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. 2018 Oct;9(10):867-878.
doi: 10.1007/s13238-017-0384-8. Epub 2017 Mar 10.

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

Yanjing Song  1   2 , Chuan Tong  3 , Yao Wang  3 , Yunhe Gao  1   2 , Hanren Dai  3 , Yelei Guo  3 , Xudong Zhao  1   2 , Yi Wang  1   2 , Zizheng Wang  2 , Weidong Han  4   5 , Lin Chen  6
Affiliations

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

Yanjing Song et al. Protein Cell. 2018 Oct.

Abstract

Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 and CD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.

Keywords: CD137; HER2; cancer stem cell; chimeric antigen receptor; gastric cancer; immunotherapy.

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Figures

Figure 1
Figure 1
Generation, expansion, and characterization of HER2-directed chimeric antigen receptor T cells. (A) Schematic representation of anti-HER2 CAR gene sequence (not drawn to scale). (B) Average expansion (-fold) of CART-HER2, mock T, and NT T cells, produced using PBMCs from three healthy donors; the cells were cultured in vitro for 10–14 days. Mean and SDs are shown for three different T-cell lines. (C) Phenotypic features of CART-HER2 and NT T cells, from three healthy donors, were evaluated by FACS analysis on day 12 of culture. Mean positive rates ± SD from three different T-cell lines are shown. (D) Transfection efficiency of CAR or mock gene into T cells was determined by FACS analysis using the marker GFP on day 12. The data are represented as means ± SD. *Represents P < 0.05
Figure 2
Figure 2
Specific activity of HER2-directed chimeric antigen receptor T cells against HER2 + GC cells. (A) FACS was used to test the surface expression of HER2 proteins in a series of human GC cell lines, including N87, 7901, AGS, HGC27, MGC803, BGC823, MKN45, and primary GC cells from two patients with GC. (B) HER2 expression in N87 and 7901 cells was downregulated via transduction of lentivirus-mediated short hairpin RNA-HER2. The knockdown effects of HER2 expression in sorted GFP-positive cells were evaluated by FACS analysis. (C) The levels of cytokines, released by CART-HER2, mock T, and NT T cells, were measured by enzyme-linked immunosorbent assay (ELISA) after 4-h incubation with HER2high+ and HER2KD GC cells at an effector-to-target (E/T) ratio of 20:1. (D) The levels of cytokines, released by CART-HER2 and NT T cells, were measured by ELISA after 4-h incubation with patient-derived GC cells at an E/T ratio of 20:1. The data are represented as the mean cytokine concentrations ± SD (pg/mL) from triplicate cultures. NS represents no statistical significance, *represents P < 0.05, **represents P < 0.01
Figure 3
Figure 3
Specific cytotoxicity exhibited by HER2-directed chimeric antigen receptor T cells against HER2 + GC cell line and primary GC cells ex vivo. (A) The cytotoxic activity of CART-HER2, mock T, and NT T cells against HER2high+ and HER2KD GC cells was determined using a 4-h lactate dehydrogenase (LDH) release assay in a dose-dependent manner. HER2− primary GC cells were used as controls. (B) A 4-h LDH release assay, at an effector-to-target (E/T) ratio of 20:1, was used to compare cytotoxicity between CART-HER2, mock T, and NT T cells against different GC cells (C) An 8-h LDH release assay, at an E/T ratio of 20:1, was used to compare cytotoxicity between CART-HER2, mock T, and NT T cells against different GC cells. (D) Cytotoxicity comparisons of CART-HER2 cells against different GC cells were performed between the 4-h and 8-h LDH release assays at an E/T ratio of 20:1. All of the results are expressed as the mean of triplicate values ± SD. NS represents no statistical significance, **represents P < 0.01
Figure 4
Figure 4
Antitumor efficacy of HER2-directed chimeric antigen receptor T cells against xenografts derived from HER2 + GC cell lines in mice. (A) Two million HER2high+ 7901 cells were injected subcutaneously into the unilateral axillary region of BALB/c nude mice on day 0. Five HER2high+ tumor-bearing mice per group were randomly assigned to the CART-HER2 and NT groups before treatment. When the mean tumor volume reached approximately 100 mm3 on day 12, intravenous treatment with T cells was performed. Mice with maximum tumor diameters over 2 cm were sacrificed on day 33. The tumor volume data are represented as mean ± SD (mm3). The tumor weight data are represented as mean ± SD (mg). Images of tumor samples from sacrificed mice are shown. (B) The persistence of the infused CART-HER2 cells in the peripheral blood. qPCR was performed at serial time points, after infusion with CART-HER2 cells, to determine the expression levels of CAR in the peripheral blood of mice (n = 8). (C) Correlations of CAR copy numbers in tumor tissue and blood samples, obtained after the HER2high+ mice, treated with CART-HER2 cells, were sacrificed on day 33. (D) Hematoxylin-eosin (HE) and immunohistochemical (IHC) staining for anti-CD3 were performed on tumor samples from sacrificed mice
Figure 5
Figure 5
Effective response by CART-HER2 cells to GCSCs. (A) FACS analysis was used to determine the surface expression of HER2 and CD44 proteins in gastric cancer stem cells from suspended tumor spheres. (B) Co-culture of tumor spheres with 2 × 105 CART-HER2 cells, or NT T cells, per well was performed in an ultra-low adherent 24-well plate using serum-free media. Images of the killing activity, exerted by CART-HER2 cells against tumor spheres at serial time points, are shown. (C) Ten thousand gastric cancer stem cells were injected subcutaneously into the unilateral axillary region of BALB/c nude mice on day 0. Five mice per group were randomized to the CART-HER2 and NT groups. When the mean tumor volume reached approximately 100 mm3 on day 42, intravenous treatment with T cells was performed. Mice with maximum tumor diameters over 2 cm were sacrificed on day 63. The tumor volume data are represented as mean ± SD (mm3). The tumor weight data are represented as mean ± SD (mg). Images of tumor samples from sacrificed mice are shown
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