Hirokazu Matsushita

Adoptive immunotherapy for advanced non-small cell lung cancer using zoledronate-expanded γδTcells: a phase I clinical study.

Human &ggr;&dgr; T cells can recognize and kill non-small cell lung cancer (NSCLC) cells using the V&ggr;9V&dgr;2 T-cell receptor and/or NKG2D. We have established clinical grade large-scale ex vivo expansion of &ggr;&dgr; T cells from peripheral blood mononuclear cells by culturing with zoledronate and interleukin-2 (IL-2). A phase I study was conducted to evaluate safety and potential antitumor effects of re-infusing ex vivo expanded &ggr;&dgr; T cells in patients with recurrent or advanced NSCLC. Patients peripheral blood mononuclear cells were stimulated with zoledronate (5 &mgr;M) and IL-2 (1000 IU/mL) for 14 days. Harvested cells, mostly &ggr;&dgr; T cells, were given intravenously every 2 weeks without additional IL-2, a total of 6 times. The cumulative number of transferred &ggr;&dgr; T cells ranged from 2.6 to 45.1×109 (median, 15.7×109). Fifteen patients underwent adoptive immunotherapy with these &ggr;&dgr; T cells. There were no severe adverse events related to the therapy. Immunomonitoring data showed that with increasing numbers of infusions, the number of peripheral &ggr;&dgr; T cells gradually increased. All patients remained alive during the study period with a median survival of 589 days and median progression-free survival of 126 days. According to the Response Evaluation Criteria In Solid Tumors, there were no objective responses. Six patients had stable disease, whereas the remaining 6 evaluable patients experienced progressive disease 4 weeks after the sixth transfer. We conclude that adoptive transfer of zoledronate-expanded &ggr;&dgr; T cells is safe and feasible in patients with NSCLC, refractory to other treatments.

Adoptive cytotoxic T lymphocyte therapy triggers a counter‐regulatory immunosuppressive mechanism via recruitment of myeloid‐derived suppressor cells

Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T‐cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel‐1‐specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor‐bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN‐γ, exerted anti‐tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b+Gr1+ myeloid‐derived suppressor cells (MDSCs). Notably, CD11b+Gr1intLy6G−Ly6C+ monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen‐specific CD8+ T cells. The anti‐tumor activity of the adoptively‐transferred CTLs and the accumulation of MDSCs both depended on IFN‐γ production on recognition of tumor antigens by the former. In CCR2−/− mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter‐regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment‐induced recruitment of these MDSCs would improve the efficacy of immunotherapy.

Cytotoxic T Lymphocytes Block Tumor Growth Both by Lytic Activity and IFNγ-Dependent Cell-Cycle Arrest

Matsushita and colleagues used microarray gene expression analysis coupled with fluorescent ubiquitination-based cell-cycle indicator (fucci) analysis and flow cytometry of a B16 murine model of melanoma to demonstrate that the predominant mechanism of CTL therapy in regulating tumor growth is via IFNγ-mediated cell-cycle arrest. To understand global effector mechanisms of CTL therapy, we performed microarray gene expression analysis in a murine model using pmel-1 T-cell receptor (TCR) transgenic T cells as effectors and B16 melanoma cells as targets. In addition to upregulation of genes related to antigen presentation and the MHC class I pathway, and cytotoxic effector molecules, cell-cycle–promoting genes were downregulated in the tumor on days 3 and 5 after CTL transfer. To investigate the impact of CTL therapy on the cell cycle of tumor cells in situ, we generated B16 cells expressing a fluorescent ubiquitination-based cell-cycle indicator (B16-fucci) and performed CTL therapy in mice bearing B16-fucci tumors. Three days after CTL transfer, we observed diffuse infiltration of CTLs into the tumor with a large number of tumor cells arrested at the G1 phase of the cell cycle, and the presence of spotty apoptotic or necrotic areas. Thus, tumor growth suppression was largely dependent on G1 cell-cycle arrest rather than killing by CTLs. Neutralizing antibody to IFNγ prevented both tumor growth inhibition and G1 arrest. The mechanism of G1 arrest involved the downregulation of S-phase kinase-associated protein 2 (Skp2) and the accumulation of its target cyclin-dependent kinase inhibitor p27 in the B16-fucci tumor cells. Because tumor-infiltrating CTLs are far fewer in number than the tumor cells, we propose that CTLs predominantly regulate tumor growth via IFNγ-mediated profound cytostatic effects rather than via cytotoxicity. This dominance of G1 arrest over other mechanisms may be widespread but not universal because IFNγ sensitivity varied among tumors. Cancer Immunol Res; 3(1); 26–36. ©2014 AACR. See related commentary by Riddell, p. 23

Intraperitoneal injection of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of malignant ascites due to gastric cancer

Malignant ascites caused by peritoneal dissemination of gastric cancer is chemotherapy‐resistant and associated with poor prognosis. We conducted a pilot study to evaluate the safety of weekly intraperitoneal injections of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of such malignant ascites. Patient peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and interleukin‐2 (1000 IU/mL). After 14 days culture, Vγ9Vδ2 T‐cells were harvested and administered intraperitoneally in four weekly infusions. The day before T‐cell injection, patients received zoledronate (1 mg) to sensitize their tumor cells to Vγ9Vδ2 T‐cell recognition. Seven patients were enrolled in this study. The number of Vγ9Vδ2 T‐cells in each injection ranged from 0.6 to 69.8 × 108 (median 59.0 × 108). There were no severe adverse events related to the therapy. Intraperitoneal injection of Vγ9Vδ2 T cells allows them access to the tumor cells in the peritoneal cavity. The number of tumor cells in the ascites was significantly reduced even after the first round of therapy and remained substantially lower over the course of treatment. IFN‐γ was detected in the ascites on treatment. Computed tomography revealed a significant reduction in volume of ascites in two of seven patients. Thus, injection of these antitumor Vγ9Vδ2 T‐cells can result in local control of malignant ascites in patients for whom no standard therapy apart from paracentesis is available. Adoptively transferred Vγ9Vδ2 T‐cells do indeed recognize tumor cells and exert antitumor effector activity in vivo, when they access to the tumor cells.

Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

Human γδ T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity. The majority of γδ T cells in peripheral blood have the Vγ9Vδ2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions. Therefore, γδ T cells are attractive candidate effector cells for cancer immunotherapy. Vγ9Vδ2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis; and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway. In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of γδ T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and γδ T cells activation. Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to γδ T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransformed cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating γδ T cells with aminobisphosphonates. Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. Monocytes that accumulate IPP become antigen-presenting cells and stimulate Vγ9Vδ2 T cells in the peripheral blood. Based on these mechanisms, we developed a technique for large-scale expansion of γδ T cell cultures using zoledronate and interleukin-2 (IL-2). Other methods for expansion of γδ T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP) or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). All of these methods allow ex vivo expansion, resulting in large numbers of γδ T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded γδ T cells display CD27(-)CD45RA(-) effector memory phenotype and thier function can be evaluated by IFN-γ production assay.

γδ T cell therapy for the treatment of non-small cell lung cancer

γδ T cells are attractive effector cells for cancer immunotherapy as they can secrete cytokines abundantly and exert potent cytotoxicity against a wide range of cancer cells. They comprise 1-5% of peripheral blood T cells, the majority expressing the Vγ9Vδ2 T cell receptor that recognizes phosphoantigens. Direct in vivo activation of Vγ9Vδ2 T cells in cancer patients as well as adoptive transfer of ex vivo expanded Vγ9Vδ2 T cells has been investigated in several clinical trials. We previously established a large-scale in vitro expansion method for Vγ9Vδ2 T cells using zoledronate and interleukin-2 (IL-2). We found that Vγ9Vδ2 T cells from patients with advanced cancer as well as from healthy donors underwent extensive proliferation under these conditions. Such cultured Vγ9Vδ2 T cells retained cytokine secretion capacity and mediated cytotoxicity against a variety of cancer cell lines. Recently, we conducted a phase I clinical study to evaluate safety and potential anti-tumor effects of re-infusing ex vivo expanded γδ T cells in patients with advanced or recurrent non-small-cell lung cancer (NSCLC) refractory to or intolerant of current conventional treatments. There were no severe adverse events related to the therapy. All patients remained alive during the study period with a median survival of 589 days and median progression-free survival of 126 days. Six patients had stable disease (SD), whereas the remaining six evaluable patients experienced progressive disease (PD) four weeks after the sixth transfer. We conclude that adoptive transfer of zoledronate-expanded γδ T cells is safe and feasible in patients with NSCLC, refractory to other treatments.

Vaccination with Antigen-Transfected, NKT Cell Ligand–Loaded, Human Cells Elicits Robust In Situ Immune Responses by Dendritic Cells

Both innate and adaptive immunity are crucial for cancer immunosurveillance, but precise therapeutic equations to restore immunosurveillance in patients with cancer patients have yet to be developed. In murine models, α-galactosylceramide (α-GalCer)-loaded, tumor antigen-expressing syngeneic or allogeneic cells can act as cellular adjuvants, linking the innate and adaptive immune systems. In the current study, we established human artificial adjuvant vector cells (aAVC) consisting of human HEK293 embryonic kidney cells stably transfected with the natural killer T (NKT) immune cell receptor CD1d, loaded with the CD1d ligand α-GalCer and then transfected with antigen-encoding mRNA. When administered to mice or dogs, these aAVC-activated invariant NKT (iNKT) cells elicited antigen-specific T-cell responses with no adverse events. In parallel experiments, using NOD/SCID/IL-2rγc(null)-immunodeficient (hDC-NOG) mouse model, we also showed that the human melanoma antigen, MART-1, expressed by mRNA transfected aAVCs can be cross-presented to antigen-specific T cells by human dendritic cells. Antigen-specific T-cell responses elicited and expanded by aAVCs were verified as functional in tumor immunity. Our results support the clinical development of aAVCs to harness innate and adaptive immunity for effective cancer immunotherapy.

An Immunogram for the Cancer-Immunity Cycle: Towards Personalized Immunotherapy of Lung Cancer.

Introduction: The interaction of immune cells and cancer cells shapes the immunosuppressive tumor microenvironment. For successful cancer immunotherapy, comprehensive knowledge of antitumor immunity as a dynamic spatiotemporal process is required for each individual patient. To this end, we developed an immunogram for the cancer‐immunity cycle by using next‐generation sequencing. Methods: Whole exome sequencing and RNA sequencing were performed in 20 patients with NSCLC (12 with adenocarcinoma, seven with squamous cell carcinoma, and one with large cell neuroendocrine carcinoma). Mutated neoantigens and cancer germline antigens expressed in the tumor were assessed for predicted binding to patients’ human leukocyte antigen molecules. The expression of genes related to cancer immunity was assessed and normalized to construct a radar chart composed of eight axes reflecting seven steps in the cancer‐immunity cycle. Results: Three immunogram patterns were observed in patients with lung cancer: T‐cell–rich, T‐cell–poor, and intermediate. The T‐cell–rich pattern was characterized by gene signatures of abundant T cells, regulatory T cells, myeloid‐derived suppressor cells, checkpoint molecules, and immune‐inhibitory molecules in the tumor, suggesting the presence of antitumor immunity dampened by an immunosuppressive microenvironment. The T‐cell–poor phenotype reflected lack of antitumor immunity, inadequate dendritic cell activation, and insufficient antigen presentation in the tumor. Immunograms for both the patients with adenocarcinoma and the patients with nonadenocarcinoma tumors included both T‐cell–rich and T‐cell–poor phenotypes, suggesting that histologic type does not necessarily reflect the cancer immunity status of the tumor. Conclusions: The patient‐specific landscape of the tumor microenvironment can be appreciated by using immunograms as integrated biomarkers, which may thus become a valuable resource for optimal personalized immunotherapy.

Vaccination with NY-ESO-1 overlapping peptides mixed with Picibanil OK-432 and montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

We conducted a clinical trial of an NY-ESO-1 cancer vaccine using 4 synthetic overlapping long peptides (OLP; peptides #1, 79–108; #2, 100–129; #3, 121–150; and #4, 142–173) that include a highly immunogenic region of the NY-ESO-1 molecule. Nine patients were immunized with 0.25 mg each of three 30-mer and a 32-mer long NY-ESO-1 OLP mixed with 0.2 KE Picibanil OK-432 and 1.25 mL Montanide ISA-51. The primary endpoints of this study were safety and NY-ESO-1 immune responses. Five to 18 injections of the NY-ESO-1 OLP vaccine were well tolerated. Vaccine-related adverse events observed were fever and injection site reaction (grade 1 and 2). Two patients showed stable disease after vaccination. An NY-ESO-1-specific humoral immune response was observed in all patients and an antibody against peptide #3 (121–150) was detected firstly and strongly after vaccination. NY-ESO-1 CD4 and CD8 T-cell responses were elicited in these patients and their epitopes were identified. Using a multifunctional cytokine assay, the number of single or double cytokine-producing cells was increased in NY-ESO-1-specific CD4 and CD8 T cells after vaccination. Multiple cytokine-producing cells were observed in PD-1 (−) and PD-1 (+) CD4 T cells. In conclusion, our study indicated that the NY-ESO-1 OLP vaccine mixed with Picibanil OK-432 and Montanide ISA-51 was well tolerated and elicited NY-ESO-1-specific humoral and CD4 and CD8 T-cell responses in immunized patients.

Neoantigen Load, Antigen Presentation Machinery, and Immune Signatures Determine Prognosis in Clear Cell Renal Cell Carcinoma

In ccRCC the abundant neoepitopes associated with more effective antitumor immune responses were counterbalanced by a strongly immunosuppressive microenvironment. Therefore, combining blockade of immunosuppressive molecular pathways with immunotherapies targeting neoantigens may achieve synergistic antitumor activity. Tumors commonly harbor multiple genetic alterations, some of which initiate tumorigenesis. Among these, some tumor-specific somatic mutations resulting in mutated protein have the potential to induce antitumor immune responses. To examine the relevance of the latter to immune responses in the tumor and to patient outcomes, we used datasets of whole-exome and RNA sequencing from 97 clear cell renal cell carcinoma (ccRCC) patients to identify neoepitopes predicted to be presented by each patients autologous HLA molecules. We found that the number of nonsilent or missense mutations did not correlate with patient prognosis. However, combining the number of HLA-restricted neoepitopes with the cell surface expression of HLA or β2-microglobulin(β2M) revealed that an A-neohi/HLA-Ahi or ABC-neohi/β2Mhi phenotype correlated with better clinical outcomes. Higher expression of immune-related genes from CD8 T cells and their effector molecules [CD8A, perforin (PRF1) and granzyme A (GZMA)], however, did not correlate with prognosis. This may have been due to the observed correlation of these genes with the expression of other genes that were associated with immunosuppression in the tumor microenvironment (CTLA-4, PD-1, LAG-3, PD-L1, PD-L2, IDO1, and IL10). This suggested that abundant neoepitopes associated with greater antitumor effector immune responses were counterbalanced by a strongly immunosuppressive microenvironment. Therefore, immunosuppressive molecules should be considered high-priority targets for modulating immune responses in patients with ccRCC. Blockade of these molecular pathways could be combined with immunotherapies targeting neoantigens to achieve synergistic antitumor activity. Cancer Immunol Res; 4(5); 463–71. ©2016 AACR.