Antitumor activity of NKG2D CAR-T cells against human colorectal cancer cells in vitro and in vivo

Abstract

Colorectal cancer is one of the most common malignancies worldwide, as it is often diagnosed at an advanced stage. Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable success and emerged as one of the most promising therapeutic strategies in multiple malignancies. The purpose of this study was to investigate the anti-tumor activity of NKG2D CAR-T cells against human colorectal cancer cells. A non-viral third-generation NKG2D CAR was constructed, and subsequently transduced into T cells to obtain the NKG2D CAR-T cells. In vitro, NKG2D CAR-T cells showed cytotoxicity against human colorectal cancer cells in a dose-dependent manner compared with untransduced T cells. In addition, IL-2 and IFN-γ secreted by these cells were significantly higher than those by untransduced T cells. In vivo, NKG2D CAR-T cells significantly suppressed tumor growth, reduced tumor sizes and extended overall survival of mice in a xenograft model of HCT-116 cells. Furthermore, human NKG2D-positive lymphocytes infiltration could be found in the tumor sections of NKG2D CAR-T cells-treated mice. There were no severe pathological changes found in vital organs in any of the treatment groups. NKG2D CAR-T cells showed excellent killing effect and represented a promising immunotherapeutic strategy against human colorectal cancer.

Keywords: Chimeric antigen receptor; NKG2D; colorectal cancer; minicircle DNA.

Figure 1

Schema of NKG2D CAR minicircle DAN non-viral vector construction. NKG2D CAR contains a CD8α leader signal sequence sequence, an extracellular domain (ECD) of human NKG2D receptor, a hinge region of CD8α, a transmembrane domain (TMD) of CD28, and an intracellular domain (ICD) of CD28, 4-1BB and CD3ζ. This gene fragment was cloned into the parental cloning vector pMC.CMV-MCS-EF1-GFP-SV40polyA. After transformation into ZYCY10P3S2T minicircle production E. coli, the parental cloning vector underwent a recombination event between the PhiC321 attB and attP sites, resulting in two products --- the NKG2D CAR minicircle DNA vector and the miniplasmid backbone. In the process, L-arabinose was added into the media to induce the I-SceI endonuclease expression, resulting in degradation of the miniplasmid backbone.

Figure 2

Detection of NKG2DLs expression on two human colorectal cancer cells. LS174T and HCT-116 cells were stained with the specific antibody (human MICA PE-conjugated antibody, human MICB PE-conjugated antibody, human ULBP-1 PE-conjugated antibody, human ULBP-2/5/6 PE-conjugated antibody, and human ULBP-3 PE-conjugated antibody, filled histogram) or isotype control antibody (mouse IgG2B PE-conjugated antibody and mouse IgG2A PE-conjugated antibody, open histogram). MFI ratios of specific staining versus staining of isotype control are detailed in the histograms.

Figure 3

Characterization of NKG2D-specific CAR-T cells. A. Expression of NKG2D CAR and GFP on T cells or NKG2D CAR-T cells detected by flow cytometry. Left, isotype control. Middle, T cells control. Right, NKG2D CAR-T cells. B. Fluorescence microscopy images of GFP expression in CAR-T cells were observed by fluorescence microscopy for 24, 48, or 72 hours (Magnification × 200). C. The expression level of exogenous CD3ζ was analyzed by Western blotting. D. In vitro fold expansion of T cells following stimulation of Human T-Activator CD3/CD28 Dynabeads was assayed using cell counting assays.

Figure 4

Phenotype Changes of NKG2D-specific CAR-T cells. A. Expression of CD4 on T cells or NKG2D CAR-T cells detected by flow cytometry. B. Expression of CD8 on T cells or NKG2D CAR-T cells detected by flow cytometry. Left, isotype control. Middle, T cells control. Right, NKG2D CAR-T cells.

Figure 5

In vitro anti-tumor capacity of NKG2D CAR-T cells. A. The cytotoxicity of T cells (black line) and NKG2D CAR-T cells (gray line), against LS174T (left) or HCT-116 (right) was analyzed by LDH release assay at the indicated E:T ratios. B. Levels of IL-2 (left) and IFN-γ (right) secreted by T cells and NKG2D CAR-T cells were analyzed by ELISA after co-cultured with LS174T or HCT-116 cells for 24 hours. Results are expressed as means ± SD of triplicate experiments. Differences between the T cells and NKG2D CAR-T cells at each E:T ratio were evaluated using Student’s t-test. *P < 0.05 vs. T cells. **P < 0.01 vs. T cells.

Figure 6

Anti-tumor capacities of NKG2D CAR-T cells in vivo. A. Schematic of the experimental protocol. NOD/SCID mice were inoculated subcutaneously with 1 × 10⁶ HCT-116-Luc cells on day-12, and subsequently treated by the tail vein injection with 1 × 10⁷ NKG2D CAR-T cells (NKG2D CAR-T cells group) on day 0 and 7. B. In vivo bioluminescence imaging was used to measure tumor growth of mice on day 20. C. Tumor volumes were measured by calipers every 5 days. Data are expressed as means ± SD. D. Overall survival of mice was presented in Kaplan-Meier curves. E. Tumor sections were stained with anti-human NKG2D specific antibody for detection of T cells (Magnification × 400).

Figure 7

Safety of NKG2D CAR-T cells in vivo. A. Body weight was measured using an electronic scale every 5 days. Data are expressed as means ± SD. B. HE staining analysis was performed to examine the pathological changes of the heart, liver, lungs, spleen, and kidneys of the mice. (Magnification × 200).