immunoblots. NCAM and GAPDH were used as loading controls. (F) Co-IP was performed by using an anti-DAP-10 antibody in cell lysate of A1847 cell-stimulated iPSC-NK populations. Protein was subjected to the analysis of DAP-10, NKG2D, and CD3z by immunoblots. (G) Total and phospho-protein analysis of Fyn-PLC pathway (15 min A1847 stimulation); Syk-Vav1-Erk pathway and NF-κB (IKKα/β and IκBα) pathway (30 min A1847 stimulation) in cell lysate of iPSC-NK populations by immunoblots. Lane 1–4: unstimulated iPSC-NK cell populations as in (E); Lane 5–8: A1847 stimulated iPSC-NK cell populations as in (F). (H and I) Cytolysis ability of iPSC-NK populations, or T cell populations against (H) K562meso or (I) A1847 cells were quantified using the IncuCyte real-time imaging system over a 24 hour time-course. Percentage of caspase 3/7 event stained cells over the total pre-labeled cells were measured. Statistics by two-tailed Student t-test, * P<0.05, ** P<0.01. See also Figure S4 and S6.
Figure 4
Figure 4. CAR4-expressing iPSC derived NK cells display superior anti-ovarian cancer activity in vivo
(A) Schematic of in vivo studies using luciferase (luc)-expressing mesohigh A1847 cells in a mouse xenograft model treated with PB-NK cells and iPSC-NK populations and cytokine administration. (B) Tumor burden was determined by weekly bioluminescent imaging (BLI). (C and D) Images of representative time points are shown. Quantification of tumor burden on (C) day 7, and (D) day 28 was plotted based on BLI total flux (photons/sec). (E) Tumor burden of each group was monitored for 49 days post NK population infusion. The BLI data is plotted, mean ± S.D are shown. Statistic: two-tailed Student t-test, T-CAR(meso)-iPSC-NK vs. CAR4(meso)-iPSC-NK. (F) Kaplan-Meier curve representing the percent survival of the experimental groups: Tumor only, or treated with PB-NK cells, iPSC-NK cells, T-CAR(meso)-iPSC-NK cells, or CAR4(meso)-iPSC-NK cells. Statistic: two-tailed Log-rank test. (G–I) Flow cytometric quantification of CD45+CD56+CD3- NK cell population from (G) peripheral blood, (H) spleen, and (I) peritoneal fluid. Each dot represents one recipient mouse. Median ± S.D. is shown. Statistical analysis by two-tailed one-way ANOVA. *P<0.05, ** P<0.01, *** P<0.001. See also Figure S7A.
Figure 5
Figure 5. Comparison of CAR-expressing T cells to CAR-expressing NK cells
(A) NSG mice were inoculated intraperitoneally with 2 × 105 luc+ A1847 cells. 3 days later, mice received 225cGy radiation following with 1.0 × 107 of iPSC-NK or T cell populations infusion intraperitoneally. Cytokines were administrated as indicated in previous for the following 21 days in mice that received iPSC-NK population treatment only. Tumor burden was determined by weekly BLI imaging until day 21. Images of representative time points are shown. (B and C) Flow cytometric quantification of (C) CD45+CD3+ T cell populations and (B) CD45+CD56+CD3- iPSC-NK populations from peripheral blood. Each dot represents one recipient mouse. (D) BLI data of tumor burden of each group monitored for 21 days post immune effector cell infusion (dpi). (E) Body weight of each group was monitored and plotted weekly in the time-course for 21 days post immune effector cell infusion. Mean ± S.D. is shown. Statistical analysis by two-tailed one-way ANOVA, T cells vs. iPSC-NK cells, T CAR(meso)-T cells vs. CAR4(meso)-iPSC-NK. (F–H) Levels of (F) hIFN-γ, (G) hTNF-α, and (H) hIL-6 were measured on plasma collected from peripheral blood in different days post immune effector cells infusion by ELISA assay. Median ± S.D. is shown. Statistic: two-tailed one-way ANOVA. (I) Kaplan-Meier curve represents the percent survival of the experimental groups. Statistical analysis by two-tailed Log-rank test. n.d.: not detected, NS: not significant, *P<0.05, ** P<0.01, *** P<0.001. See also Figure S7B–S7D.
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