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Abstracts J Immunother Volume 33, Number 8, October 2010 archived tissues. Therefore, we sought to identify a molecular tissue-profile that reproduces the flow cytometry-based quantification of NK-content of TIL. Using qRT-PCR, transcript levels of lymphocyte markers, chemokines and cytokines were quantified in RCC tissues with predetermined NK cell-content of TIL. Transcript levels of NKp46, perforin, CX3CL1 and its receptor CX3CR1 were found to correlate significantly with the flow cytometry-based classification. Patients with tumors showing high CX3CR1 transcript level had significantly better survival than patients with low CX3CR1 transcript levels. The new gene profile, being easily amenable to clinical translation, offers the possibility to investigate the relevance of NK cells in the prediction of cancer progression or the response to immunotherapy. FIGURE 1. Five-year survival data of 66 glioblastoma patients treated with pritumumab. CR indicates complete response; MR, moderate response; PG, progressive disease; PR, partial response; ST, stable disease. antibody with human tissues showed the antigen, altered tumorassociated vimentin, to be highly restricted to various cancers and not normal cells and tissues. In various clinical trials in Japan 249 patients with brain cancer were treated with pritumumab. The overall response rate was between 25-30% with several survivors beyond 5-years post-treatment. In one Phase II study of 66 patients the survival rate of complete and partial responders was over 70%. The patients were on a low dose regimen of 1 mg given twice a week for a course of 24 weeks for a total dose of 48 mgs per course. Pritumumab appears to be a safe and effective therapy in patients with malignant gliomas (Fig. 1). Gene Expression Signature Allows Prediction of NK cell Content in Tissue and Survival of Renal Cell Carcinoma Patients Judith Hosse*, Alexander Buchnerwz, Rainer Riesenbergw, Petra U. Prinz*, Sabine I. Siegerty, Robert KammererJ, Peter J. Nelsonz, Elfriede Noessner*. *Helmholtz Zentrum München-German Research Center for Environmental Health, Institute of Molecular Immunology; w Ludwig Maximilians University, Tumor Immunology Laboratory, LIFE Center; zLudwig Maximilians University, Department of Urology, University Clinic Grosshadern; yLudwig Maximilians University, Institute of Pathology; zLudwig Maximilians University, Clinical Biochemistry, Medical Policlinic, Munich; JFriedrich-Loeffler-Institut, Institute of Immunology, Tübingen, Germany. Renal cell carcinoma (RCC) is considered immunogenic based on the observation of spontaneous regression and response to immunotherapy. However, only subgroups of patients are responders and the reason why these patients respond whereas others exhibit tumor progression under identical cytokine therapies is largely unknown. RCC tissues are densely infiltrated with immune effector cells but how the infiltrate relates to tumor growth-control or patient’s responsiveness to therapy is unknown. We previously showed that RCC tumors can be separated into two groups according to their percentage of NK cells within tumor-infiltrating lymphocytes (TIL): one group has a high (NKhigh), the other a low (NKlow) percentage of NK cells (cut-off: 20% NK within TIL). Additionally, the functional quality of NK cells differed, with NK cells from NKhigh tumors gaining cytolytic activity by short-term incubation with low-dose IL-2. Based on this quick responsiveness to IL-2, we considered that the amount of NK cells among TIL may be a predictive marker for clinical outcome and could correlate with survival and response to immunotherapy. Testing this hypothesis requires the analysis of a large patient cohort, including patients with relevant follow-up data after IL-2 immunotherapy. The current procedure for NKhigh/NKlow discrimination utilizes flow cytometry of TIL isolated from fresh post-surgery tissue and, thus, is not compatible with the analysis of Enhanced Anti-tumor Effect of Combination Therapy With Anti-CD40 Antibody and the mTOR Kinase Inhibitor AZD8055 Qun Jiang*, Jonathan M. Weiss*, John R. Ortaldo*, Timothy Back*, Sylvie Guichardw, Anjan Thakurtaw, Robert H. Wiltrout*. *NCI-Frederick, Frederick; w AstraZeneca, Rockville, MD. mTOR signaling, which plays a central role in controlling cell proliferation, survival, mobility and angiogenesis, is considered an important target for new anticancer drugs development. The involvement of the CD40/CD154 signaling pathway has also been reported to play an important role in regulating anti-tumor immune responses. In this study, using a first-in-class orally bioavailable mTOR kinase inhibitor, AZD8055, which, unlike rapamycin, can inhibit both mTORC1 and mTORC2, we evaluated the anti-tumor efficacy of mTOR kinase inhibitor treatment alone in a murine liver metastatic renal cell carcinoma (Renca) model, and compared biological responses achieved by administering AZD8055 as a single agent to those obtained in combination with agonistic anti-CD40 antibody. In vitro survival assays showed that Renca cell apoptosis could be induced by AZD8055. In vivo, although AZD8055 alone modestly inhibited Renca tumor development in the liver, its use in combination with anti-CD40 induced significantly greater anti-tumor responses. The combination treatment also resulted in an increase and activation of macrophages, dendritic cells, NK cells and CD8 T cells in liver. AZD8055/anti- CD40 treatment also increased the level of systemic Th1 cytokines, including IL-12, IFN-g, TNFa. IFN-g production by CD8+ T cells and TNFa production by macrophages were also found to be elevated in the AZD8055/anti-CD40 combination treatedgroup, compared with any single treatment group. Using IFN-g KO mice, we demonstrated that the AZD8055/anti-CD40- induced anti-tumor response was dependent on IFN-g. Furthermore, the expression of Th1-associated chemokines, RANTES, MIG and IP- 10, were significantly induced in the combination treated-group. These data indicate that the combination of mTOR kinase targeting agents with immunotherapy results in synergistic immune responses and suggest that this combination approach could be an area of further development in renal cancer therapy. Developing a Neoadjuvant Murine Surgical Model of Cancer Using Vesicular Stomatitis Virus Agnieszka Kus*w, Lisa Mackenzie*, Chantal Lemay*w, Kelley Parato*, Harold Atkins*, John Bell*w, Rebecca Auer*. *Ottawa Hospital Research Institute; w University of Ottawa, Ottawa, ON, Canada. Introduction: Surgery is the primary treatment modality for most solid tumours. Despite complete resection, the development of metastatic disease limits its curative potential and provides the rationale for neoadjuvant (preoperative) therapies. Oncolytic Viruses (OVs) are replicating therapeutics that are selected or engineered to grow in malignant cell types and are capable of killing the infected target, while leaving normal, adjacent cells unharmed. OV infection creates localized inflammation and 898 | www.immunotherapy-journal.com r 2010 Lippincott Williams & Wilkins
J Immunother Volume 33, Number 8, October 2010 Abstracts additionally the presentation of foreign viral proteins together with tumour antigens, promoting adaptive anti-tumour immune responses. We have shown induction of an immune response by using Vesicular Stomatitis Virus (VSV) as an adjuvant in a tumour lysate vaccine setting. We hypothesize that the treatment of tumours prior to surgical resection will generate an in situ anti-tumour immune response which may protect immune competent hosts from recurrence or metastasis. Methods: Donor mice are injected with 1 10 4 6 CT26lacZ tumour cells subcutaneously into both flanks on day -14. Mice are euthanized on day 0 at which point the tumours are removed. Recipient mice receive 1 1 mm CT26lacZ tumour implants subcutaneous in the flank. Mice are treated with PBS or 5 10 4 8pfu VSV51ÄGMCSF intravenous prior to complete surgical resection of the flank tumour. Mice are then challenged with 1 10 4 6 CT26lacZ cells subcutaneous on the opposite flank, and tumour growth and rate is recorded. Results: We developed a CT26lacZ surgical model of cancer, where mice that received a 1 1 mm tumour implant from donor mice grew the challenge tumour more consistently compared to mice that received a subcuteneous injection of CT26lacZ cells (no treatment). We found that mice treated with PBS or VSVD51GMCSF prior to surgical resection were not protected against their challenge tumour. Mice that were treated with VSVD51GMCSF without surgical removal of the primary tumour were protected against the challenge tumour. On the contrary, mice that were treated with VSVD51GMCSF and received a mock surgery or complete surgical resection were not protected against their challenge tumour. Conclusions and Discussion: Our data demonstrates that surgical stress abrogates protection against the challenge tumour and therefore the anti-tumour immune response generated by VSVD51GMCSF treatment. Surgical stress can induce immunosuppression or tumour growth facilitation. We are currently characterizing this period of surgical stress, and we are also aiming to overcome this stressed state by altering viral treatment regimens and route of administration. The Immune Enhancing Effects of IL-7 on Human T Cells are Due to Activation of Stat Signaling but Not Repression of Cbl-b Guenther Lametschwandtner*, Thomas Gruberw, Gottfried Baierw, Dominik Wolfz, Isabella Haslinger*, Manfred Schuster*, Hans Loibner*. *Apeiron Biologics AG, Vienna; w Experimental Cell Genetics Unit; zTyrolean Cancer Research Institute, Innsbruck Medical University, Innsbruck, Austria. The E3 ubiquitin ligase Cbl-b plays a key role for anti-tumor immune responses, as cbl-b deficient mice are protected against different tumors and cbl-b deficient CD8 T cells efficiently reject tumors. We have recently shown that transient silencing of cbl-b in human T cells was able to reproduce the phenotype of cbl-b deficient murine T cells, validating cbl-b as target for cancer immunotherapy. Very recently, IL-7 has been described to control cbl-b expression and adjuvant IL-7 improved antitumor responses in murine models. We have therefore investigated whether IL-7 treatment of human T cells induces comparable biological effects as described for murine T cells. Cbl-b silencing or IL-7 treatment enhanced proliferation of TCR-stimulated CD4 and CD8 cells and induced increased production of IFN-g, TNF-a and IL-2. However, IL-7 treatment of cbl-b silenced T cells induced similar effects, suggesting mechanistic independence from cbl-b. Accordingly, IL-7 treatment of human CD4 and CD8 T cells did not modulate cbl-b expression. We next tested the role of STAT factors, as IL-7 is a member of the common cytokine receptor g- chain family (gc), which are known to signal via STAT transcription factors. Indeed, IL-7 induced STAT5 phosphorylation in human CD4 and CD8 T cells although with kinetic differences: in CD8 T cells STAT5 phosphorylation peaked earlier and declined more rapidly as compared to CD4 T cells. In agreement, the enhancement of cytokine production showed similar differences. IL-2, another member of the gc cytokine family, is clinically used as immune stimulant in cancer therapy. Thus, we have directly compared the effects of IL-2 and IL-7 on human immune cells. Like IL-7, IL-2 did not modulate cbl-b expression, but both cytokines induced STAT5 phosphorylation and enhanced proliferation and cytokine production. However, IL-7 failed to support the growth of human CD8 cells as efficiently as IL-2 did. Moreover, IL-2 but not IL-7 was able to activate human NK cells, thus suggesting that IL-7 will not have superior effects over IL-2 in clinically applied immune therapies against tumors. In conclusion, we show here that IL-7 does not modulate cbl-b expression in human T cells and therefore findings in the murine system using IL-7 might not be directly translatable into the clinic. In contrast, interventions targeting cblb have identical effects in murine and human T cells and thus seem to be a more promising strategy to enhance immune cell-mediated reactivity targeting tumors in vivo. Ovarian Cancer Cells Ubiquitously Express HER-2 and can be Distinguished from Normal Ovary by Genetically Redirected T Cells Evripidis Lanitis*w, Ian Hagemannz, Degang Song*, Lin Zhang*, Richard Carrolly, Raphael Sandaltzopoulosw, George Coukos*, Daniel J. Powell Jr*. *OCRC, UPENN; zPathology & Lab Medicine, UPENN; yAFCRI, UPENN, Philadelphia, PA; w MBG, Democritus University of Thrace, Alexandroupolis, Greece. Background: HER-2-specific T cells can be induced by vaccination or generated de novo by genetic engineering, however it remains uncertain to what extent T cell-based HER-2-directed immunotherapy can be utilized for the treatment of advanced ovarian cancer. Objective: To validate HER-2 as a well-suited tumor antigen for widespread T cell-based adoptive immunotherapy of ovarian cancer. Methods: HER-2 expression was first evaluated using immunohistochemical analysis (IHC) in 50 high-grade ovarian serous carcinomas. To determine the relative expression of HER-2 in ovarian cancer cell lines, patient tumor samples and normal ovarian surface epithelial cells (OSE), Q-PCR, FACS and western blot was performed. Human T cells were genetically engineered to express the C6.5 HER-2-specific chimeric immune receptor (CIR). HER-2-redirected T cells were tested for their capacity to recognize and kill HER-2 expressing tumors and OSE cells. Results: IHC analysis showed HER-2 expression in 52% of primary OvCas; 26 cases had HER-2 expressed at one or more tumor sites while HER-2 was undetectable in 24 samples. However, Q-PCR, FACS and western blot analysis demonstrated HER-2 expression in all established ovarian tumors (13/13) and short-term cultured tumors (7/7). Consistent with these results, all tumor cells derived from primary ascites (24/24) and solid tumor (12/12) expressed HER-2, albeit at variable levels. Compared to tumor, all (n = 4) normal OSE expressed lower but detectable HER-2 levels. Genetically redirected T cells recognized and reacted against all ovarian cancer cell lines (14/14), primary ascites (5/5) and solid tumor (5/5) tested, however little or no reactivity was observed against normal OSE (1/4). Conclusions: Our results show that IHC under represents the frequency of OvCas that express/overexpress HER-2 which may exclude patients with low HER-2 expressing tumors from receiving HER-2 targeted therapy. Utilizing more sensitive detection methods, we found that OvCas ubiquitously express HER-2, and generally at higher levels than normal ovary tissue. Importantly, all HER-2 expressing tumors are recognized by HER-2-redirected T cells and the latter are sensitive to even low levels of HER-2 expressed by OvCas. Importantly the CIR is able to distinguish recognition of ovarian cancer from normal targets, despite the fact that the normal cells do express HER-2 and therefore may minimize the potential for ‘‘off target’’ reactivity. These findings provide the rationale for the development of HER-2-redirected T cell-based immunotherapeutic approaches in women with ovarian carcinoma. r 2010 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 899
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