Nicotinamide Nucleoside Enhances The Anti-tumor Efficiency Of GPC3-CAR T Cells In Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is one of the main causes of cancer-related death in the world,and the incidence and mortality of cancer in China are still in the forefront in recent years.Although the current treatment methods are mainly surgery,chemotherapy,targeted and hepatic arterial chemoembolization,the prognosis is still poor,and there is a lack of effective treatment for patients with advanced or recurrent liver cancer.Chimeric antigen receptor(CAR)modified T cells(CAR)is a new immunotherapy method,which has a good effect in the treatment of relapsed or refractory B cell malignancies and has been widely used in clinic.Clinical trials of GPC3-targeting CAR-T cells for liver cancer are ongoing,but their efficacy is limited.The use of CAR-T cell therapy in solid tumors remains a challenge.Factors such as tumor vascular barrier and tumor immunosuppressive microenvironment limit the use of CAR-T cell therapy in solid tumors,including liver cancer.Therefore,it is important to overcome the inhibition of tumor microenvironment and improve the efficacy of CAR-T cell therapy in solid tumors.It has been proved that tumor microenvironment with low oxygen,acidity and nutrition can cause metabolic disorder of T cells,leading to decrease of NAD~+level in T cells,thus affecting the activation and anti-tumor function of T cells.Nicotinamide adenine dinucleotide(NAD~+)is an important molecule in glycolysis,tricarboxylic acid cycle and other important cellular metabolic pathways,as well as an important mediator in cellular energy metabolism.NAD~+is biosynthesized through de novo synthesis,Preiss-handler pathway and rescue pathway.Niacinamide phosphoribose transferase(NAMPT)is a key enzyme in the NAD~+rescue pathway,and the level of NAD~+can be significantly reduced by using the NAMPT inhibitor FK866.Nicotinamide nucleoside(NR),one of the precursor substrates of nicotinamide adenine dinucleotide(NAD~+),can safely and effectively increase human NAD~+levels after ingestion.NAD~+supplementation with nicotinamide(NAM),the precursor of NAD~+,can enhance the killing ability of CD19-targeted CAR-T cells against hematologic tumors.However,whether NAD~+supplementation with NR can enhance the killing ability of CAR-T cells against solid tumors has not been reported.Therefore,this study aimed to construct hepatocellular carcinoma GPC3-targeted CAR-T cells,simulate the low NAD~+expression state of T cells in tumor microenvironment in vitro,detect the effect of NR supplementation on T cells,and detect the killing effect of oral NR on GPC3-targeted CAR-T cells on hepatocellular carcinoma in vivo in mice.Materials and methods1.T cells were treated with different concentrations of FK866 in vitro,and the apoptosis of T cells was detected by flow cytometry.After T cells were treated with NAMPT inhibitor FK866 and/or NR,the level of NAD~+in T cells was detected by liquid chromatography-mass spectrometry(LC-MS).2.The expression of GPC3 in hepatoma cell lines SK-HEP-1,Hep G2 and Huh7 was detected by flow cytometry and Western blot.3.T cells were isolated from peripheral blood of healthy subjects,and lentivirus infection was used to construct third-generation CAR-T cells targeting GPC3 with CD28 and 4-1BB costimulation domains.The infection efficiency was detected by flow cytometry and Western blot.4.GPC3-CAR T cells were pretreated with FK866 and/or NR in vitro to detect the killing effect of GPC3-CAR T cells against Hep G2 and Huh7 cell lines.5.By using NSG mice,Huh7 subcutaneous tumor and Huh7 GL abdominal metastatic tumor were constructed to explore the effect of NR in GPC3-CAR T cells on the therapeutic effect of HCC subcutaneous tumor and abdominal metastatic tumor.Results1.NAMPT inhibitor FK866 can reduce the level of NAD~+in T cells and FK866 does not affect the viability of T cells,which can be used to simulate the low NAD~+state of T cells in tumor microenvironment in vitro;at the same time,supplementation of NR can reverse the inhibition of FK866 on T cell and increase the level of NAD~+in T cells.2.GPC3 was highly expressed and moderately expressed in hepatoma cell lines Hep G2 and Huh7,while negative in SK-HEP-1 cells.3.The results of flow cytometry and Western blot showed that the third generation GPC3-CAR T was constructed successfully,and the positive rate was 36.4%.4.The results of cell experiment in vitro showed that supplementation of NAD~+could reverse the decrease of killing effect of FK866 on GPC3-CAR T and improve the anti-tumor effect of CAR-T cells.5.The results of animal experiments in vivo showed that the supplementation of NAD~+precursor NR could improve the killing ability of CAR-T cells to tumor and prolong the survival time of mice.ConclusionNR,the precursor of NAD~+,can increase the level of NAD~+in T cells.And the supplementation of NR can enhance the anti-tumor effect of CAR-T cells in hepatocellular carcinoma.