T Cells Expressing A LMP1-specific Chimeric Antigen Receptor Mediate Antitumor Effects Against EBV-Associated Malignancies In Vitro And In Vivo

BackgroundEpstein-Barr Virus (EBV) is a ubiquitous double-stranded DNA virus that is a member of γ-herpeps. EBV infect>90%of human through contact with oral secretions. EBV-infected cells are rapidly eliminated by immune system with cytoxic T-cells (CTLs), both CD8+and CD4+, and natural killer (NK) cells, but a few virus remains in an asyptomatic latent state within resting B-cells for lifetime. Under conditons of host’s cellular immune system fails to control EBV-induced B-cell proliferation, infected B cells transform from the latent state into malignant cells. EBV is associated with B lymphomas, including Hodgkin’s disease (HD) and Burkitt lymphpomas (BL), Nasal pharyngeal cancer (NPC) and Posttransplant Lymphoproliferative Disorders (PTLD).EBV-associated malignancys are usually difficult to be removed by surgery. Although radiotheraputic and chemotherputic methods are effecive for those malignancys, they could not eliminate tumor cells completely, especially those in circulatory system and metastatic focuses. Therefore, seeking for effective targeted therapy and immune therapy has gradually become a research direction, by considering EBV as the risk factor of EBV-associated malignancy.Chimeric antigen receptor (CAR) modified T cell therapy is a novel strategy of combining the advantages of T cell based therapy and antibody based tumor specificity. CART cells may be the best method in adopitive antitumor immunotherapy with the ability to specially recognize and kill tumor cells. First generation CARs encode antibody-based external receptor structures and cytosolic domains that encode signal transduction modules composed of the immunoreceptor tyrosine based activation motif such as TCRζ or FcRy. The first generation CARs with poor expression and persistence were observed in vivo, as phase I clinical trail. Based on principles of T cell activation, CD28signaling domain provided costimulation when engineered in cis with the TCRζ domain into second CAR generation design, as well as the members of tumor necrosis factor receptor family such as4-1BB(CD137) and OX-40(CD134) into third generation CAR design. Many trails are currently ongoing to test second and third generation CARs and these clinical studies shows notable success for cancer therapy.The target antigens of CAR is the hinge of recognizing and killing tumor cells, which are overexpression on the surface of tumor cells, while with low or no expression on normal cells. The latent membrane protein-1(LMP1) encoded by Epstein-Barr virus (EBV) is an oncoprotein which acts by constitutive activation of various signalling pathways, including NF-κB. It leads cell growth intrinsic to the cancer cell as well as having extrinsic affects upon the tumour microenvironment. LMP1as a foreign antigen may be a good therapeutic target in the treatment of EBV associated disease. Therefore, it is of important significance for the Targeted LMP1immunotherapy to the EBV-associated malignancy with high expression of LMP1.ObjectivesTo redirect T cell express the2nd generation and3rd generation CARs specific for LMP1; to evaluate the antitumor and proliferation activity in vitro and in vivo; to analysis the mechanisms of kill tumor cell by LMP1/CAR T cell.Methods1. To evaluate the LMP1expression on human Nasopharyngeal carcinoma and lymphoma cell lines by flow cytometry. LCL cells were preparated by using EBV infect human B lymphocyte, and were detected by anti-CD40, anti-CD19, anti-CD.80and anti-CD86antibodies.2. To construct the2nd generation CAR specific for LMP1and transduce human T lymphocytes using retrovirus and lentivirus encoding LMP1CAR; to evaluate the LMP1CAR expression on human T cells by flow cytometry.3. To investigate the function of T cells expressing LMP1specfic CAR in vitro using cytotoxity assay, IFN-y and IL-2release assay.4. To investigate the function of T cells expression LMP1specfic CAR in murine model.5. To construct the3rd generation CAR specific for LMP1and transduce human T lymphocytes using lentivirus encoding LMP1CAR.6. To investigate the function of T cells expression LMP1specfic3rd generation CAR in vitro using cytoxity assay, IFN-y release assay and proliferation assay.7. To investigate the function of T cells expression LMP1specfic3rd generation CAR in murine model including tumor size, H&E and IHC.Results1. LMP1is expressed in SUNE1-LMP1, HNE-LMP1, C666-1, B95-8, LCL, CIR-neo, RPMI6666and Raji, while low or no expressed in HNE2, SUNE1, Romas, Daudi. This study has prepared a LCL cell line with CD40-positive rate of97%, CD19-positive rate of90%, CD80-positive rate of85%, CD86-positive rate of80%.2. The2nd generation were constructed successfully confirmed by sequencing and western blotting. The efficiency of infected T cells by lentivirus is between62%and75%, while the efficiency is between30%and45%by retrovirus. LMP1CAR was detected on T cells by FACS and western blotting.3. T cells expressing anti-LMP12nd CAR specifically recognize LMP1+tumor cell lines and show cytotoxic against human LMP1+tumor cell lines but not LMP1negative cell lines in vitro. The T cells also secrete IFN-y, IL-2depend with LMP1+cell lines.4. In LMP1xenograft model, T cells substantially inhibited the growth of tumors, while control T cells did not. Statistical analysis of the tumor growth curve revealed significant differences between the HELA/CAR and control groups. 5. The anti-LMP13rd generation CAR were constructed successfully confirmed by sequencing, and aboout72%T cells could expressed anti-LMP13rd generation CAR (LMP1-134and LMP1-137) by lentivrius infection.6. The3rd generation CART cells also can recognize and kill LMP1+tumor cell, but there is no significant difference. LMP1-137T cells secrete more IFN-γ than LMP1-28T cells and LMP1-134T cells, but LMP1-134T cells and LMP1-137T cells secrete more IL-2than LMP1-28T cells and with stronger proliferation capacity.7. The anti-LMP13rd generation CART cells as well as the2nd generation CART cells can inhibit tumor growth through intravenous injection and LMP1-137T cells work best. The results of HE stain show that the tumors of LMP1/CART groups with more necrosis and human lymphocyte which confirmed by IHC.Conclusions1. Lentivirus has better transfection efficiency than retrovirus.2. The anti-LMP12nd and3rd generation CART cells can recognize and kill LMP1positve tumor cells, and3rd generation CART cell inhibit tumor growth better than2rd generation CART cell in vivo.3. Anti-LMP1CART cells which contain CD134or CD137sequence can secrete more IL-2and have stronger proliferation capacity.