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Adoptive transfer of MAGE-A4 T-cell receptor gene−transduced lymphocytes in patients with recurrent esophageal cancer.

Disease progression and overall survival of 10 patients who received transfer of TCR-transduced lymphocytes.

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. Clin Cancer Res; 21(10); 2268–77. ©2015 AACR.

Peptides which have homologue sequences with MAGE-A4143-151 peptide.

BackgroundBecause 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.MethodsWe 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.ResultsIn 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.ConclusionsThe 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 numberNCT02366546.

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.

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.

Background Adoptive transfer of TCR-redirected T cells has been reported to exhibit efficacy in some patients with melanoma and sarcoma. However, cytokine release syndrome (CRS) or its relations to tumor response has not been well documented. This study aimed to evaluate clinical responses in association with the cell kinetics and CRSs after transfer of high-affinity NY-ESO-1 TCR-gene transduced T cells in cancer patients. Methods We developed a novel-type affinity-enhanced NY-ESO-1-specific TCR and an originally-developed retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1/TCR sequence was mutated for high affinity with replacements of G50A and A51E in CDR2 region. This is a first-in-human clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It was designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3 + 3 cohort design. Cyclophosphamide (1,500mg/m2) were administered prior to the TCR-T cell transfer as pre-conditioning. Results Nine patients were treated with the TCR-T cells that expanded in peripheral blood with a dose-dependent manner, associated with rapid proliferation within 5 days after infusion. Three patients receiving 5x109 cells developed early-onset CRSs, with elevated levels of serum IL-6, IFN-γ. The CRSs on day1 or 2 were well managed with tocilizumab treatment. Three synovial sarcoma patients exhibited tumor shrinkage and partial responses, and they all had high-expression of NY-ESO-1 in the tumor samples, namely, 75% or more. Exploratory analysis revealed that multiple chemotactic cytokines including CCL2 and CCL7, and IL-3 increased in the serum from the patients with CRS. The proportions of effector-memory phenotype T cells in the infused cell-product were significantly associated with CRS development. Conclusions The affinity-enhanced NY-ESO-1/TCR-T cell transfer exhibited early-onset CRS in association with in vivo cell proliferation and sequential tumor responses in the patients with high-NY-ESO-1-expressing synovial sarcoma. Clinical trial identification NCT02366546. Legal entity responsible for the study The authors. Funding Japan Agency for Medical Research and Development. Disclosure All authors have declared no conflicts of interest.

2530 Background: Adoptive transfer of TCR-redirected T cells has been reported to exhibit efficacy in some of melanoma and sarcoma patients. However, there have not been well known about cytokine release syndrome (CRS) or its relations to tumor responses. This study evaluates clinical responses in association with the cell kinetics and CRSs after transfer of high-affinity NY-ESO-1 TCR-gene transduced T cells in NY-ESO-1-expressiong cancer patients (NCT02366546). Methods: We developed a novel-type affinity-enhanced NY-ESO-1-specific TCR and an originally-developed retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1/TCR sequence is mutated for high affinity with replacements of G50A and A51E in CDR2 region. This is a first-in-man clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It was designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3+3 cohort design. Cyclophosphamide (1,500mg/m2) were administered prior to the TCR-T cell transfer as pre-conditioning. Results: 9 patients were treated with the NY-ESO-1/TCR-T cell transfer. The TCR-T cells expanded in peripheral blood with a dose-dependent manner, associated with rapid proliferation within 5 days after the cell transfer. 3 patients receiving 5x109 cells developed early-onset CRSs, with elevations of serum IL-6, IFN-γ. The CRSs developed on day1 or 2 after the cell transfer. They were well managed with tocilizumab treatment. 3 synovial sarcoma patients exhibited tumor shrinkages of partial responses, and they all had high-expression of NY-ESO-1 in the tumor samples, namely, 75% or more. Exploratory analysis revealed that multiple chemotactic cytokines including CCL2 and CCL7, and IL-3 increased in the serum from the patients with CRS. The proportions of effector-memory phenotype T cells in the infused cell-product were significantly associated with CRS development. Conclusions: The affinity-enhanced NY-ESO-1/TCR-T cell transfer exhibited early-onset CRS in association with in vivo cell proliferation and sequential tumor responses in the patients with high-NY-ESO-1-expressing synovial sarcoma. Clinical trial information: NCT02366546.

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) Vβ 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 Vβ-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.

Adoptive cell transfers of receptor gene-engineered T cells include chimeric antigen receptor-gene transduced T (CAR-T) cell therapy and TCR-gene transduced T (TCR-T) cell therapy. In CD19-CAR-T cell therapy, high incidence of cytokine release syndrome (CRS) is associated with in vivo CAR-T cell proliferation and its clinical efficacy. In human TCR-T cell therapies, there have not been well known about CRS and its association with in vivo T cell kinetics or tumor responses. We have been developing a novel-type affinity-enhanced NY-ESO-1-specific TCR, and an original retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1 TCR is mutated for high affinity with replacements of G50A and A51E in CDR2 region, which is restricted with HLA-A*02:01 and A*02:06. We extensively examined potential cross-reactivities to different antigen-peptides in preclinical studies, and the high-affinity NY-ESO-1 TCR did not recognize analogous peptides. The new generation retroviral TCR-vector provides enhanced expression of transduced tumor-specific TCRs and an inhibition effect of formations of self-reactive TCRs. This is a first-in-man clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It is designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3+3 cohort design. Cyclophosphamide with/without fludarabine were administered prior to the TCR-T cell transfer as pre-conditioning. Six patients were treated with the NY-ESO-1 TCR-T cell transfer, and evaluated for the safety and in vivo cell kinetics. The TCR-T cells appeared in peripheral blood with a dose-dependent manner, associated with in vivo proliferation in an early phase. In three patients given 5x108 cells, no toxicities were seen. Two patients receiving 5x109 cells developed early-phase CRS (G2), with elevations of serum IL-6 and IFN-gamma. They were managed the treatment of anti-IL-6 receptor monoclonal antibody, tocilizumab. In a patient who developed CRS, an event of lung injury (G3) occurred, which was associated with marked infiltration of the NY-ESO-1 TCR-T cells. It was successfully treated with steroid. Two synovial sarcoma patients exhibited tumor responses of PRs. In one patient, progression-free survival lasted more than 8 months. In summary, the affinity-enhanced NY-ESO-1 TCR-T cell transfer exhibited CRSs in association with in vivo cell proliferation and sequential tumor responses. Disclosures No relevant conflicts of interest to declare.

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. Clin Cancer Res; 21(10); 2268–77. ©2015 AACR.

P450nor, nitric oxide reductase from Fusarium oxysporum, was expressed in the soluble fraction of Escherichia coli cells by modifying the N-terminal codons and utilizing the pCW vector. The modified P450nor purified to electrophoretic homogeneity had spectral and enzymatic properties identical to those of native P450nor obtained from the fungi. Residues 239 to 247 of the modified P450nor were replaced with Lys by site-directed mutagenesis. Thr-243 to His- and Arg-mutants were also created. Among 11 mutants, only the Thr-243 to Lys-mutant exhibited an absorption spectrum characteristic of a nitrogenous ligand-bound form of P450 at pH 8.0 in the ferric state, but the spectrum was altered to that of the wild-type P450nor as the pH was lowered. Other mutants had spectra typical of the low- and high-spin mixed form of P450 in the ferric state. In the ferrous state, all mutants showed the same spectrum as the wild-type P450nor. Nitric oxide reductase activity was considerably decreased by the replacement of Thr-243 with Lys, His, or Arg or Ala-239 with Lys. These findings indicate that Thr-243 is located more closely to the heme iron than other residues in the putative distal helix of P450nor and plays an important role in the catalytic activity, but a specific difference in the structure of the heme pocket from other P450s is suggested.

A unique cytochrome P-450 (P-450) is involved in fungal denitrification, acting as a nitric oxide reductase. The phylogenetic classification of the P-450 (P-450nor) into the group of bacterial P-450s along with its involvement in the anaerobic process are of evolutional interest. The corresponding gene, CYP 55, of the fungus Fusarium oxysporum was isolated and sequenced. The P-450nor gene contained 7 introns that consisted of 49 to 55 bp. The presence of the introns suggested that horizontal transfer of the gene from bacteria to the fungus, if it has occurred, was an early event in evolution. Besides a TATA box, several inverted repeats were found in the 5'-upstream flanking region. One inverted repeat exhibited the same sequence as the binding site of FNR (fumarate and nitrate reduction), a DNA-binding, O2 (dioxygen)-sensor protein that positively regulates expressions of many hypoxic genes in Escherichia coli and other bacteria. The result suggested that the expression of P450nor is regulated in response to oxygen tension by an FNR-like system. Northern blot analyses showed that both nitrate and nitrite are the actual inducers of P-450nor and that its expression is predominantly regulated at the transcriptional level. These results raise the interesting possibility that the expression of the fungal denitrification system is regulated, as in the case of bacterial nitrate respiration, by a set of mechanisms, i.e., a combination of an FNR-like system and a system responding to nitrate/nitrite.

Wilms' Tumor 1 (WT1) is expressed in a majority of MDS and AML cells and mRNA of WT1 in peripheral blood and bone marrow is monitored as a marker of minimal residual disease of AML and MDS. Several WT1 protein-derived epitopes that are recognized by cytotoxic T lymphocytes (CTLs) along with HLA molecules are determined. In vitro study and WT1 peptide vaccine trials have demonstrated that WT1-specfic CD8+ T cells with cytotoxic activity can be induced. Adoptive T cell therapy using ex vivo expanded WT1-specific CTLs or WT1-specific T-cell receptor (TCR)-gene transduced cells are potentially effective to refractory MDS and AML. Antigen-specific TCR-gene transfer may cause serious autoimmune disease by mispairing of introduced and endogenous TCR chains that recognize auto-antigens. We established a retroviral vector system encoding siRNAs for endogenous TCR genes to eliminate TCR-mispairing. Using the siRNA-encoding viral vector, we have conducted a first-in-man trial of WT1-specfic TCR-gene transduced T cell transfer. In the trial, we evaluate the safety of the TCR T cell transfer in patients with MDS and AML, and assess in vivo kinetics of the transferred cells. The study was designed as cell-dose escalation with three cohorts of 2x108, 1x109, and 5x109cells per infusion. Peripheral blood mononuclear cells were collected from each patient. Then, the cells were cultured with IL-2, anti-CD3 antibody, and RetroNectin®. Proliferating lymphocytes were infected with a retroviral vector, MS3-WT1-siTCR, which was constructed from DNA encoding WT1235-243/HLA*A24:02 specific TCR-α and -β chains and siRNAs for endogenous TCR genes. After 13-14 days in culture, the lymphocytes were harvested and frozen until infusion. Patients were enrolled to the clinical trial if they were refractory AML or MDS ineligible for allogeneic stem cell transplant, positive for HLA-A*24:02, had performance status of 0 to 2, and had normal organ function. WT1-TCR T cells were infused intravenously twice on days 0 and 28. Modified WT1235-243peptide (300μg) emulsified with Montanide, was given subcutaneously on day 2 and 16 after the second infusion. To date, 5 patients (4 MDS and 1 AML cases) with a median age of 69 years, received WT1-TCR T cells. Three received 2x108 cells (cohort 1) and 2 received 1x109 cells (cohort 2) per infusion, respectively. We did not see any severe adverse events related to the cell infusion or peptide vaccination. No renal or mesothelial damages were observed. We then assessed transduced TCR-gene copy numbers in peripheral blood samples collected at multiple pre-determined time points until day 58.TCR-gene marked cells were detected in all patients after the cell infusion. They appeared immediately after the infusion, reaching peak levels between 1 and 3 days. Then, the levels gradually declined. After the second infusion, which was followed by peptide vaccination, the cells appeared in the similar way to the first cycle. The peptide vaccine did not seem to affect the peripheral cell kinetics. Dose-dependent kinetics were shown between the cohort 1(2 x108 cells) and the cohort 2 (1x109cells). In two patients, transient decline of peripheral abnormal cells that were MDS-related erythroblasts, and decrease of bone marrow blasts were observed, respectively. Although the clinical trial is still ongoing, transfer of WT1-TCR-gene transduced lymphocyte to MDS and AML patients is safe and tolerable. TCR- T cells appeared in peripheral blood with cell-dose dependent manner. Disclosures Fujiwara: Celgene: Honoraria, Other: Travel, Acomodations, Expenses. Akatsuka:Takara Bio. Inc.: Other: Advisor to the CAR project. Tomura:TAKARO BIO INC.: Employment. Nukaya:TAKARA BIO INC.: Employment. Takesako:TAKARA BIO INC.: Employment.

Introduction: Engineering an antigen receptor gene in patients' lymphocytes is one promising strategy to create antigen-specific lymphocytes without senescent phenotypes. The strategy provides an opportunity to extend the application of adoptive T cell therapy for cancer patients. However, this concept has not been tested in epithelial cancer patients. Material and methods: MAGE-A4-specific TCR α and β chains were isolated from a human T cell clone that recognizes MAGE-A4 143-151 peptide in an HLA-A*24:02 restricted manner. This T cell clone did not show any cross reactivity to the peptides with a homology to the MAGE-A4 epitope. A retroviral vector that encodes these TCR chains without any artificial modification was constructed; the lymphocytes transduced with the retroviral vector killed the MAGE-A4 expressing tumor in vitro and inhibited the tumor growth in the NOG immmunodeficient mice. Patients were eligible if they had previously-treated recurrent MAGE-A4-expressing esophageal cancer, and were positive for HLA-A*24;02. Lymphocytes harvested from the patients were infected with the retroviral vector. The TCR-gene transduced T lymphocytes were once transferred to the patients without lymphocyte-depleting treatment, and MAGE-A4 peptide was given 2 and 4 weeks after. The cell doses were divided into 3 cohorts of 2x108 1x109 and 5x109, with a dose-escalating design, by evaluating the safety. Results: 10 patients received the TCR-gene transduced T lymphocytes. No adverse events related to the cell transfer were observed. The TCR-gene transduced lymphocytes were detected in their peripheral blood in all 10 patients, which showed a dose-dependent appearance during the first 14 days, reaching peak and plateau levels from 3 to 7 days, and declined within 14 days. The cells persisted at 0.5% to 1% level in the peripheral mononuclear cells from day 14 to 63 after the cell transfer. In 6 patients whose blood samples had been collected for over 6 months, 3 patients maintained stable levels as long as 16 months, maintaining the immune reactivity to MAGE-A4-expressing tumor cells. In one patient, whose esophageal tumor was biopsied after the transfer, the TCR-gene transduced cells were detected in the tumor site. 7 patients developed tumor progressions within 2 months after the transfer. Their overall survivals were ranged from 3 to 18+ months, with a median of 10. 3 patients who had minimal tumor lesions at baseline have been free from disease-progression for 12, 15, and 19 months, respectively. Conclusion: Wild-type TCR-gene transduced lymphocytes targeting MAGE-A4 antigen were safely given to refractory esophageal cancer patients. The cells persisted in their peripheral blood in a dose-dependent manner in the early phase, and they have been stably persisting over 6 months. Three patients are free from disease progression more than a year. These results encourage us to proceed to further phase trials. Citation Format: Shinichi Kageyama, Hiroaki Ikeda, Naoko Imai, Mikiya Ishihara, Yoshihiro Miyahara, Shugo Ueda, Takeshi Ishikawa, Hiroaki Naota, Kohshi Ohishi, Taizo Shiraishi, Naoki Inoue, Masashige Tanabe, Tomohide Kidokoro, Hirofumi Yoshioka, Daisuke Tomura, Ikuei Nukaya, Junichi Mineno, Kazutoh Takesako, Naoyuki Katayama, Hiroshi Shiku. Adoptive transfer of wild-type TCR gene transduced T lymphocytes targeting MAGE-A4 antigen to patients with refractory esophageal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT212. doi:10.1158/1538-7445.AM2014-CT212

The application of adoptive immunotherapy with tumor-specific T cells has been limited because of the short life span of the transferred T cells unless the host has been manipulated. Engineering the antigen receptor gene in patients’ lymphocytes is one promising strategy to create antigen-specific lymphocytes without senescent phenotypes. The strategy provides an opportunity to broaden the types of cancer to be treated. However, this concept has not been tested in the epithelial cancer patients. We completed a phase I clinical trial of TCR gene therapy targeting MAGE-A4 to treat esophageal cancer patients without lympho-depleting pre-conditioning. The trial was designed as a cell-dose escalation consisting of three cohorts, 2x10E8, 1x10E9 and 5x10E9 cells/patient. The treatment was tolerable with no adverse events associated with transferred cells. In all ten patients of the 3 cell-doses, the transferred lymphocytes were detected in their peripheral blood in a dose-dependent manner during the first 14 days. In 4 patients, the infused cells have been persisting more than 5 months after the transfer. The T cell clones were established from the transferred lymphocytes that were harvested more than 100 days after the transfer. These clones sustained the reactivity to the antigen-expressing tumor cells. Three patients showed SD or long tumor free status. These results suggest that this approach may extend the availability of adoptive T cell therapy for epithelial cancer patients by providing tumor-reactive and long surviving lymphocytes reducing the risk of intensive pre-treatments.

The cDNAs and genes for two isozymes of cytochrome P450nor of the fungus Cylindrocarpon tonkinense , P450nor1 and P450nor2, were cloned and sequenced. Their deduced amino acid sequences respectively showed 83 and 70% identity to that of P450nor of Fusarium oxysporum , and 69% identity to each other. The genes for P450nor1 and P450nor2 were termed, respectively, CYP 55A2 and CYP 55A3 . The cDNA for P450nor1 contained a targeting-like presequence upstream the N-terminus of mature protein whereas that for P450nor2 did not, suggesting their different intracellular localisations. We also succeeded in expressing these P450nor isoforms in the host-vector system of the yeast Saccharomyces cerevisiae . We purified one of the recombinant proteins, P450nor of F oxysporum . Little difference could be observed between the native and recombinant proteins in catalytic and spectroscopic properties. We constructed chimeric proteins of P450nor of F oxysporum and P450nor2 which are different in their specificity against the electron donors: reduced pyridine nucleotides. The specificity of chimeric proteins against NADH/NADPH showed that the specificity is determined by the N-terminal half of protein. We found a consensus amino acid sequence between three isoforms of P450nor, A-X-G-X-X-A, similar to the NAD-binding motif G-X-G-X-X-G/A in the region that corresponds to the B′-helix in P450cam.

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.

Levels of transcripts initiated at a hut promoter in Bacillus subtilis were analysed. The addition of histidine to the culture medium increased the level of the transcript sixfold. In the presence of histidine and glucose together, the level of the transcript was reduced to the level in the absence of induction. Furthermore, addition of a mixture of 16 amino acids to cultures of induced cells and of catabolite-repressed cells decreased levels of the transcript 16-fold and 2.6-fold, respectively. Thus, it appears that at least three regulatory mechanisms associated with induction, catabolite repression, and amino acid repression, control the transcriptional activity of the hut promoter. Expression of the hut promoter-lacZ fusions that contained various regions of the hutP gene and deletion analysis of the hutP region revealed a cis-acting sequence associated with catabolite repression that was located between positions +204 and +231 or around position +203.

A cDNA clone for the nitrate/nitrite-inducible cytochrome P-450 (P-450) of the fungus Fusarium oxysporum (tentatively termed P-450dNIR) was isolated by an immunoscreening method. Sequence determination revealed a polypeptide of 403 amimo acid residues (Mr = 44,371), which was shown to contain the full-length sequence of the fungal P-450. The amino terminus region of the predicted sequence contained neither the signal-like, hydrophobic domain that is commonly observed in microsomal P-450s nor the tagging prosequence that is essential for localization of mitochondrial P-450s. Further, the sequence exhibited higher homologies against those of soluble bacterial P-450s, in particular P-450s of Streptomyces, rather than those of eukaryotic P-450s including yeast and fungal P-450s. These results are highly indicative that P-450dNIR is the first soluble P-450 derived from eukaryotic organisms. The unique features might be related to the novel function of P-450dNIR, which is involved in a dissimilatory reduction of nitrite by the fungus. P-450dNIR was classified into a new family, P-450LV, and the corresponding gene of the fungus was named CYP55.

Meeting abstracts Engineering the antigen receptor gene in patients' lymphocytes is one promising strategy to create antigen-specific lymphocytes without senescent phenotypes. The strategy provides an opportunity to extend the application of adoptive T cell therapy for cancer patients. However