Tetsuro Sasada

Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery

Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(D,L-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp100(25-33) and TRP2(180-188), were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freunds adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2(180-188) and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.

Peptide-Induced Negative Selection of Thymocytes Activates Transcription of an NF-ΚB Inhibitor

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-kappa B, I kappa BNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to I kappa B family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of I kappa BNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. I kappa BNS blocks transcription from NF-kappa B reporters, alters NF-kappa B electrophoretic mobility shifts, and interacts with NF-kappa B proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of I kappa BNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.

Biologic activity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic stem cell transplantation

Through an immune-mediated graft-versus-leukemia effect, allogeneic hematopoietic stem cell transplantation (HSCT) affords durable clinical benefits for many patients with hematologic malignancies. Nonetheless, subjects with high-risk acute myeloid leukemia or advanced myelodysplasia often relapse, underscoring the need to intensify tumor immunity within this cohort. In preclinical models, allogeneic HSCT followed by vaccination with irradiated tumor cells engineered to secrete GM-CSF generates a potent antitumor effect without exacerbating the toxicities of graft-versus-host disease (GVHD). To test whether this strategy might be similarly active in humans, we conducted a Phase I clinical trial in which high-risk acute myeloid leukemia or myelodysplasia patients were immunized with irradiated, autologous, GM-CSF-secreting tumor cells early after allogeneic, nonmyeloablative HSCT. Despite the administration of a calcineurin inhibitor as prophylaxis against GVHD, vaccination elicited local and systemic reactions that were qualitatively similar to those previously observed in nontransplanted, immunized solid-tumor patients. While the frequencies of acute and chronic GVHD were not increased, 9 of 10 subjects who completed vaccination achieved durable complete remissions, with a median follow-up of 26 months (range 12–43 months). Six long-term responders showed marked decreases in the levels of soluble NKG2D ligands, and 3 demonstrated normalization of cytotoxic lymphocyte NKG2D expression as a function of treatment. Together, these results establish the safety and immunogenicity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic HSCT, and raise the possibility that this combinatorial immunotherapy might potentiate graft-versus-leukemia in patients.

A Critical Role for CD2 in Both Thymic Selection Events and Mature T Cell Function

To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2−/− H-2b mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2Kb molecule were compared on a wild-type or CD2−/− background. In N15tg RAG-2−/− CD2−/− mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4−CD8− double-negative thymocytes at the CD25+CD44− stage. Moreover, mature N15tg RAG-2−/− CD2−/− T cells are ∼100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-γ production. Repertoire analysis shows substantial differences in Vα usage between non-tg C57BL/6 (B6) and B6 CD2−/− mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.

Overcoming the hurdles of randomised clinical trials of therapeutic cancer vaccines

Most of the recent randomised clinical trials of therapeutic cancer vaccines have failed to demonstrate a meaningful therapeutic benefit to patients over existing treatments. Furthermore, some clinical trials have demonstrated a detrimental effect on patients, resulting in poorer outcomes. These unexpected results have shed light on several important issues to be solved for further development of cancer vaccines. As has been discussed with respect to the use of granulocyte-macrophage colony-stimulating factor (GM-SCF) as an adjuvant, the failures of clinical trials may be explained, in part, by a vaccine-specific adverse event, i.e. the induction of an inconvenient immune response that inhibits pre-existing host immunity. This hypothesis may be supported by the fact that randomised trials of personalised peptide vaccines that were selected in consideration of pre-existing host immunities in individual patients resulted in clear benefit to patients. The development of reliable biomarkers for the selection of appropriate patients and vaccine antigens would thus be pivotal to prevent such vaccine-specific adverse events. This article discusses possible ways to overcome the hurdles of randomised clinical trials of therapeutic cancer vaccines based on a review of recently conducted clinical trials.

Adoptive Transfer of Invariant NKT Cells as Immunotherapy for Advanced Melanoma: A Phase I Clinical Trial

Purpose: Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFNγ production, and iNKT IFNγ production may stratify for survival. Previous clinical trials using iNKT cell activating ligand α-galactosylceramide have shown clinical responses. Therefore, a phase I clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB–IV melanoma. Experimental Design: Residual iNKT cells [<0.05% of patient peripheral blood mononuclear cell (PBMC)] were purified from autologous leukapheresis product using an antibody against the iNKT cell receptor linked to magnetic microbeads. iNKT cells were then expanded with CD3 mAb and IL2 in vitro to obtain up to approximately 109 cells. Results: Expanded iNKT cells produced IFNγ, but limited or undetectable IL4 or IL10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1–2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMCs after some infusions, particularly when GM-CSF was also given. IFNγ responses to α-galactosylceramide were increased in PBMCs from some patients after infusions, and delayed-type hypersensitivity responses to Candida increased in 5 of 8 evaluated patients. Three patients have died, three were progression-free at 53, 60, and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters. Conclusions: Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with antitumor potential. Clin Cancer Res; 23(14); 3510–9. ©2017 AACR.

Involvement of the TCR Cβ FG Loop in Thymic Selection and T Cell Function

The asymmetric disposition of T cell receptor (TCR) Cβ and Cα ectodomains creates a cavity with a side-wall formed by the rigid Cβ FG loop. To investigate the significance of this conserved structure, we generated loop deletion (βΔFG) and βwt transgenic (tg) mice using the TCR β subunit of the N15 CTL. N15βwt and N15βΔFG H-2b animals have comparable numbers of thymocytes in S phase and manifest developmental progression through the CD4−CD8− double-negative (DN) compartment. N15βΔFG facilitates transition from DN to CD4+8+ double-positive (DP) thymocytes in recombinase activating gene (RAG)-2−/− mice, showing that pre-TCR function remains. N15βΔFG animals possess ∼twofold more CD8+ single-positive (SP) thymocytes and lymph node T cells, consistent with enhanced positive selection. As an altered Vα repertoire observed in N15βΔFG mice may confound the deletions effect, we crossed N15αβ TCR tg RAG-2−/− with N15βΔFG tg RAG-2−/− H-2b mice to generate N15αβ RAG-2−/− and N15αβ.βΔFG RAG-2−/− littermates. N15αβ.βΔFG RAG-2−/− mice show an 8–10-fold increase in DP thymocytes due to reduced negative selection, as evidenced by diminished constitutive and cognate peptide-induced apoptosis. Compared with N15αβ, N15αβ.βΔFG T cells respond poorly to cognate antigens and weak agonists. Thus, the Cβ FG loop facilitates negative selection of thymocytes and activation of T cells.

The TCR Cβ FG Loop Regulates αβ T Cell Development

The TCRβ chain constant domain contains an unusually elongated, solvent-exposed FG loop. This structural element forms one component of an αβ TCR cavity against which CD3εγ may abut to facilitate Ag-specific signaling. Consistent with this notion, in the present study we show that N15αβ TCR transfectants expressing a FG loop-deleted chain (βΔFG) stimulate less tyrosine protein phosphorylation than those bearing a wild-type β-chain (βwt) upon TCR cross-linking. Furthermore, coimmunoprecipitation studies suggest a weakened association between the CD3εγ heterodimer and the β-chain in TCR complexes containing the βΔFG variant. To further investigate the biologic role of the Cβ FG loop in development, we competitively reconstituted the thymus of Ly5 congenic or RAG-2−/− mice using bone marrow cells from βwt or βΔFG transgenic C57BL/6 (B6) mice. Both βwt and βΔFG precursor cells generate thymocytes representative of all maturational stages. However, βΔFG-expressing thymocytes dominate during subsequent development, resulting in an excess of βΔFG-expressing peripheral T cells with reduced proliferative and cytokine production abilities upon TCR stimulation. Collectively, our results show that the unique Cβ FG loop appendage primarily controls αβ T cell development through selection processes.

Thymic selection is influenced by subtle structural variation involving the p4 residue of an MHC class I-bound peptide.

The T lineage repertoire is shaped by opposing processes of positive and negative selection. To probe the specificity of selection, N15 TCR‐transgenic (tg) recombinase‐activating gene (RAG)‐2–u2009/u2009– H‐2b mice recognizing the VSV8 octapeptide RGYVYQGL bound to Kb were utilized in conjunction with VSV8 variants differing only at the central p4 position. The V4I mutant octamer, like VSV8, induces negative selection of immature double‐positive thymocytes on the β2‐microglobulin (β2M)+u2009/u2009+ background and is a strong agonist for mature N15 T cells. In contrast, V4L or V4norvaline octamers promote positive selection in N15tg RAG‐2–u2009/u2009– β2M–u2009/u2009– H‐2b fetal thymic organ culture and are weak agonists for N15 T cells. Hence, the absence of a p4 side chain Cβ‐methyl group results in positive selection of the N15 TCR. Hydrophobicity of the p4 residues also modulates thymocyte fate: the positively selecting norvaline and leucine variants have one and two Cγ‐methyl groups, respectively, while the weakly selecting γ‐methylleucine p4 contains three Cγ‐methyl groups. Moreover, the most hydrophobic octamer containing p4 cyclohexylglycine substitution fails to select. Thus, for N15 and presumably other MHC class I‐restricted TCR, there is a high degree of structural specificity to peptide‐dependent thymic selection processes.

Prognostic value of SUVmax measurements obtained by FDG-PET in patients with non-small cell lung cancer receiving chemotherapy.

[(18)F]Fluorodeoxyglucose (FDG) uptake has been shown to correlate well with tumor proliferation rates. In patients with non-small cell lung cancer (NSCLC) receiving chemotherapy, we analyzed the relationships between the maximum standardized uptake value (SUVmax) obtained by FDG positron emission tomography (FDG-PET) and other clinical factors, and examined whether or not SUVmax could predict progression-free survival (PFS) and/or overall survival (OS). This retrospective study involved 62 consecutive NSCLC patients (35 male and 27 female: median age, 65 years). All patients underwent FDG-PET examination before treatment. As the first-line treatment, the patients received chemotherapy with (n=15) or without (n=47) radiotherapy. Survival curves were obtained by the Kaplan-Meier method, and differences in survival between subgroups were analyzed by the log-rank test and the Cox proportional hazards model. Significant correlations were observed between SUVmax and gender (P=0.006), histology (P<0.001), smoking status (P=0.049), stage (P=0.015), and treatment modality (P=0.008), but not other factors, including age (P=0.402) and performance status (P=0.421). The median SUVmax was 5.1 (25-75th percentile: 3.45-7.0) in patients with adenocarcinoma and 8.3 (25-75th percentile: 6.9-9.9) in those with other types of NSCLC. Adenocarcinomas showed significantly lower SUVmax than the other tumor types (P<0.001). Cox analysis adjusting for possible confounding factors, including gender, smoking status, histology and stage, demonstrated that the hazard ratios increased as the SUVmax increased in terms of both PFS (P=0.008) and OS (P=0.045), indicating that SUVmax predicts outcome independently of other clinical factors, such as histology and stage. Our findings indicate that FDG-PET examination can provide information useful for prognostication in NSCLC.