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Abstracts J Immunother Volume 33, Number 8, October 2010 with a decrease in T regulatory cells, after treatment have been observed. The anti-tumor effects of GM-CSF and IL-12 microsphere treatment have been shown to be dependent on IFN-g. However, further studies demonstrated that the immune response was transient and that the T regulatory cells rebounded rapidly. Recent data from our laboratory suggested that indoleamine 2, 3- dioxygenase (IDO), an IFN-g-inducible immune-suppressive enzyme, may play a role in the post-therapy T regulatory cell resurgence. Since dendritic cells (DCs) are central to both induction of an immune response, and have been shown to be significant producers of IDO, the effect of IL-12 and GM-CSF microsphere treatment on this potent antigen presenting cell was explored. We found that intra-tumoral injection of IL-12 and GM-CSF microsphere resulted in rapid recruitment of DC to tumors with subsequent migration to tumor-draining lymph nodes (TDLN). Post-treatment DCs displayed increased CD86 expression, a pro-inflammatory cytokine profile and effective CD8 + and CD4 + T cell priming in vitro (immunogenic). By Day 7 posttreatment however, the priming ability of these DCs was completely lost (tolerogenic). Further analysis revealed that day 7 DCs expressed high levels of IDO, inhibition of which resulted in the rescue of the ability to prime T cells. GM-CSF and IL-12 mediated induction of immunogenic DCs was completely abrogated in IFN-g knockout mice, establishing the critical role of this cytokine in post-therapy immune activation. Importantly, TDLN DCs failed to upregulate IDO and IDO-inhibition did not restore priming function to day 7 DCs in IFN-g knockout mice, revealing that IFN-g was also responsible for the induction of tolerogenic function in DCs. These results establish the dichotomous role of IFN-g in the regulation of IL-12-mediated antitumor immunity and identify DC as the primary conduit that mediate these effects. Furthermore, these data support the hypothesis that blocking IDO in therapeutic regimen designed to induce TH1 responses may prove useful by extending the window of T cell priming and activation. F19 Labeling of Human a-Type-1-Polarized Dendritic Cells and Standard Dendritic Cells for In Vivo Trafficking by Fluorine MRI Brooke M. Helfer*, Anthony Balducci*, Robbie Mailliard*w, Eric Ahrenszy, Pawel KalinskiJ, Amy Wesa*. *Research and Development, Celsense Inc; w Infectious Deiseases and Microbiology, University of Pittsburgh; zBiological Sciences; yPittsburgh NMR Center for Biomedical Sciences; JDepartment of Surgery, Carnegie Mellon Univesrsity, Pittsburgh, PA. Human dendritic cells (DC) have great clinical promise as an immunotherapeutic intervention against cancer. A question remains to the dispersion and persistence of DC following administration in assessing therapeutic potency. Here we utilize a perfluoropolyether (PFPE) magnetic resonance imaging (MRI) tracer agent that aims to address these questions in the assessment of a-type-1-polarized dendritic cells (aDC1). aDC1 producing high levels of interleukin-12p70 with putative strong lymph node homing capabilities, are currently being evaluated clinically for anti-cancer activity. 19F MRI tracking is an attractive approach to use for clinical DC tracking because administration of a fluorine label, presents a non-invasive, highly specific image and paired with proton MRI provides clear and quantitative cell localization within anatomic context. Previously, standard DC were evaluated as a ‘‘proof of principle’’ assay in a murine xenograft model to track the migration of human sDC in-vivo by 19F MRI. Mature DC but not immature DC were observed to migrate into the region of draining lymph nodes 18 hours post injection by MRI. Single cell lymph node preparations further determined the presence of DC by flow cytometry. Standard DC were also compared to aDC1 in both function and phenotype, following cellular labeling with PFPE. Standard DC and aDC1 had similar labeling efficiencies with PFPE, which had no effect on the DC’s ability to stimulate T cell expansion, produce IL-12p70, process antigen or express costimulatory and polarizing factors. These data support the clinical translation of PFPE to safely track and quantify the administration and migration of human aDC1 by non-invasive 19F MRI for cancer patients. Polarized Dendritic Cells in the Immunotherapy of Established Cancer: Roles of Signal 3 and Signal 4 Pawel Kalinski. Surgery, University of Pittsburgh, Pittsburgh, PA. Cancer vaccines have been shown capable of prolonging survival of cancer patients, but their ability to induce regression of established tumors remains low. The use of ex-vivo-generated dendritic cells (DCs) helps to sidestep the dysfunction of endogenous DCs in patients with advanced cancer and to deliver the key signals needed for effective anti-tumor responses. Recently, we and others have shown that different DC populations can deliver specialized ‘‘signal 3’’ regulating the acquisition of desirable effector functions by T cells, and an additional ‘‘signal 4’’ that regulates T cell homing properties. Moreover, ex-vivo instruction of DCs can be used to preferentially activate CTLs, Th1- and NK cells, while limiting the undesirable activation of Treg cells. Type-1-polarized DCs (DC1) are characterized by strongly-enhanced, rather than ‘‘exhausted’’, ability to produce IL-12p70 and other CTL-, Th1-, and NK cellactivating factors. A single round of in vitro sensitization with DC1s loaded with tumor-related antigens induces 40 to 70-fold higher numbers of functional CTLs against multiple tumor-related antigens and multiple types of cancer cells (melanoma, glioma, CLL, CTCL, prostate, colorectal, endometrial and ovarian cancers), when compared with immature DCs and nonpolarized mature DCs. DC1s are particularly effective in inducing effector functions in CD8 + T cells, NK cells, and Th1-type CD4 + Th cells (delivery of ‘‘signal 3’’). They also induce a switch in the expression of chemokine receptors on naïve T cells (delivery of ‘‘signal 4’’), promoting T cell responsiveness to tumor-produced chemokines. DC1-based vaccines are being currently evaluated in phase I/II clinical trials for patients with different forms of advanced cancer. Autologous Whole-Tumor Antigen Combinatorial Immunotherapy for Recurrent Ovarian Cancer Lana E. Kandalaft*, Daniel J. Powell*, Lori Smith*, Sarah Adams*, John Liao*, Andrea Hageman*, Janos Tanyi*, Qunrui Ye*, Andrew Best*, Drew Torigianw, Christina S. Chu*, Stephen C. Rubin*, Marnix Boschz, George Coukos*. *Ovarian Cancer Research Center, School of Medicine; w Radiology, University of Pennsylvania, Philadelphia, PA; zNorthwest Biotherapuetics, Bethesda, MD. Background: Despite surgical and chemotherapeutic advances, the death rate from ovarian cancer has not changed over the past two decades, warranting the investigation of novel therapeutic strategies. We report the pilot application of combinatorial immunotherapy comprising dendritic cell (DC)-based autologous whole tumor antigen vaccination in combination with antiangiogenesis therapy in patients with recurrent ovarian cancer. Method: Six patients with recurrent progressive stage III and IV ovarian cancer with available tumor lysate from secondary debulking surgery underwent priming with intravenous bevacizumab and oral metronomic cyclophosphamide (bev/cy 2 doses), followed by vaccination with DCVax-L, an autologous DC preparation pulsed with autologous tumor lysate (5 to 10 10e6 DC per dose, 3 doses) plus bevacizumab (2 doses). Feasibility, safety, and biological and clinical efficacy were evaluated. Results: Therapy was feasible and well tolerated in all six subjects. Following bevacizumab and oral metronomic cyclophosphamide, vaccination with DCVax-L produced few grade 1 toxicities and elicited tumor-specific T cell responses in four of the six patients. Two of these four patients experienced partial objective clinical 878 | www.immunotherapy-journal.com r 2010 Lippincott Williams & Wilkins
J Immunother Volume 33, Number 8, October 2010 Abstracts response after completion of vaccine while the remaining two demonstrated stable disease. Among the former, one subject progressed through bev/cy, but had objective response to DCVax-L. Humoral responses were elicited in the 2 patients that experienced objective response, and an HLA-A0201-restricted, Her2-specific T cell response was documented following vaccination in one patient. The over all clinical benefit achieved in this study is 60% (2PR, 2SD and 2PD). Interpretation: Our results suggest the use of combinatorial cellular immunotherapy comprising bev/cy with DC vaccination with whole tumor antigen for the treatment of patients with recurrent ovarian cancer was well tolerated and warrants further investigation. Targeting Soluble Tumor Associated Antigens CEA and HER2 to Exosomes Enhances Antigen Specific Immune Reponses H.K. Lyerly*, Jean Bernard Le Pecqw, Zach Hartman*, Tim Clay*, Alain Delcayrew, Yanal Murad*. *Comprehensive Cancer Center, Duke University, Durham, NC; w Exothera, Menlo, CA. Patients with cancer may have detectable levels of tumor associated antigens (TAAs) in their circulation, but usually have minimal adaptive immune responses to those antigens. The immunogenicity of soluble protein differs from protein associated with membrane vesicles (exosomes), and this difference may explain the minimal immune response directed against circulating self or TAAs. Adaptive immunity to model TAAs has been enhanced in experimental animal models by directing antigen expression into exosomes. We sought to enhance the immunogenicity of common circulating TAAs by generating recombinant adenoviral vectors expressing the TAA coupled to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/ lactadherin which targets them to exosomes. We created novel adenoviral vectors expressing one of two non-mutated TAAs, including two often found in the circulation of cancer patients, carcinoembyronic antigen (CEA), and the extracellular domain (ECD) of HER2. We compared these novel vectors to vectors expressing the nondirected antigens in animal models to determine if exosomal targeting enhanced immunogenicity. We saw robust improvement in antigen specific immune responses to each of these antigens and conclude that the mode of secretion can influence the immunogenicity of TAAs expressed by viral vectors. This finding may explain their lack of immunogenicity of circulating TAAs, and may be used to enhance future antitumor vaccination protocols. Comparative Analysis of Cytotoxic T Lymphocyte Cell Response of Dendritic Cells Loaded with Hepatocellular Carcinoma -Derived RNA or Cell Lysate Ke Pan, Hui Wang, Jing-jing Zhao, Jian-chuan Xia. State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China. The choice of the tumor antigen preparation used for dendritic cells (DCs) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing HCC cell lines ability, as well as higher frequency of IFN-g production of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cells proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-g and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy. A Peptide Derived from EBV-gH Glycoprotein That Reproduces Several Inhibitory Effects of EBV on Monocyte Derived Dendritic Cells Carlos A. Parra-Lo´pez*, Darnel A. Marchenaw, Mauricio Urquiza Martínezz, Johanna Melo Cárdenasz, Magnolia Vanegasz, Manuel E. Patarroyo Murilloz. *Microbiology and Immunology, Universidad Nacional de Colombia; w Immunology and Oncology Research Group, Fundacioń Salud de los Andes; zVirology Group, Fundacioń Instituto de Inmunologıá de Colombia (FIDIC), Bogota´, Colombia. By interacting with B cells, epithelial cells and monocytes, Epstein- Barr virus (EBV) has been implicated in developing several malignant tumors such as nasopharyngeal carcinoma, Burkitt’s lymphoma and the X-associated lymphoproliferative disease. EBV interact with monocyte-derived dendritic cells (MoDCs) mainly through EBV proteins gp350/220 and gp42/gH/gp25 (gp42/gH/gL) complex which binds to CD21 (CR2), MHC Class II and the integrins avb6 and avb8. EBV produces in MoDCs different effects including inhibition of development, apoptosis and induction of cytokines that both stimulate and inhibit activation of Th1 CD4+ T cells (IL-12 and IL-10/IL-6). The glycoprotein gp85 also named EBV-gH protein has a role in the binding of EBV to MoDCs through peptides 11435 (181TYKRVTEKGDEHVLSLVFGK200) and 11438 (241YFVPNLKDMFSRAVTMTAAS260). Here we describe that an analogue peptide from 11438 with a stable alphahelix (herein named P33210) has similar effects on MoDCs as EBV does. Cells cultured with this peptide remarkably displayed a delay in the MoDCs development evidenced by induction of apoptosis, sustained CD14 expression and impaired expression of DC-SIGN, CD83 and HLA-DR. Interestingly, in MoDCs from several normal donors, P33210 induced high levels of IL-12p70 synthesis in complete absence of IL-10 that persisted longer than that elicited by EBV. Altogether, these results suggest that peptide P33210 may be a tool to dissect activation pathways leading to apoptosis/ IL-12 production and/or inhibition of MoDCs maturation induced by EBV. Induction of Systemic and Therapeutic Antitumor Immunity Using Intratumoral Injection of Bone-Marrow Derived Dendritic Cells Genetically Modified to Express IL-23 Marimo Sato-Matsushita*, Kimiyasu Yoneyama*w, Takafumi Nakamura*, Yuko Kitagawaw, Hideaki Tahara*. *Institute of Medical Science, The University of Tokyo; w Department of Surgery, Keio University School of Medicine, Tokyo, Japan. Purpose: We have reported that the systemic administration of IL-23 induces potent antitumor immunity primarily mediated when the Th1-type response is fully promoted in the presence of endogenously expressed IL-12. In this study, we investigated whether bone marrow- derived dendritic cells (BM-DCs) adenovirally transduced with genes encoding murine IL-23 have therapeutic benefits for antitumor immunotherapy. Experiment Design and Results: We made RDG fiber-mutant adenovirus (Ad) vectors encoding IL-23 or EGFP. The MCA205 fibrosarcoma was intradermally inoculated to C57BL/6, and the mice were intratumorally injected with BM-DCs transduced with Ax3CAmIL23/RGD (Ad-IL-23-DCs) on day 8. The tumors of mice treated with AD-IL-23-DCs resulted in significant growth suppression when compared to that with BM-DCs transduced Ad- EGFP-F/RGD (Ad-EGFP-DCs). Ad-IL-23-DCs treatment induced MCA-205-specific and potent CTL responses in draining lymph node. Furthermore, The NK activity also increased in splenocytes at levels greater than those of Ad-EGFP-DCs. In addition, The significant induction of IFN-g and IL-17 and decrease of Foxp3+CD4+Tregs in TIL were strongly suggested in the mice injected with Ad-IL-23-DCs when compared with those of Ad-EGFP-DCs. Conclusion: This strategy designed to deliver genetically modified DCs to tumor sites is associated with systemic and therapeutic antitumor immunity and could be an alternative approach to those r 2010 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 879
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