Enhanced Tumour Infiltration and Functionality of anti-GPC3 CAR-T Cells in Hepatocellular Carcinoma Through Locoregional Administration

Despite the rapid development of anti-cancer immunotherapy, the prognosis of hepatocellular carcinoma (HCC) patients is far from satisfactory. GPC3 CAR-T cell therapy has shown varied patient response. One of the potential reasons of low response rates is due to the limited CAR-T cell infiltration. Therefore, locoregional GPC3 CAR-T therapy has been a promising option, with potential to enhance the treatment efficacy. In this study, the therapeutic efficacy and underlying mechanisms of locoregional GPC3 CAR-T cell delivery were explored using orthotopic mouse HCC xenografts. CAR-T cell injection was performed locoregionally (through portal vein) and systemically (through tail vein). Compared to the systemic treatment, locoregional CAR-T cell therapy could better control the tumour growth and was associated with improved liver function. In addition, increased percentage of tumour infiltrating CAR-T cells was observed in the locoregional therapy group, with those CAR-T cells gaining increased cytotoxicity, chemotaxis and less exhaustion phenotype. Furthermore, portal vein injection also exhibited a better tumour inhibition ability over tail vein injection on a metastatic model which simultaneously bore both orthotopic and extrahepatic tumour lesions. In the current study, we have demonstrated that locoregional administration of CAR-T cells is associated with increased tumour infiltration and better therapeutic efficacy. Moreover, our results provide insights that locoregional CAR-T cell therapy could also be employed to the late-stage patients with distant metastasis.Despite the rapid development of anti-cancer immunotherapy, the prognosis of hepatocellular carcinoma (HCC) patients remains unsatisfactory. Chimeric antigen receptor (CAR) T cell therapy against HCC has been developed by targeting glypican 3 (GPC3), however, clinical trials for the anti-GPC3 CAR-T cell therapy showed varied patient responses with limited CAR-T cell infiltration. Locoregional administration has been a promising option for CAR-T therapy against solid tumours, yet it potential has not been examined for HCC treatment. In this study, we investigated into locoregional administration of anit-GPC3 CAR-T cells in preclinical models and examined the therapeutic efficacies using multiple HCC xenografts mouse models. Markedly, comparison of CAR-T cell injections performed locoregionally (through portal vein) and systemically (through tail vein) into mice carrying orthotopic HepG2 tumours demonstrated much enhanced inhibition of the tumour growth by the locoregional CAR-T therapy. Consistently, tumour infiltration of the CAR-T cells was significantly enhanced by the portal vein injection compared to tail vein injection. Flow cytometry and single cell-RNA analysis further revealed increased cytotoxicity, enhanced chemotaxis, and less exhaustion phenotype among the tumour-infiltrating CAR-T cells in the portal vein injection group. Treatment with accelerating CAR-T dosage resulted in further improved functionality of CAR-T cells and treatment efficacy, associating with improved liver functions. Markedly, portal vein injection also exhibited a better tumour inhibition ability over tail vein injection in a metastatic model simultaneously bearing orthotopic and extrahepatic tumour lesions. Collectively, our study demonstrated that locoregional CAR-T therapy is associated with increased tumour infiltration and better therapeutic efficacy, promising to treat the late-stage patients with distant metastasis.