Safety and persistence of WT1-specific T-cell receptor gene-transduced lymphocytes in patients with AML and MDS.

Wilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR-gene transduced T-cell (TCR-T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.

Donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene: detailed immunological function following add-back after haplo-identical transplantation.

BACKGROUND AIMS: Haplo-identical hematopoietic stem cell transplantation (HSCT) with add-back of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK cells) is one of the most widely applied promising new gene therapy approaches. However, the immunological status of added-back TK cells after HSCT has yet to be well characterized. METHODS: We investigated TK cells through the use of flow cytometry, T-cell receptor (TCR) Vß repertoire spectratyping and linear amplification-mediated polymerase chain reaction followed by insertion site analysis in a patient enrolled in our clinical trial. RESULTS: A comparison of onset with remission of acute graft-versus-host disease confirmed that TK cells were predominantly eliminated and that proliferative CD8(+) non-TK cells were also depleted in response to ganciclovir administration. The TCR Vß-chain repertoire of both TK cells and non-TK cells markedly changed after administration of ganciclovir, and, whereas the TCR repertoire of non-TK cells returned to a normal spectratype long after transplantation, that of TK cells remained skewed. With the long-term prophylactic administration of acyclovir, TK cells oligoclonally expanded and the frequency of spliced variants of TK cells increased. Known cancer-associated genes were not evident near the oligoclonally expanded herpes simplex virus (HSV)-TK insertion sites. CONCLUSIONS: We demonstrate obvious differences in immunological status between TK cells and non-TK cells. In addition, we speculate that long-term prophylactic administration of acyclovir increases the risk of oligoclonal expansion of spliced forms of TK cells.

NY-ESO-1-specific redirected T cells with endogenous TCR knockdown mediate tumor response and cytokine release syndrome.

BACKGROUND: Because of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive. METHODS: We conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide. RESULTS: In vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels. CONCLUSIONS: The trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS. TRIAL REGISTRATION NUMBER: NCT02366546.

Infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene for recurrent hematologic malignancies after allogeneic hematopoietic stem cell transplantation.

The infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK-cells) is a promising strategy for the treatment of hematologic malignancies relapsing after allogeneic hematopoietic stem cell transplantation. Here we report the results of a phase I clinical trial designed to examine the feasibility, safety, and efficacy of donor lymphocyte infusion (DLI) of TK-cells. Three patients (two with malignant lymphomas, one with acute myeloid leukemia) were enrolled in the trial and received a single DLI of 1 × 10(7) or 5 × 10(7) TK-cells/kg. No local or systemic toxicity related to the gene-transfer procedure was observed. Two patients achieved stable disease. No patient had severe graft-versus-host disease requiring systemic steroid and/or ganciclovir administration. TK-cells were detected in the peripheral blood of all three patients by PCR, but did not persist longer than 28 days. Analysis of cytotoxic T lymphocyte activity detected no immune response against TK-cells by the recipient's own T cells. Flow cytometric analysis showed low proliferative activity and cytotoxic function of TK-cells. In conclusion, DLI of TK-cells was safely performed in all three patients. Our analysis suggests the probable cause of rapid disappearance of TK-cells to be insufficient in vivo expansion of TK-cells in these patients.

Adoptive Transfer of MAGE-A4 T-cell Receptor Gene-Transduced Lymphocytes in Patients with Recurrent Esophageal Cancer.

PURPOSE: Preparative lymphodepletion, the temporal ablation of the immune system, has been reported to promote persistence of transferred cells along with increased rates of tumor regression in patients treated with adoptive T-cell therapy. However, it remains unclear whether lymphodepletion is indispensable for immunotherapy with T-cell receptor (TCR) gene-engineered T cells. EXPERIMENTAL DESIGN: We conducted a first-in-man clinical trial of TCR gene-transduced T-cell transfer in patients with recurrent MAGE-A4-expressing esophageal cancer. The patients were given sequential MAGE-A4 peptide vaccinations. The regimen included neither lymphocyte-depleting conditioning nor administration of IL2. Ten patients, divided into 3 dose cohorts, received T-cell transfer. RESULTS: TCR-transduced cells were detected in the peripheral blood for 1 month at levels proportional to the dose administered, and in 5 patients they persisted for more than 5 months. The persisting cells maintained ex vivo antigen-specific tumor reactivity. Despite the long persistence of the transferred T cells, 7 patients exhibited tumor progression within 2 months after the treatment. Three patients who had minimal tumor lesions at baseline survived for more than 27 months. CONCLUSIONS: These results suggest that TCR-engineered T cells created by relatively short-duration in vitro culture of polyclonal lymphocytes in peripheral blood retained the capacity to survive in a host. The discordance between T-cell survival and tumor regression suggests that multiple mechanisms underlie the benefits of preparative lymphodepletion in adoptive T-cell therapy.

Cloning and Expression of the Cytochrome P-450nor2 cDNA from Cylindrocarpon tonkinense.

Cytochrome P-450nor is involved in the fungal denitrification and acts as a nitric oxide reductase. The cDNA library from Cylindrocarpon tonkinense was constructed with lambdagtll, and screened with antibodies. From the positive clones, the P-450nor2 cDNA fragments were recovered, and subcloned into the expression vector pYES2, then expressed in the yeast system. Western blot analysis showed that the expressed protein was hybridized with the antibody. Enzyme assay indicated that the expressed protein had the activities of P-450nor2, which reduced NO to form N(2)O, employing NADH or NADPH as the sole electron donor.