Abstract
BackgroundMore favorable treatment against epithelial ovarian cancer (EOC) is urgently needed because of its insidious nature at an early stage and a low rate of five-year survival. The current primary treatment, extensive surgery combined with chemotherapy, exhibits limited benefits for improving prognosis. Chimeric antigen receptor T (CAR-T) cell technology as novel immunotherapy has made breakthrough progress in the treatment of hematologic malignancies, and there were also benefits shown in a partial solid tumor in previous research. Therefore, CAR-T cell technology may be a promising candidate as an immunotherapeutic tool against EOC. However, there are some weaknesses in targeting one antigen from the previous preclinical assay, such as on-target off-tumor cytotoxicity. The dual-target CAR-T cell may be a better choice.MethodsWe constructed tandem PD1-antiMUC16 dual-CAR, PD1 single-CAR, and anti-MUC16 single-CAR fragments by PCR and genetic engineering, followed by preparing CAR-T cells via lentiviral infection. The expression of CAR molecules on single and dual CAR-T cells was detected by flow cytometry. The killing capacity and activation of CAR-T cells were measured by cytotoxic assays and cytokines release assays in vitro. The therapeutic capacity of CAR-T cells was assessed by tumor-bearing mice model assay in vivo.ResultsWe successfully constructed CARs lentiviral expression vectors and obtained single and dual CAR-T cells. CAR-T cells demonstrated robust killing capacity against OVCAR-3 cells in vitro. Meanwhile, CAR-T cells released plenty of cytokines such as interleukin-2(IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α(TNF-α). CAR-T cells showed a therapeutic benefit against OVCAR-3 tumor-bearing mice and significantly prolonged the survival time. Dual CAR-T cells were shown to be two to four times more efficacious than single CAR-T cells in terms of survival time.ConclusionAlthough exhibiting a similar ability as single CAR-T cells against OVCAR-3 cells in vitro, dual CAR-T cells demonstrated enhanced killing capacity against OVCAR-3 cells as compared to single CAR-T cells in vivo and significantly prolonged the survival time of tumor-bearing mice. PD1-antiMUC16 CAR-T cells showed more potent antitumor activity than single CAR-T cells in vivo. The present experimental data may support further research work that will have the potential to lead to clinical studies.
Highlights
More favorable treatment against epithelial ovarian cancer (EOC) is urgently needed because of its insidious nature at an early stage and a low rate of five-year survival
Construction of dual-target Chimeric antigen receptor T (CAR-T) cells by Lentiviral vector transduction According to the above protocol, we designed the PD1antiMUC16 CAR molecule structure, which comprised Programmed cell death-1 (PD1)-antiMUC16 or PD1 or anti-Mucin 16 (MUC16) extracellular Single chain antibody fragment (scFv) fragment, a hinge region, a transmembrane domain, followed by intracellular 4-1BB co-stimulation domain and CD3ζ domain
Via testing by agarose gel electrophoresis, PD1, antiMUC16, PD1-antiMUC16, and plasmid skeleton fragment bands were observed at 510 bp,1500 bp,2000 bp, and 7435 bp, respectively (Fig. 2a), and original gel electrophoresis was in Additional file 1 (Figure S1)
Summary
More favorable treatment against epithelial ovarian cancer (EOC) is urgently needed because of its insidious nature at an early stage and a low rate of five-year survival. The current primary treatment, extensive surgery combined with chemotherapy, exhibits limited benefits for improving prognosis. Chimeric antigen receptor T (CAR-T) cell technology as novel immunotherapy has made breakthrough progress in the treatment of hematologic malignancies, and there were benefits shown in a partial solid tumor in previous research. More than 50% of serous carcinoma is the primary type of EOC [4], and it is diagnosed at stage III (51%) or stage IV (29%) due to the absence of specific early symptoms [3]. The current primary treatment of EOC is extensive surgery combined with platinum-based or taxane-based chemotherapy, there are limited benefits for improving prognosis [2,3,4]. CAR-T cell therapy as one of the representative adoptive immunotherapies, has made unprecedented progress in the treatment of hematologic malignancies. Because of the deficiency of tumor-specific targets and physiologic barrier, it is challenging for the patients with solid tumors to receive benefits [6]
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