Kenta Narumi

Proinflammatory Proteins S100A8/S100A9 Activate NK Cells via Interaction with RAGE

S100A8/A9, a proinflammatory protein, is upregulated in inflammatory diseases, and also has a tumor-promoting activity by the recruitment of myeloid cells and tumor cell invasion. However, whether the expression of S100A8/A9 in tumors predicts a good or poor prognosis is controversial in the clinical setting. In this study, to clarify the in vivo role of S100A8/A9 in the tumor microenvironment, we s.c. inoculated Pan02 cells stably expressing S100A8 and S100A9 proteins (Pan02-S100A8/A9) in syngeneic C57BL/6 mice. Unexpectedly, after small tumor nodules were once established, they rapidly disappeared. Flow cytometry showed that the number of NK cells in the tumors was increased, and an administration of anti-asialoGM1 Ab for NK cell depletion promoted the growth of Pan02-S100A8/A9 s.c. tumors. Although the S100A8/A9 proteins alone did not change the IFN-γ expression of NK cells in vitro, a coculture with Pan02 cells, which express Rae-1, induced IFN-γ production, and Pan02-S100A8/A9 cells further increased the number of IFN-γ+ NK cells, suggesting that S100A8/A9 enhanced the NK group 2D ligand-mediated intracellular activation pathway in NK cells. We then examined whether NK cell activation by S100A8/A9 was via their binding to receptor of advanced glycation end product (RAGE) by using the inhibitors. RAGE antagonistic peptide and anti-RAGE Ab inhibited the IFN-γ production of NK cells induced by S100A8/A9 proteins, and an administration of FPS-ZM1, a RAGE inhibitor, significantly enhanced the in vivo growth of Pan02-S100A8/A9 tumors. We thus found a novel activation mechanism of NK cells via S100A8/A9–RAGE signaling, which may open a novel perspective on the in vivo interaction between inflammation and innate immunity.

Suppression of Tregs by anti-glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon-α gene therapy for pancreatic cancer

We have reported that interferon (IFN)‐α can attack cancer cells by multiple antitumor mechanisms including the induction of direct cancer cell death and the enhancement of an immune response in several pancreatic cancer models. However, an immunotolerant microenvironment in the tumors is often responsible for the failure of the cancer immunotherapy. Here we examined whether the suppression of regulatory T cells (Tregs) within tumors can enhance an antitumor immunity induced by an intratumoral IFN‐α gene transfer. First we showed that an intraperitoneal administration of an agonistic anti‐glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model. The anti‐GITR mAb was then combined with the intratumoral injection of the IFN‐α‐adenovirus vector. The treatment with the antibody synergistically augmented the antitumor effect of IFN‐α gene therapy not only in the vector‐injected tumors but also in the vector‐uninjected tumors. Immunostaining showed that the anti‐GITR mAb decreased Foxp3+ cells infiltrating in the tumors, while the intratumoral IFN‐α gene transfer increased CD4+ and CD8+ T cells in the tumors. Therefore, the combination therapy strongly inclined the immune balance of the tumor microenvironment in an antitumor direction, leading to a marked systemic antitumor effect. The CCR5 expression on Tregs was downregulated in the antibody‐treated mice, which may explain the decrease of tumor‐infiltrating Tregs. The combination of Treg‐suppression by GITR mAb and the tumor immunity induction by IFN‐α gene therapy could be a promising therapeutic strategy for pancreatic cancer.

Administration route-dependent induction of antitumor immunity by interferon-alpha gene transfer.

Type I interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis, and immunomodulation. We have demonstrated that intratumoral injection of an IFN‐α‐expressing adenovirus effectively induces cell death of cancer cells and elicits a systemic tumor‐specific immunity in several animal models. On the other hand, reports demonstrated that an elevation of IFN in the serum following an intramuscular delivery of a vector is able to activate antitumor immunity. In this study, we compared the intratumoral and systemic routes of IFN gene transfer with regard to the effect and safety of the treatment. Intratumoral injection of an IFN‐α adenovirus effectively activated tumor‐responsive lymphocytes and caused tumor suppression not only in the gene‐transduced tumors but also in distant tumors, which was more effective than the intravenous administration of the same vector. The expression of co‐stimulatory molecules on CD11c+ cells isolated from regional lymph nodes was enhanced by IFN gene transfer into the tumors. Systemic toxicity such as an elevation of hepatic enzymes was much lower in mice treated by intratumoral gene transfer than in those treated by systemic gene transfer. Our data suggest that the intratumoral route of the IFN vector is superior to intravenous administration, due to the effective induction of antitumor immunity and the lower toxicity. (Cancer Sci 2010)

In vivo delivery of interferon-α gene enhances tumor immunity and suppresses immunotolerance in reconstituted lymphopenic hosts

T cells recognize tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP); however, HP-driven antitumor responses gradually decay in association with tumor growth. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study we examined whether a tumor-specific immune response induced by IFN-α could enhance and sustain HP-induced antitumor immunity. An intratumoral IFN-α gene transfer resulted in marked tumor suppression when administered in the early period of syngeneic hematopoietic stem cell transplantation (synHSCT), and was evident even in distant tumors that were not transduced with the IFN-α vector. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c+ cells in the tumors and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c+ cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including interleukin (IL)-6. The CD11c+ cells rescued effector T-cell proliferation from regulatory T-cell-mediated suppression, and IL-6 may have a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.

A targeting ligand enhances infectivity and cytotoxicity of an oncolytic adenovirus in human pancreatic cancer tissues

The addition of a targeting strategy is necessary to enhance oncolysis and secure safety of a conditionally replicative adenovirus (CRAd). We have constructed an adenovirus library displaying random peptides on the fiber, and have successfully identified a pancreatic cancer-targeting ligand (SYENFSA). Here, the usefulness of cancer-targeted CRAd for pancreatic cancer was examined as a preclinical study. First, we constructed a survivin promoter-regulated CRAd expressing enhanced green fluorescent protein gene (EGFP), which displayed the identified targeting ligand (AdSur-SYE). The AdSur-SYE resulted in higher gene transduction efficiency and oncolytic potency than the untargeted CRAd (AdSur) in several pancreatic cancer cell lines. An intratumoral injection of AdSur-SYE significantly suppressed the growth of subcutaneous tumors, in which AdSur-SYE effectively proliferated and spread. An ectopic infection in adjacent tissues and organs of intratumorally injected AdSur-SYE was decreased compared with AdSur. Then, to examine whether the targeting ligand actually enhanced the infectivity of CRAd in human pancreatic cancer tissues, tumor cells prepared from surgical specimens were infected with viruses. The AdSur-SYE increased gene transduction efficiency 6.4-fold higher than did AdSur in single cells derived from human pancreatic cancer, whereas the infectivity of both vectors was almost the same in the pancreas and other cancers. Immunostaining showed that most EGFP(+) cells were cytokeratin-positive in the sliced tissues, indicating that pancreatic cancer cells but not stromal cells were injected with AdSur-SYE. AdSur-SYE resulted in a stronger oncolysis in the primary pancreatic cancer cells co-cultured with mouse embryonic fibroblasts than AdSur did. CRAd in combination with a tumor-targeting ligand is promising as a next-generation of oncolytic virotherapy for pancreatic cancer.

Intratumoral interferon-α gene transfer enhances tumor immunity after allogeneic hematopoietic stem cell transplantation

One of the major challenges in the treatment of solid cancers by allogenic hematopoietic stem cell transfer (alloHSCT) is the specific enhancement of antitumor immunity. Interferon (IFN) is a cytokine with pleiotropic biological functions including an immunomoduration, and our preclinical studies have shown that an intratumoral IFN-α gene transfer induced strong local tumor control and systemic tumor-specific immunity. In the present study, we examined whether the IFN-α gene transfer could enhance recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. First, when a mouse IFN-α adenovirus vector (Ad-mIFN) was injected into subcutaneous xenografts of syngeneic renal and colon cancer cells, tumor growth was significantly suppressed in a dose-dependent manner. A significant tumor cell death and infiltration of immune cells was recognized in the Ad-mIFN-injected tumors, and the dendrtic cells isolated from the tumors showed a strong Th1-oriented response. The antitumor effect of Ad-mIFN was then examined in a murine model of minor histocompatibility antigen-mismatched alloHSCT. The intratumoral IFN-α gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors which did not receive the vector injection. A cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to IFN-α. Graft-versus-host disease was not exacerbated serologically or clinically in the mice treated with IFN-α. This combination strategy deserves evaluation in future clinical trials for human solid cancers.

Development of a Novel Efficient Method To Construct an Adenovirus Library Displaying Random Peptides on the Fiber Knob

Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 10(4) level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.

Syngeneic Hematopoietic Stem Cell Transplantation Enhances the Antitumor Immunity of Intratumoral Type I Interferon Gene Transfer for Sarcoma

Sarcoma at advanced stages remains a clinically challenging disease. Interferons (IFNs) can target cancer cells by multiple antitumor activities, including the induction of cancer cell death and enhancement of immune response. However, the development of an effective cancer immunotherapy is often difficult, because cancer generates an immunotolerant microenvironment against the host immune system. An autologous hematopoietic stem cell transplantation (HSCT) is expected to reconstitute a fresh immune system, and expand tumor-specific T cells through the process of homeostatic proliferation. Here we examined whether a combination of autologous HSCT and IFNs could induce an effective tumor-specific immune response against sarcoma. First, we found that a type I IFN gene transfer significantly suppressed the cell growth of various sarcoma cell lines, and that IFN-β gene transfer was more effective in inducing cell death than was IFN-α in sarcoma cells. Then, to examine the antitumor effect in vivo, human sarcoma cells were inoculated in immune-deficient mice, and a lipofection of an IFN-β-expressing plasmid was found to suppress the growth of subcutaneous tumors significantly. Finally, the IFN gene transfer was combined with syngeneic HSCT in murine osteosarcoma models. Intratumoral IFN-β gene transfer markedly suppressed the growth of vector-injected tumors and inhibited formation of spontaneous lung and liver metastases in syngeneic HSCT mice, and an infiltration of many immune cells was recognized in metastatic tumors of the treated mice. The treated mice showed no significant adverse events. A combination of intratumoral IFN gene transfer with autologous HSCT could be a promising therapeutic strategy for patients with sarcoma.

Interaction of natural killer cells with neutrophils exerts a significant antitumor immunity in hematopoietic stem cell transplantation recipients.

Autologous hematopoietic stem cell transplantation (HSCT) can induce a strong antitumor immunity by homeostatic proliferation (HP) of T cells and suppression of regulatory T cells following preconditioning‐induced lymphopenia. However, the role of innate immunity including natural killer (NK) cells is still not understood. Here, first, we examined whether NK cells exert an antitumor effect after syngeneic HSCT in a murine colon cancer model. Flow cytometry showed that NK cells as well as T cells rapidly proliferated after HSCT, and the frequency of mature NK cells was increased in tumor during HP. Furthermore, NK cells undergoing HP were highly activated, which contributed to substantial tumor suppression. Then, we found that a large number of neutrophils accumulated in tumor early after syngeneic HSCT. It was recently reported that neutrophil‐derived mediators modulate NK cell effector functions, and so we examined whether the neutrophils infiltrated in tumor are associated with NK cell‐mediated antitumor effect. The depletion of neutrophils significantly impaired an activation of NK cells in tumor and increased the fraction of proliferative NK cells accompanied by a decrease in NK cell survival. The results suggested that neutrophils in tumor prevent NK cells from activation‐induced cell death during HP, thus leading to a significant antitumor effect by NK cells. This study revealed a novel aspect of antitumor immunity induced by HSCT and may contribute to the development of an effective therapeutic strategy for cancer using HSCT.

Vascular Endothelial Growth Factor-D–Mediated Blockade of Regulatory T Cells within Tumors Is Induced by Hematopoietic Stem Cell Transplantation

Lymphopenia-induced homeostatic proliferation of T cells after autologous hematopoietic stem cell transplantation (HSCT) skews the T cell repertoire by engaging tumor-associated Ags, leading to an induction of antitumor immunity. However, how HSCT alters the immunosuppressive microenvironment in the tumors is unknown. In this study, we first analyzed the kinetics of regulatory T cells (Tregs) in the tumors after syngeneic HSCT. Unexpectedly, the frequency of CD4+ cells expressing Foxp3 was increased in the spleens, whereas the frequency was clearly decreased in the tumors after HSCT. The origin of reconstituted CD4+ and Foxp3+ cells in the tumors was mainly from the expansion of transferred splenic T cells. Then, to examine the mechanism of Treg suppression after HSCT, we isolated CD11c+ cells from tumors. A large amount of Treg-inhibitory cytokine IL-6 was secreted from the CD11c+ cells in the tumors, but not in the spleens in the recipient mice. Furthermore, to understand what factor affects the activity of CD11c+ cells in the tumors after HSCT, we analyzed the expression of various cytokines/chemokines with mouse cytokine Ab arrays, and noticed that VEGF-D concentration was increased in the tumors in the early period after HSCT. The CD11c+ cells produced IL-6 in response to VEGF-D stimulation, and an administration of VEGF receptor-3 neutralizing Ab significantly suppressed the production of IL-6 from CD11c+ cells accompanied with the increase of Tregs in the tumors of HSCT recipients. Autologous HSCT creates an environment that strongly supports the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the suppression of Tregs.