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Nishi-Tokyo-shi, Japan

Nicol A.J.,University of Queensland | Tokuyama H.,University of Queensland | Tokuyama H.,Tokyo Medical University | Mattarollo S.R.,University of Queensland | And 4 more authors.
British Journal of Cancer | Year: 2011

Background: Adoptive transfer of ex vivo expanded autologous Vγ9V2 T cells may be of therapeutic benefit for cancer because of their potent direct cytotoxicity towards tumour cells, synergistic cytotoxicity when combined with aminobisphosphonates and enhancement of antibody-dependent cell-mediated cytotoxicity. Methods: To determine the feasibility and clinical safety of therapy with ex vivo expanded, activated Vγ9V2 T cells in combination with zoledronate, we enrolled 18 subjects with advanced solid tumours into a phase I clinical study. Administered indium 111-oxine-labelled Vγ9V2 T cells were tracked in a cohort of patients. Results: Administered Vγ9V2 T cells had an activated effector memory phenotype, expressed chemokine receptors predictive of homing to peripheral tissues and were cytotoxic in vitro against tumour targets. Adoptively transferred Vγ9V2 T cells trafficked predominantly to the lungs, liver and spleen and, in some patients, to metastatic tumour sites outside these organs. No dose-limiting toxicity was observed, but most patients progressed on study therapy. However, three patients administered Vγ9V2 T cells while continuing previously ineffective therapy had disease responses, suggesting an additive effect. Conclusion: Therapy with aminobisphosphonate- activated Vγ9V2 T cells is feasible and well tolerated, but therapeutic benefits appear only likely when used in combination with other therapies. © 2011 Cancer Research UK All rights reserved.

Yamashiro H.,Medinet Medical Institute | Yoshizaki S.,Yokohama City University | Tadaki T.,Medinet Medical Institute | Egawa K.,Medinet Medical Institute | Seo N.,Hamamatsu University School of Medicine
Journal of Leukocyte Biology | Year: 2010

The BTN molecule consists of three subfamilies, BTN1, BTN2. and BTN3, and possesses interesting properties for biological regulation. Although the biological significance of BTN1 and BTN2 has been progressively clarified, the receptor function of BTN3 remains to be elucidated as a result of the absence of appropriate agonists. To clarify the participation of BTN3 in immune regulation, BTN3-specific mAb, referred to as 34-7 and 232-5, were generated from BTN3 gene-immunized mice. The 232-5 mAb, specific to the extracellular domain of the BTN3 molecule, stained almost all populations of human PBMCs, including T, NK, NKT, and B cells. Notably, treatment with the 232-5 mAb resulted in phosphorylation of BTN3A3 molecules, leading to attenuated proliferation and cytokine secretion by CD4+ and CD8+ T cells in a CD4+ CD25+ Treg cell-independent manner, demonstrating the agonistic property of the 232-5 mAb in BTN3-mediated negative signal transduction. The magnitude of the cell surface expression of BTN3 molecules correlated inversely with lymphocyte activity, suggesting that BTN3 molecules contribute to the maintenance of the immune system. Taken together, our findings provide new insights for the role of BTN3 as an inhibitor of excessive cellular immune responses. © Society for Leukocyte Biology.

Kuwada E.,Medinet Medical Institute | Tadaki T.,Medinet Medical Institute | Kambara K.,Medinet Medical Institute | Egawa K.,Medinet Medical Institute | And 2 more authors.
Biotechnology and Applied Biochemistry | Year: 2011

Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated β-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery. © 2011 International Union of Biochemistry and Molecular Biology, Inc.

Takahara M.,Medinet Medical Institute | Kanemura Y.,Osaka National Research Institute | Nieda M.,Medinet Medical Institute | Maekawa R.,Medinet Medical Institute | And 2 more authors.
Biotherapy | Year: 2010

In this study, we examined the ability of dendritic cells (DC) electroloaded with autologous tumor lysate to induce antigen-reactive IFN-γ-producing cells in comparison with the DC co-cultured with the tumor lysate. In four patients with glioblastoma, tumor lysate was prepared from surgically resected autologous tumor. Monocytes (CD14 positive) and lymphocytes (CD14 negative) were separated from peripheral blood mononuclear cells (PBMC). DC, which were differentiated from monocytes, were loaded with autologous tumor lysate by electroporation or co-culture, and then were mixed and cultured with autologous lymphocytes. The lymphocytes after cultivation were analyzed for their immune response against autologous tumor lysate by EUSPOT assay. The results showed that DC electroloaded with tumor lysate could elicit higher induction of tumor lysate-reactive IFN-γ-producing cells than DC co-cultured with the lysate in all four patients. In addition, the treatment of such DC with zoledronate enhanced the induction of antigen-reactive IFN-γ-producing cells.

Yoshikawa T.,Exploratory Oncology Research and Clinical Trial Center | Takahara M.,Medinet Medical Institute | Tomiyama M.,Medinet Medical Institute | Nieda M.,Medinet Medical Institute | And 2 more authors.
International Journal of Oncology | Year: 2014

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T-cell expansion. To induce expansion of γδ T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of γδ T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3-specific CTLs was approximately 24- to 170,000-fold. These CD8+ cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8- cells, including γδ T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8+ cells, CD8- cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of γδ T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.

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