Adoptive Immunotherapy For Cancer Using Chimeric Antigen Receptor Redirected Tcells: Preclincal Study

BACKGROUDAdoptive T cell therapy involves the ex vivo selection and expansion of effector T cells for the treatment of patients with cancer. Currently, there are two main strategies employed to endow T cells with the specificity and power to kill tumor. One strategy is isolating and indentifying functional lymphocytes (tumour infiltrating lymphocytes, TILs) directly from tumor biopsies and then subsequently expanded ex vivo by IL-2-driven expansion regimes. Another strategy is introducing chimeric antigen receptor (CAR) specific to tumor associated antigen (TAA) into T cells and subsequently expanded in vitro. The essential components of a CAR are an antigen targeting domain fused to an intracellular signalling domain anchored to the surface of the T cell by a transmembrane domain. The antigen binding domain most commonly involves a single chain antibody fragment (scFv) consisting of the antigen recognition components of a monoclonal antibody. The signalling domains used have focused on the key signaling molecules CD3ζchain. Since the first generation of CAR using only extracellular antigen-binding domain and CD3ζsignaling portion, studies have focused on the use of costimulatory domains including intracellular-signaling chains of CD28 and 4-1BB (CD137) so that maximal T-cell activation can be efficiently optimized and the anti-tumor responses can be maintained upon antigen-receptor ligation.Folate receptor alpha (FRa) expression is highly restricted in normal adult tissues, but upregulated in a wide range of human cancer types including epithelial ovarian cancer, breast cancer, renal cell cancer, lung adenocarcinoma as well as colon cancer and nasopharyngeal cancer. FRa represent an attractive candidate for targeted immunotherapy. Thus, redirecting T cell specificity for FRa using chimeric immuce receptor is a novel approach for cancer therapy and is currently being investigated in preclinical and clinical trials.OBJECTIVESTo redirect T cell express the first generation and second generation CARs specific for FRa; to evaluate the role of costimulatory molecule 4-1BB (CD 137) in the CARs mediate adoptive T cell therapy for cancer in vivo.METHODS1. To evaluate the FRa expression on human ovarian cancer cell lines by flow cytometry.2. To construct the first and second generation CAR specific for FRa and transduce human T lymphocytes using lentivirus encoding FRa CAR; to evaluate the FRa CAR expression on human T cells by flow cytometry.3. To investigate the function of T cells expressing FRa specific CAR in vitro using IFN-g release assay, cytokine beads array and cytotoxity assay.4. To investigate the function of T cells expressing FRa specific CAR in murine model including Winn assay, subcutaneous, metastatic intraperitoneal as well as lung-involved human ovarian cancer model.5. To compare the antitumor activity of intratumor (IT), intravenous (IV) and intraperitoneal (IP) routes of administration CAR modified T cells in mice.6. To evaluate the persistence of transfferd T cells in GFP, z and MOv19-BBζand anti-CD 19-BBζgroup in mice.RESULTS1. Most of the human ovarian cancer cell line as well as breast cancer cell lines express FRa.2. First and second generation CARs specific to FRa are successfully generated and highly expressed on human T lympyhocytes surface determined by flow cytomytry.3. T cells expressing FRa CAR specifically recognize FRa+tumor cell lines and predominantly secret Thl cytokines including IL-2, TNF-a, IFN-y but low level of Th2 cytokines such as IL-4 and IL-10. T cells expressing FRa CAR also showed specifically cytotoxic against human FRa+tumor cell lines but not FRa negative cell lines in vitro.4. In vivo, T cells expressing first generation FRa CAR (FR-z) and second generation CAR (FR-BBz) inhibit the tumor grow out in Winn assay and FR-BBz is superior to MOv19-ζ(p=0.026). In established model, FR-z expressing T cells only delayed the tumor growth but FR-BBz T cells can rapidly eradicate the large, well established tumor (p<0.0001) and the eradication is antigen specific because the CD 19 specific CAR T cells had no effect on tumor growth. In metastatic intraperitoneal model, whether delivered by IP or IV routes of administration, MOvl9-BBζT cells can inhibt the ascites formation and improve the overall survival(IP group median survival:68 days;IV group median survival:52 days), while the control T cells expressing anti-CD 19-BBz can not (IP group median survival:9 days;IV group median survival:12 days). In the lung-involved metastatic model, the T cells exrepssing FR-BBz can resulted in rapid regression of lung metastasis in all treated animals 14 days p.i.and 80%(4/5) of mice had no evidence of recurrence after 2 months. By contrast, disease progression occurred in all mice receiving anti-CD19-BBζT cells (p<0.01).5. Human CD4+and CD8+T cell counts were highest in mice receiving FR-BBz CAR T cells, whether delivered by IT, IP orⅣroutes of administration, compared to gfp, FR-Δz treatment groups. Notably, human T cell counts in mice receiving FR-BBz CAR T cells by injection was significantly higher than in the parallel FR-z CAR group (P< 0.01), indicating a role for CD137 in T cell survival in vivo.6. Mice receiving FR-BBz CAR T cells had significantly higher human CD4+ and CD8+T cell counts than mice in anti-CD 19 CAR or gfp groups (p=0.009), indicating that tumor antigen recognition drives the survival of the adoptively transferred T cells in vivo. Interestingly, T cell persistence was reproducibly higher in mice receiving anti-CD 19-BBz CAR T cells compared to gfp T cells (p= 0.012), suggesting that persistence of CAR T cells can be promoted in part through a 4-1BB (CD137)-driven process that does not require scFv engagement with antigen.CONCLUSIONS1. Engineered T cells expressing chimeric antigen receptor specific to tumor associated antigen thepary for cancer is a novel and powerful approach.2. This study demonstrate that direct and specific recognition and killing of human ovarian cancers by genetically-engineered human lymphocytes redirected by FRa-specific CARs.3. FR-BBz CAR T cell infusion mediated tumor regression in models of subcutaneous, metastatic intraperitoneal, and lung-involved human ovarian cancer.4. Chimeric antigen receptor containing 4-1BB (CD 137) costimulatory molecules showed enchanced anti-tumor activity and survival of transferred T cell in vivo.5. Clinical investigation of the safety of this novel T cell-based approach to FRαoverexpressed cancer treatment is warranted. BackgroundOverexpression of HER2/neu is identified in many cancer types including breast cancer, ovarian cancer, pancreatic cancer, colon cancer, non-small cell lung cancer (NSCLC), prostate cancer, osteosarcoma and osteosarcoma lung metastasis. HER2/neu overexpression correlated to poor prognosis and shorter overall survival of tumor patients.Thus, HER2/neu become an accepted tumor marker and attractive target for tumor immunotherapy.ObjectivesTo construct the HER2/neu specific chimeric antigen receptor containing CD3zeta signaling domain.To evaluate the anti-tumor activity of T cell expressing HER2/neu specific chimeric antigen receptor.Methods1. Sequence HER2/neu specific single-chain antibody (scFv) and repair the mutation points.2. Construction of HER2/neu specific chimeric antigen receptor and introduce the HER2/neu CAR into T cells using lentiviral technology.The transduce effiency was determined by flow cytometry.3. Evaluate the function of engineered T cells in vitro including cytokine release assay and cytotoxity assay.Results1. The anti-HER2/neu scFv has 6 mutation points and succefully modified by Multi Site-Directed Mutagenesis Kit.2. HER2/neu specific CAR was successfully constructed in lentiviral vector and human T lymphocytes can be engineered to express high level of HER2/neu specific CAR.3. T lymphocytes expressing HER2/neu specific chimeric antigen receptor can recognize the HER2/neu (+) tumor cell lines and secret Thl cytokines and can specifically kill HER2/neu (+) tumor cells.ConclusionsHuman T lymphocytes expressing HER2/neu specific CAR can specifically recognize and kill HER2 positive tumor cells. Our results provide an experimental basis for cancer immunotherapy. BackgroundThe potential of cancer adoptive celluar immunotherapy is promising. A crucial step for this strategy is the rapid, large-scale ex vivo expansion of tumor-reactive T cells. During the establishment of this approach, we noted large differences between results using different culture media to expand T cells.ObjectivesTo compare of three defined culture media, RPMI, AIM-V and OpTmizer, for T-cell expansion, phenotype, lentiviral transduction efficiency.MethodsPeripheral blood T cells were cultured in RPMI, AIM-V or OpTmizer without human AB serum or with 1% and 5% human AB serum.We examined the influence of culture medium on T-cell growth, phenotype, lentiviral transduction efficiency after activation using anti-CD3/CD28 beads. We also evaluated the function of transduced T cells by encoding FRαchimeric antigen receptor (CAR) lentivirus in different medium with 5% human AB serum.ResultsWithout serum supplement, only OpTmizer was a good culture medium for T-cell expansion following anti-CD3/CD28 beads stimulation. When supplemented with 1% human ABserum, OpTmizer and AIMV are better than RPMI for T-cell expansion following anti-CD3/CD28 beads stimulation. When supplemented with sufficient serum (5%), numbers of T cells obtained were similar in the different medium, but their phenotype and lentivirus transduction efficiency were quite different. RPMI and OpTmizer can expand more CD8+T cells and resulted in less CD4+FOXP3+Treg cells.The lentiviral transduction efficiency of human T cells is higher in AIMV medium compared to PRMI and OpTmizer and resulted in more cytokine release when stimulated by FRα(+) tumor cells.ConclusionsOur studies suggest that T-cell growth, phenotype, lentivirus transduction efficiency depends on different cluture medium. Thus, the selection of appropriate culture media to support efficient expansion of the type of T cell desired is crucial for engineered T cells cancer immunotherapy.