Luminescence imaging during proton-beam irradiation is promising as an effective method for range estimation in proton therapy.

Purpose: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4 + T cells in HLA-A2402-positive patients with therapyrefractory HER2-expressing cancers. Experimental Design: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 Ag of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4 + T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein.Results: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade1transient skin reaction at the sites of vaccination. HER2-specific CD8 + and/or CD4 + T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom bothT-cell responses were detected in these patients. In four patients with CD8 + T-cell responses, two patients reacted to previously identified HER2 63-71 peptide and the other two reacted only to 146HER2 mRNA-transduced cells. Conclusions: CHP-HER2 vaccine was safe and induced HER2-specific CD8 + and/or CD4 + T-cell immune responses.

Purpose: For identification of CTL epitopes useful for cancer vaccines, it is crucial to determine whether cognate epitopes are presented on the cell surface of target cancer cells through natural processing of endogenous proteins. For this purpose, we tried to use the cellular machinery of both mice and human to define naturally processed CTL epitopes derived from two ''cancer germ line''genes, MAGE-A4 and SAGE. Experimental Design: We vaccinated newly produced HLA-A2402 transgenic mice with DNA plasmids encoding target antigens. Following screening of synthesized peptides by splenic CD8 + Tcells of vaccinated mice, we selected candidate epitopes bound to HLA-A2402.We then examined whether human CD8 + T cells sensitized with autologous CD4 + PHA blasts transduced by mRNA for the cognate antigens could react with these selected peptides in an HLA-A2402-restricted manner. Results: After DNAvaccination, murine CD8 + Tcells recognizing MAGE-A4 143-151 or SAGE [715][716][717][718][719][720][721][722][723] in an HLA-A2402-restricted manner became detectable. Human CTLs specific for these two peptides were generated after sensitization of HLA-A2402-positive CD8 + Tcells with autologous CD4 + PHA blasts transduced with respective mRNA. CTL clones were cytotoxic toward tumor cell lines expressing HLA-A2402 and cognate genes.Taken together, these CTL epitopes defined in HLA-A24 transgenic mice are also processed and expressed with HLA-A2402 in human cells. The presence of SAGE 715-723 -specific precursors was observed in HLA-A2402-positive healthy individuals. Conclusions:Two novel HLA-A2402-restricted CTL epitopes, MAGE-A4 143-151 and SAGE 715-723 , were identified. Our approach assisted by cellular machinery of both mice and human could be widely applicable to identify naturally processed CTL epitopes.

The recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a single chain variable fragment (scFv) specific to the WT1/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination.

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