Pluripotent stem cell-derived CAR-macrophage cells with antigen-dependent anti-cancer cell functions

Abstract

The Chimera antigen receptor (CAR)-T cell therapy has gained great success in the clinic. However, there are still major challenges for its wider applications in a variety of cancer types including lack of effectiveness due to the highly complex tumor microenvironment, and the forbiddingly high cost due to the personalized manufacturing procedures. In order to overcome these hurdles, numerous efforts have been spent focusing on optimizing Chimera antigen receptors, engineering and improving T cell capacity, exploiting features of subsets of T cell or NK cells, or making off-the-shelf universal cells. Here, we developed induced pluripotent stem cells (iPSCs)-derived, CAR-expressing macrophage cells (CAR-iMac). CAR expression confers antigen-dependent macrophage functions such as expression and secretion of cytokines, polarization toward the pro-inflammatory/anti-tumor state, enhanced phagocytosis of tumor cells, and in vivo anticancer cell activity. This technology platform for the first time provides an unlimited source of iPSC-derived engineered CAR-macrophage cells which could be utilized to eliminate cancer cells.

Keywords: Anti-cancer cell functions; Antigen-dependent activation; Chimera antigen receptor (CAR); Induced pluripotent stem cells (iPSC)-derived macrophage cells (iMac).

Fig. 1

CAR-expressing iPSCs can differentiate into CAR-macrophage cells. a Overview of deriving CAR-iMacs from CAR-iPSCs. b Flow cytometry analysis of iPSC-derived cells at different stages of differentiation with stage-specific markers. c qRT-PCR showing pluripotent marker gene and key macrophage marker gene expression at different stages of CAR-iPSC differentiation. n = 3, error bar: standard error of the mean. d Hierarchical clustering of transcriptomes of CAR-iPSCs, their differentiated cells, and primary and untransduced iPSC-differentiated macrophages in different states. e Principal component analysis (PCA) of the same samples as in d. f Top GO terms enriched in genes up-regulated on day 28 differentiated CAR-iMac cells compared with CAR-iPSCs, and the right panel is an example of GSEA analysis of the GO terms. NES normalized enrichment score. P = 0: P value is a very small number. g UMAP plot showing separation between human iPSCs and CAR-iMac cells. h UMAP plot showing subpopulation clustering of CAR-iMac cells. Ten clustered C0–C9 were identified and labeled as 0–9 with different colors. i Heatmap showing blasting the C0-C9 clusters of cells illustrated in g against a human single-cell atlas database containing single-cell RNA-seq data of hundreds of cell types including macrophages (https://scibet.cancer-pku.cn). j Trajectory analysis of differentiated cells along a pseudotime axis. k Heatmap showing averaged expression of M1 or M2 signature pathway genes in different clusters of cells illustrated in i. l Heatmaps to compare (benchmark) the 10 clusters (C0-C9) of CAR-iMac cells against previously published M1 or M2 polarized macrophages using metabolism genes. Human iPS cells differentiated macrophages polarized by IFN-γ and LPS; IPS_M2: Human iPS cells differentiated macrophages polarized by IL-4; HM_M1: Human PBMC-derived macrophages polarized by IFN-γ and LPS; HM_M2: Human PBMC-derived macrophages polarized by IL-4

Fig. 2

CAR-iMac cells showed antigen-dependent phagocytosis and anticancer cell functions in vitro and in vivo. a Confocal microscopy pictures showing phagocytosis of K562 or K562-CD19 cells (red) by CAR (CD19)-iMac cells (green). b Flow cytometry showing phagocytosis of K562 or K562-CD19 cells by CAR (CD19)-iMac cells. c Western blotting showing phosphorylation of ERK and NF-κB P65 in CAR (CD19)-iMac cells in the indicated conditions. d qRT-PCR showing cytokine gene mRNA expression when CAR (CD19)-iMac cells were incubated with K562 or K562-CD19 cancer cells for 24 h. n = 3, error bar: standard error of the mean. e Top GO terms enriched in genes up-regulated in CAR-iMac cells. Right panel is GSEA analysis of “positive regulation of cytokine production.” f Top KEGG pathways enriched in genes up-regulated in CAR-iMac cells. Right panel is GSEA analysis of “antigen processing and presentation.” g Confocal microscopic images showing phagocytosis of OVCAR3 cells (red) by iMac or CAR (meso)-iMac cells (green). h Flow cytometry showing phagocytosis of OVCAR3 ovarian cancer cells by iMac or CAR (meso)-iMac cells. i GO term analysis with RNA-seq data showing the up-regulated genes in CAR (meso)-iMac cells. Right panel is GSEA analysis of “cytokine activity gene.” j qRT-PCR showing cytokine gene mRNA expression when iMac or CAR (meso)-iMac cells were incubated with OVCAR3 cells for 24 h. n = 3. Error bar: standard error of the mean. k 3 × 10⁶ DiR dye-labeled iMac cells were intraperitoneally injected into NSG mice. n = 3. Error bars represent standard error of the mean. l 4 × 10⁵ of luciferase-expressing ovarian cancer cells (HO8910) were intraperitoneally injected into NSG mice. Mice were treated 4 h later with I.P. injection of PBS, 4 × 10⁶ iMac or 4 × 10⁶ CAR (meso)-iMac cells. Bioluminescence showing tumor development on the indicated days. Statistical analysis was calculated via one-way ANOVA with multiple comparisons between the PBS group and the CAR-iMac group. *P < 0.05; **P < 0.01. m Quantification of tumor burden (total flux) by bioluminescent imaging on day 4, 11, 14, and day 25 after CAR-iMac treatment was plotted. Data are presented as the median ± SD, with statistical significance calculated via one-way ANOVA with multiple comparisons. *P < 0.05; **P < 0.01, ns not significant