A Novel Split CAR-T Targeting Glypican-3 In Anti-hepatocellular Carcinoma:The Roles And Mechanisms

Background Glypican-3(GPC3)is a glycoprotein that is highly expressed on the surface of hepatocellular carcinoma(HCC),and it has not been expressed in normal tissues or underexpressed in recent years.Cloned antigen receptor T cells(chimeric antigen receptor T cells,CAR-T)have achieved remarkable results in the treatment of hematological tumors,and have been clinically tested in various solid tumors.Among them,many clinical trials of CAR-T for hepatocellular carcinoma have targeted GPC3.However,due to the rigid structure of the receptor,traditional CAR-T cells have some inherent limitations,such as uncontrollable over-activation and triggering severe cytokine release syndrome.Therefore,CAR-T cells are in the process of treating solid tumors.There are great safety risks in this,which severely impair the widespread application of CAR-T cells in the clinic.How to improve the safety of CAR-T cell therapy,maintain its effectiveness,and improve its mechanism are urgent problems to be solved.Hepatocellular carcinoma(HCC)is the fifth most commonly occurring cancer worldwide 1.In 2012,there were 782,000 new cases of HCC globally,83%of which occurred in less developed regions 2.Although surgery is currently the most effective treatment for HCC,the tumor recurrence rate is very high after tumor resection,and the age-standardized five-year relative survival rate of HCC is only 10.1%3.Due to the difficulty of early diagnosis,most HCC patients are diagnosed as an advanced stage at the initial visit and lose the opportunity for curative treatment such as hepatectomy or ablation,making HCC the second leading cause of cancer-related death in adult males due to the lack of effective therapies 4.Sorafenib and Lenvatinib,the two clinically approved targeted drugs for first-line treatment of patients with unresectable HCC,could only extend the overall survival by 2 to 3 months 5,6.Thus,there is an urgent need for new methods of treating HCC.Reports have shown that activation of the host immune system produces significant anti-tumor effects 7.On the other hand,by releasing immune checkpoints that inhibit anti-tumor responses,a variety of cancer patients can achieve unprecedented long-term tumor response 8.For example,blocking the immune checkpoints by anti-cytotoxic T lymphocyte-associated protein 4(CTLA-4)or the programmed cell death 1(PD-1)antibodies,either alone or in combination,unleashes the patient's immune response and generates therapeutic effects in a variety of cancers 9,including HCC 10.Although that is promising,the main premise to induce anti-tumor effects by immune checkpoint blockade is the preexistence of tumor-specific infiltrating T cells,which are not always available 11.Moreover,the release of non-specific T cells by immune checkpoint blockade may result in autoimmune destruction 12.In this regard,immunotherapy targeting tumor-specific or tumor-associated antigens is less likely to cause autoimmune diseases.Human glypican-3(hGPC3)is a member of the heparan sulfate(HS)proteoglycan family and attached to the cell membrane by a glycosylphosphatidylinositol(GPI)anchor 13.A number of studies have shown that hGPC3 is highly expressed in HCC,but its expression in normal tissues is limited,making it an attractive target for immunotherapy of HCC 14.The chimeric antigen receptor(CAR)is a synthetic antigen receptor that redirects T cell specificity and function.Patient-derived CAR-T cell therapy is a promising approach for cancer immunotherapy and has achieved tremendous success in hematological malignancies 15.The results from mouse model and patient-derived xenografts(PDX)of HCC indicated that CAR-T cells targeting hGPC3 could effectively eliminate hGPC3 positive tumors,providing an encouraging candidate for HCC treatment 16,17.Actually,a dozen of clinic tries using hGPC3-targeting CAR-T cells to treat HCC have been registered and carried out 18.However,conventional CARs have a rigid structure and typically compose of a fixed antigen-specific single-chain variable fragment(scFv)and intracellular signaling domains(Figure 1A).When conventional CAR-T cells were activated by encountering the antigen,the expanded progeny cells retained all the features of parental cells and could be activated,proliferate,and produce lots of cytokines.For example,CAR-T cells would expand rapidly up to 7,000 times after transferred into patients and result in severe tumor lysis syndrome(TLS)and fatal cytokine release syndrome(CRS)19.Therefore,there is an urgent need for a better system to improve the safety of CAR-T cell therapy.Here we described a novel,split anti-hGPC3 CAR-T system composed of two components(Figure 1 B):the anti-hGPC3 scFv-SpyTag consists of an anti-hGPC3 scFv linked to a 13 amino acid peptide SpyTag 20,and the signaling molecule SpyCatcher-CAR consists of extracellular SpyCatcher linked to the intracellular domains of 4-1BB and CD3?.SpyCatcher could bind to SpyTag specifically and covalently 20.The in vitro and in vivo cytotoxicity and cytokine release results demonstrated that our split anti-hGPC3 CAR-T cells could control the growth of hepatocellular carcinoma with decreased cytokine release compared to conventional CAR-T cells.This novel split anti-hGPC3 CAR system represents a more versatile and safer application for HCC treatment without compromising CAR-T cells efficacyObjective This study gradually explores to separate the extracellular antigen recognition domain from the intracellular signal conversion domain by establishing a novel,split,two-component insertion into the antigen receptor.Therefore,the activation and proliferation of CAR-T cells can be regulated by regulating the extracellular free antigen recognition structure,thereby achieving the purpose of improving the safety of clinical treatment of CAR-T cells.Methods We divided the traditional CAR into two parts and redesigned the CAR dedicated to hGPC3.To compare the relative cytotoxicity and cytokines release activity of our SpyCAR-T cells with conventional CAR-T cells,the second-generation of GPC3-specific conventional CAR-T cells were constructed,produced and designated as aGPC3-CAR.The same anti-GPC3 scFv sequence from clone GC33 was used as the antigen recognition domain in aGPC3-CAR.Lentiviral particles of Spy CAR and ?GPC3-CAR were generated and used to transduce sorted T cells from the same donor.The transduction efficiency of aGPC3-CAR-T cells detected by staining with FITC-labeled protein L was comparable to SpyCAR-T cells.The frequencies of CD8+ T cells were also comparableThe cytotoxicity and cytokine release of isolated anti-hGPC3 CAR-T cells were evaluated for various HCC cell lines in vitro and in vivo using co-culture assays and xenograft mouse models,and isolated from traditional CAR-T cells.Results The results of in vitro experiments show that the new type of dividing GPC3 established by CAR-T cells can recognize and divide GPC3+HepG2 and Huh7 cells in a dose-dependent manner.What's more important:Similar to traditional CAR-T cells,the inflammatory cytokines(including IFN-?,TNF-?,IL-6 and GM-CSF)produced by activated CAR-T cells of our GPC3 are very low.many.In vivo experiments in immunodeficient mice show that after subcutaneous transplantation of HepG2 cells,our split GPC3 into CAR-T cells can effectively inhibit the growth of hepatocellular carcinoma,but the levels of cytokines released are significantly lower than traditional CAR-T cells.Conclusion In this study,we described for the first time the use of split GPC3-specific CAR-T cells to treat HCC,which can inhibit tumor growth and reduce cytokine release,providing improved safety of CAR-T cells in clinical treatment.