自主研发的新型CAR-T结构 – 原能细胞科技集团
➤ Chimeric antigen receptors (CARs) are hybrid molecules that combine antigen-binding properties of monoclonal antibodies (mAb) and signaling elements of lymphocyte activating molecules。
➤ Glypican-3 (GPC3), a membrane-bound proteoglycan, is expressed in several solid tumors including hepatocellular carcinoma (HCC), hepatoblastoma (HB), embryonal sarcoma, malignant rhabdoid tumor (MRT), yolk sac tumor, Wilm’s tumor, squamous cell carcinoma of the lung and liposarcoma.
➤ GPC3 is an attractive target for immunotherapy since it is not expressed at detectable levels in non-malignant tissues including normal or cirrhotic liver.
Expression of GPC3-CAR on the surface of T cells
GPC3 CAR expression on T cell surface determined by flow cytometry. One representative donor data has been shown. Mock T cells served as controls.
(A) Tumor cell lysis was measured with LDH release assay at indicated effector to target ratios and coculture for 4 hrs or 18 hrs (B) against GPC3-positive solid tumor cell lines HepG2, Huh-7 and GPC3-negative immortal hepatic cell line L-02. Representative results from 3 independent experiments were shown. One-way ANOVA with Tukey’s test analysis for G3-28z-41BBL vs G3-28BBz: *P <0.05; **P <0.01; ***P <0.001.
Differential cytokine release of GPC3 CAR-T cells in vitro and in vivo
(A) In vitro cytokine release by GPC3 CAR-T cells. GPC3-positive HepG2, Huh-7 and GPC3-negative L-02 cells were co-cultured with G3-28BBz/G3-28z-41BBL CAR or Mock T cells for 24 hrs at 1:1 and indicated cytokine levels in tissue culture supernatant were measured by Multiplex Luminex. (B) In vivo cytokine release by GPC3 CAR-T cells. HuH-7 tumor-bearing NCG mouse was injected single dose of GPC3 CAR-T or Mock T cells at 1x107 CAR-T cells per mouse i.v., at Day 3 and Day 7, the serum were collected and pooled for cytokines measurement by Multiplex Luminex. Black, Mock T cell; Grey, G3-28BBz CAR-T cell; Deep grey, G3-28z-41BBL CAR-T cell. Mean and SD were shown. *P <0.05; **P <0.01; ***P <0.001, ****P <0.0001, 1-way ANOVA with Tukey’s test analysis.
Schematic map of CAR constructs。 LTR, long terminal repeat; S, signal sequence of CD8; VL-L-VH, single chain variable regions of monoclonal anti-GPC3 antibody; CD8stem, part of the extracellular region and all of the transmembrane of CD8; CD28, intracellular signaling domain of CD28; 41BB, intracellular signaling domain of 4-1BB; CD3z, the entire cytoplasmic region of the TCR-ζ molecule; IRES, Internal Ribosome Entry Site; 41BBL, entire 4-1BB ligand molecule。
GPC3 CAR-T cells antitumor activity in vivo
(A) Growth curve of Huh-7 xenografts treated with the indicated GPC3 CAR-T cells or Mock T cells at high dose (1x107 cells/mouse). The residual tumors treated with G3-28BBZ and G3-28Z-41BBL CAR-T cells were significantly smaller than those in Mock T cells groups at Day 17 (****, P < 0.0001). (B) Survival curve of Huh-7 tumor-bearing mice after treatment with high dose GPC3 CAR-T cells. Comparison of GPC3 CAR-T cell groups vs Mock T cell group by Log-rank (Mantel-Cox) test, ***p<0.001. (C) Growth curve of Huh-7 xenografts treated with the indicated GPC3 CAR-T cells or Mock T cells at low dose (1x106 cells/mouse). (D) Survival curve of Huh-7 tumor-bearing mice after treatment with low dose GPC3 CAR-T cells. *p<0.05, Log-rank (Mantel-Cox) test.