Satoshi Kokura

3085 Background: Wilms’ tumor gene (WT1) is overexpressed in almost all malignancies including PDAC, and is supposed to be most promising tumor-associated antigen in cancer immunotherapies. To investigate superiority of WT1 vaccine in combination with gemcitabine (GEMWT1) compared to GEM alone (GEM) for patients (pts) with advanced PDAC, we designed the randomized phase II trial. Methods: HLA-A*02:01 and/or A*24:02-positive pts with locally advanced or metastatic PDAC without prior treatment, or recurrence after surgery were enrolled. Pts were randomly assigned at a ratio of 1:1 to GEMWT1 or GEM. Pts in GEM were allowed to receive GEM + WT1 vaccine after disease progression. The primary end point was overall survival (OS) rate at 1 year. Secondary end points were progression free survival (PFS), WT1-specific immunogenic response, and safety. WT1 vaccine, which was composed of HLA-restricted 9-mer WT1 peptide (3mg/body) and Montanide ISA51 adjuvant, was intradermally injected on days 1 and 15, and GEM was administrated at 1,000 mg/m² on days 1, 8, and 15 in a 28-day cycle until disease progression. Results: A total of 91 pts were enrolled and 85 evaluable finally (GEMWT1, n = 42; GEM, n = 43). GEMWT1 improved 1-yr OS rate and prolonged PFS compared to GEM (Table). These were remarkable in pts with metastatic PDAC. WT1-specific immunity was assessed with delayed-typed hypersensitivity (DTH) to WT1 peptide and tetramer assay of WT1-specific cytotoxic lymphocytes (WT1-CTLs). Median PFS was 195, 102, and 100 days for DTH(+), DTH(-), and GEM, respectively [HR (90% CI), 0.51 (0.31 to 0.85), 0.80 (0.50 to 1.28), and 1]. WT1-CTLs increased in almost all DTH(+) pts. Conclusions: GEM + WT1 vaccine prolonged PFS in pts with advanced PDAC. Clinical efficacy of this combination therapy was associated with the induction of WT1-CTL responses. Clinical trial information: UMIN000005248.

NK cells can destroy tumor cells without prior sensitization or immunization. Tumors often lose expression of MHC molecules and/or antigens. However, NK cells can lyse tumor cells in a non-MHC-restricted manner and independent of the expression of tumor-associated antigens. NK cells are therefore considered ideal for adoptive cancer immunotherapy; however the difficulty of obtaining large numbers of fully functional NK cells that are safe to administer deters its clinical use. This phase I clinical trial seeks to address this obstacle by first developing a novel system that expands large numbers of highly activated clinical grade NK cells, and second, determining if these cells are safe in a mono-treatment so they can be combined with other reagents in the next round of clinical trials. Patients with unresectable, locally advanced and/or metastatic digestive cancer who did not succeed with standard therapy were enrolled. NK cells were expanded ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296 induced T cells. Patients were administered autologous natural killer cell three times weekly via intravenous infusions in a dose-escalating manner (dose 0.5 × 109, 1.0 × 109, 2.0 × 109 cells/injection, three patients/one cohort). Total cell population had a median expansion of 586-fold (range 95–1102), with a significantly pure (90.96 %) NK cell population. Consequently, NK cells were expanded to approximately 4720-fold (range 1372–14,116) with cells being highly lytic in vitro and strongly expressing functional markers such as NKG2D and CD16. This NK cell therapy was very well tolerated with no severe adverse events. Although no clinical responses were observed, cytotoxicity of peripheral blood was elevated approximately twofolds up to 4 weeks post the last transfer. We successfully generated large numbers of activated NK cells from small quantities of blood without prior purification of the cells. We also determined that the expanded cells were safe to administer in a monotherapy and are suitable for the next round of clinical trials where their efficacy will be tested combined with other reagents. Trial Registration: UMIN UMIN000007527

Although treatment with an antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) combined with multiple therapeutic interventions has been explored, the effect of combination therapy with CTLA-4 inhibition and adoptive T-cell therapy has not been determined. In the present study, our aim was to determine whether CTLA-4 inhibition, combined with adoptive transfer of T cells at different stages of differentiation, exhibits synergistic antitumor effects in a murine colon cancer model. Mice bearing subcutaneous tumors were administered adoptive T-cell transfer of CD62Lhigh or CD62Llow cells combined with an anti-CTLA-4 antibody (α-CTLA-4) or control immunoglobulin G. Subcutaneous tumors were harvested, and the antitumor effects and helper T-cell polarization were analyzed. CTLA-4 inhibition combined with CD62Lhigh cell administration showed the strongest antitumor effect. Combination therapy increased the number of CD3+ cells within the tumor. Moreover, CTLA-4 inhibition induced polarization of T cells infiltrating the tumor toward the T helper 1 lineage, and suppressed the frequency of regulatory T cells within the tumor, particularly in combination with CD62Lhigh T-cell transfer. This is the first report demonstrating that the efficacy of α-CTLA-4 and adoptive T-cell transfer combination therapy depends on the state of differentiation of the transferred T cells. Our data support the notion that a combination of α-CTLA-4 and adoptive T-cell transfer containing an abundance of naïve phenotype cells could potentially exert antitumor effects in a clinical setting.

Ruptured esophageal varices (EV) are commonly associated with bleeding in patients with portal hypertension. As previous studies have been designed to include all viral cirrhosis patients, including those with small varices, there are no definitive reports of the risk factors for large EV (LEV) bleeding and subsequent mortality. In this study we sought to investigate these risk factors. 16 patients with initial episodes of LEV bleeding and 56 patients who had undergone initial preventive treatment for LEV were examined. The Child-Pugh score, MELD score, and their component serological factors were examined. The Child-Pugh score and the MELD score were significantly lower for preventive cases. Serum albumin concentration was significantly lower in bleeding cases, as determined by univariable and multivariable analyses. In initial LEV bleeding cases, higher MELD scores and Child-Pugh scores, and lower serum albumin concentration were associated with significantly higher one-year mortality. These results indicate that control of the MELD score, the Child-Pugh score, and serum albumin concentration is key to improving the prognosis of patients with LEV. Especially, control of serum albumin concentration may be critical for the prevention of initial episodes of LEV bleeding and improving subsequent mortality.

Previous studies have demonstrated that less-differentiated T cells are ideal for adoptive T cell transfer therapy (ACT) and that fibronectin CH296 (FN-CH296) together with anti-CD3 resulted in cultured cells that contain higher amounts of less-differentiated T cells. In this phase I clinical trial, we build on these prior results by assessing the safety and efficacy of FN-CH296 stimulated T cell therapy in patients with advanced cancer. Patients underwent fibronectin CH296-stimulated T cell therapy up to six times every two weeks and the safety and antitumor activity of the ACT were assessed. In order to determine immune function, whole blood cytokine levels and the number of peripheral regulatory T cells were analyzed prior to ACT and during the follow up. Transferred cells contained numerous less-differentiated T cells greatly represented by CD27+CD45RA+ or CD28+CD45RA+ cell, which accounted for approximately 65% and 70% of the total, respectively. No ACT related severe or unexpected toxicities were observed. The response rate among patients was 22.2% and the disease control rate was 66.7%. The results obtained in this phase I trial, indicate that FN-CH296 stimulated T cell therapy was very well tolerated with a level of efficacy that is quite promising. We also surmise that expanding T cell using CH296 is a method that can be applied to other T- cell-based therapies. UMIN UMIN000001835

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Epithelial-mesenchymal transition (EMT) is considered to be a crucial event in the development of cancer metastasis. Anoxia/reoxygenation (A/R) is known to occur in cancer tissues due to angiogenesis and changes in tissue pressure that occur during tumor growth. We investigated whether A/R induces EMT in the human colon cancer cell line HT-29. Colon cancer cells were exposed to anoxia (2 h) followed by reoxygenation (4-22 h) and evaluated for EMT changes using immunofluorescence and western blot analyses. We also investigated the expression of EMT-related transcription factors (Snail and ZEB1) using RT-PCR and evaluated the expression of NF-κB using ELISA. To determine whether NF-κB is involved in A/R-induced EMT, HT-29 cells were treated with proteasome inhibitors. Colon cancer cells exposed to A/R underwent EMT morphological changes; the cancer cells acquired a spindle-shaped phenotype. The expression of E-cadherin on the cell surface and the total amount of E-cadherin proteins were reduced after A/R. The expression of EMT-related transcription factors (Snail, ZEB1) was increased after A/R. Pretreatment with proteasome inhibitors significantly attenuated the downregulation of E-cadherin induced by A/R. These results indicate that A/R induces EMT in human colon cancer cells through an NF-κB-dependent transcriptional pathway.