B7-H3 targeted CAR-T cells show highly efficient anti-tumor function against osteosarcoma both in vitro and in vivo

Abstract

Background: Osteosarcoma (OS) mainly happens in children and youths. Surgery, radiotherapy and chemotherapy are the common therapies for osteosarcoma treatment but all their anti-tumor effects are limited. In recent years, a new cellular therapy, CAR-T, a cellular immunotherapy with genetically engineered T cells bearing chimeric antigen receptor targeting specific tumor-associated antigen, has been proved to be an effective therapy against acute lymphoblastic leukemia. Thus, CAR-T is a potentially effective therapy for osteosarcoma treatment.

Methods: A CAR gene targeting B7-H3 antigen was constructed into lentiviral vector through molecular biology techniques. Then, the CAR gene was transferred to T cells through lentiviral delivery system, and the CAR-T cells were largely expanded using in vitro culture technology. The in vitro anti-tumor effect of CAR-T cells was evaluated through Real Time Cell Analysis system (RTCA) and ELISA assay. The in vivo anti-tumor capabilities of CAR-T cells were evaluated using the patient-derived xenografts (PDX) model of osteosarcoma.

Results: The third-generation CAR-T cells we constructed could target the B7-H3 antigen, and the phenotype of CAR-T cells was consistent with normal T cells; The CAR-T cells showed superior antitumor effects both in vitro and in vivo.

Conclusion: Our study showed that B7-H3 targeted CAR-T cells had high anti-tumor efficacy against osteosarcoma both in vitro and in vivo, which proved that B7-H3 targeted CAR-T therapy is potentially effective for osteosarcoma treatment.

Keywords: B7-H3; Chimeric antigen receptor T; Osteosarcoma; Patient-derived xenografts.

Fig. 1

Expression of B7-H3 target in osteosarcoma samples and tumor cell lines. A B7-H3 expression was detected in OS tissues. OS Osteosarcoma. scale bar = 50 μm. B The surface expression of B7-H3 receptor level on tumor cell lines (Jurkat, HOS, U-2 OS, SW1353, Saos-2) detected by flow cytometry

Fig. 2

Construction of 3rd-generation CAR-T cells targeting B7-H3. A Schematic diagram of the B7-H3 CAR-T transgene. B Proportion of B7-H3 CAR infected primary human T cells determined by flow cytometry. C Histogram of the B7-H3-CAR-T rate of B7-H3 CAR infected cells and its control cells in three repeats. (mean ± SEM; ns not significant P > 0.05, *P < 0.05,**P < 0.01, ***P < 0.001)

Fig. 3

Cell phenotype of B7-H3 CAR-T cell. A Flow cytometry to detect CD3 + /CD4 + /CD8 + /CD45RA + /CCR7 + ratio in CAR-T cells and NC-T cells B Detection exhaustion biomarker of TIM3, PD1, LAG3 on CAR-T cells and NC-T cells by Flow cytometry.

Fig. 4

Functional Study of B7-H3 CAR-T cells in vitro. A The cytotoxicity of B7-H3 targeted CAR-T cells against solid tumor cell lines was analyzed by RTCA assay. The red arrow represents the time point of effector cell addition. B Comparison of killing rates under different E:T ratios on tumor cells. C Levels of IFNγ and TNFα released by B7-H3 CAR-T cells analyzed by ELISA after incubated with cells for 20 h. D Situation of effector cells and target cells (U-2 OS) displayed by optical microscopy. mean ± SEM; ns not significant P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Anti-tumor efficacy of B7-H3 CAR-T cells against PDX models of osteosarcoma in vivo. A and G Schema of the experimental events and nodes. The tumor size (B and H) and mice weight (F and J) variation with B7-H3 CAR-T cells and control T cells injection among the Observed time. C Presence of CD3 + T cells in mouse peripheral blood after 10 days of injection. D Absence of CAR-T cells in mice tumors after 10 days of injection. E Size of tumor mass differed in B7-H3 CAR-T cells, NC T cells or PBS treated mice. I Kaplan–Meier curves for survival of the PDX-OSTE0007 models are shown. (mean ± SEM; ns not significant P > 0.05, *P < 0.05,**P < 0.01, ***P < 0.001)