Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its ...effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF-sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)-related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF-induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR(-/-)) mice were used. Importantly, in both LLC and CT26 colon cancer-bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4(+)CD25(+)FoxP3(+) regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF-based cancer immunotherapy.
Hematopoietic stem cell (HSC) transplantation is a curative treatment of hematological disorders that has been utilized for several decades. Although umbilical cord blood (UCB) is a promising source ...of HSCs, the presence of an insufficient number of HSCs in these preparations limits their use, prompting need for ex vivo HSC amplification. To establish a more efficient method to expand UCB HSCs, we developed the SL-13R peptide and cultured UCB CD34+ cells with peptide in serum-free medium containing a cytokine cocktail. Following 9 days of culture with peptide, the number of total cells, CD34+CD38- cells and hematopoietic colonies significantly increased relative to control cells grown without peptide. Transplantation of these cells into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution ability. To understand how the peptide promotes HSC expansion, we identified PLEC and ERLIN2 proteins as peptide interactors and undertook loss-of-function analysis to determine whether either was required for peptide function in our culture system. PLEC knockdown UCB CD34+ cells cultured with peptide showed a decreased number of hematopoietic colonies relative to peptide-treated, non-knockdown controls. By contrast, ERLIN2 knockdown had little effect in the presence of peptide. This work suggests that PLEC functions in HSC expansion promoted by SL-13R. In summary, we have identified a novel peptide promoting expansion of UCB CD34+ cells with long-term reconstitution ability. Its use may facilitate clinical use of UCB HSCs.
Sugiyama:Science Lustre, Ltd: Equity Ownership.
Although oncolytic virotherapy is a promising anticancer therapy, antitumor efficacy is hampered by low tumor selectivity. To identify a potent and selective oncolytic virotherapy, we carried out ...large-scale two-step screening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activity against nine human non-small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positively correlated with the expression of the viral receptors, coxsackievirus and adenovirus receptor, and decay-accelerating factor, on NSCLC cells. In vitro assays revealed that the CVB3 induced apoptosis and phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injections of CVB3 elicited remarkable regression of preestablished NSCLC tumors in vivo. Furthermore, administrations of CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenografts on the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were well tolerated without treatment-related deaths. In addition, after CVB3 infection, NSCLC cells expressed abundant cell surface calreticulin and secreted ATP as well as translocated extranuclear high-mobility group box 1, which are required for immunogenic cell death. Moreover, intratumoral CVB3 administration markedly recruited natural killer cells and granulocytes, both of which contributed to the antitumor effects as shown by depletion assays, macrophages, and mature dendritic cells into tumor tissues. Together, our findings suggest that CVB3 is a potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localized and metastatic NSCLC.
The gene transduction method is a very powerful tool, not only in basic science but also in clinical medicine. Regenerative medicine is one field that has close connection with both basic and ...clinical. Recently, it has been reported that induced pluripotent stem (iPS) cells can be produced from somatic cells by a three or four gene transduction. We have also recently reported that lentiviral gene transfer of the tal1/scl gene can efficiently differentiate non-human primate common marmoset ES cells into hematopoietic cells without the support of stromal cells. In this study, we constructed a high-performance human fetal liver-derived lentiviral expression library, which contains a high number of individual clones, in order to develop a very helpful tool for understanding early hematopoiesis and/or hepatocytosis for future regenerative medicine. Our lentiviral cDNA library consisted of more than 8 x 10⁷ individual clones, and their average insert size was >2 kb. DNA sequence analysis for each individual inserted cDNAs revealed that >60% contained the full-length protein-coding regions for many genes including cytokine receptors, cytoplasmic proteins, protein inhibitors, and nuclear factors. The transduction efficiency on 293T cells was 100% and the average size of an integrated cDNA was ~1.1 kb. These results suggest that our lentiviral human fetal liver cDNA expression library could be a very helpful tool for accelerating the discovery of novel genes that are involved in early hematopoiesis and hepatopoiesis and to make the use of iPS cells more efficient in the field of regenerative medicine.
Abstract We designed a phase I trial to investigate the safety, immune responses and clinical benefits of a five-peptide cancer vaccine in combination with chemotherapy. Study subjects were patients ...positive for HLA-A2402 with locally advanced, metastatic, and/or recurrent gastrointestinal, lung or cervical cancer. Eighteen patients including nine cases of colorectal cancer were treated with escalating doses of cyclophosphamide 4 days before vaccination. Five HLA-A2402-restricted, tumor-associated antigen (TAA) epitope peptides from KOC1, TTK, URLC10, DEPDC1 and MPHOSPH1 were injected weekly for 4 weeks. Treatment was well tolerated without any adverse events above grade 3. Analysis of peripheral blood lymphocytes showed that the number of regulatory T cells dropped from baseline after administration of cyclophosphamide and confirmed that TAA-specific T cell responses were associated significantly with longer overall survival. This phase I clinical trial demonstrated safety and promising immune responses that correlated with vaccine-induced T-cell responses. Therefore, this approach warrants further clinical studies.
Abstract
Accumulated clinical studies have validated the efficacy of immunotherapies for patients with solid tumors. Although effective anti-tumor immune responses have been introduced in many ...clinical trial cases, several immunosuppressive factors such as regulatory T cells (Tregs) have been considered to inhibit induction of anti-tumor immunity.
We have developed new approach of immunotherapy in order to augment anti-tumor effect by expanding tumor-specific cytotoxic T cells (CTL), and eliminate Tregs by utilizing chemotherapy. We have recently reported the efficacy of multiplex vaccination with HLA-A*2402 restricted tumor-associated antigens peptides (TAA), preceded by the administration of low-dose cyclophosphamide (CPM) in order to eliminate Tregs. This time, we have been conducting a phase I clinical trial to study the utilization of CTL after CPM administration followed by a consequent administration of dendritic cells (DC) and IL-2. This study targets the novel HLA-A*0201 or HLA-A*2402-restricted TAA, RING finger protein 43 (RNF43), and is designed as a dose-escalation study of CTL. The purpose of this trial is to evaluate the safety and tolerability of the proposed method as primary endpoint and the efficacy including over all survival and immune responses as secondary endpoint.
11 patients with advanced solid tumors refractory to standard therapy were enrolled in this trial. They were HLA-A*2402 or A*0201 positive and exhibiting RNF43 expression levels in their tumor cells more than those in RNF43-positive control cell (HCT15). Primarily, severe adverse event greater than Grade 3 was not observed in our patients. Clinical responses of stable disease (SD) were observed in 8 out of 11 patients. Among them, 2 patients showed a decrease in the tumor markers with stabilized tumor sizes during the observed period, and 1 patient showed a mixed response. On the other hand, 3 other patients showed progressive disease (PD). The number of Tregs significantly decreased after the administration of CPM (p=0.033), and the higher decreasing rate of Tregs was related to the good clinical response. In CD107a/b mobilization assay, the ratio of RNF43-specific CD8 T cells in SD increased with time, conversely that in PD decreased (p=0.005). Also, in Intracellular Staining Assay, the ratio of IFN-g produced by RNF43-specific CD8 T cells was significantly correlated with clinical benefit (p=0.02), while TNF-α and IL-2 were not. Consequently, the combination of immunotherapy and CPM may induce tumor specific immune cells accompanied by the decreased number of Tregs.
In conclusion, this trial is tolerable and may be clinically efficient against advanced solid tumors. Further immunological assessment should be conducted in order to explore the valid biomarkers to predict clinical efficacy before treatment.
Citation Format: Yasuki Hijikata, Toshihiko Okazaki, Hiroyuki Inoue, Yoshihiro Tanaka, Kenzaburo Tani, Shinichi Kobayashi, koji Yoshida. A phase I clinical trial of RNF43 peptide-specific immune cell therapy combined with low-dose cyclophosphamide for patients with advanced solid tumors. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-172. doi:10.1158/1538-7445.AM2014-LB-172
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