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Abstracts J Immunother Volume 33, Number 8, October 2010 FIGURE 1. Accumulation of 18 [F]-FEAU in CAR+tk+ T cells. Mice were anesthesized and CAR+tk+ T cells were injected subcutaneously in the right flank. Positron emission tomography computed tomography (PET/CT) images were acquired 2 hours after intravenous administration of 18 [F]-FEAU using Inveon micro-PET/CT scanner. Images were reconstructed by 2-dimensional ordered subsets expectation maximization algorithm. PET and CT image fusion and analysis were performed using vendor software Inveon Research Workplace. the dynamic status of infused T cells. In this study, we constructed SB DNA transposon vectors encoding a panel of transgenes expressing the wild type HSV1-tk fused to hygromycin phosphotransferase (in vitro selection) and FLAG tag (expression level). Primary T cells were co-electroporated with tkHy SB transposon and a CD19-specific chimeric antigen receptor (CAR) SB transposon and propagated on CD19-specific artificial antigen presenting cells in the presence of cytocidal concentrations of hygromycin B. After 4 weeks of numeric expansion (i) 90% of the T cells were CAR+tk+, (ii) accumulated high amounts of [3H] 2 0 -fluoro-2 0 deoxy-1-b-D-arabionofuranosyl-5-ethyl-uracil (iii) were ablated in the presence of ganciclovir and (iv) exhibited redirected killing of CD19+ tumor targets. The CAR+tk+ T cells could be visualized by mPET imaging in mice (Fig. 1). This is the first report showing that SB transposition can generate CAR+tk+ T cells which can be imaged by mPET in vivo. We have adapted SB system for human application (IND # 14193), and thus this study has immediate translational application to infuse CD19-specific T cells co-expressing HSV1-tk for imaging. Expansion of Autologous Human Vg9Vd2 T Cells Ex Vivo: Potential for Adoptive T Cell Therapy of Prostate Cancer Andrew Roberts*, Maria Serrano*, Elisa Binda*, Pierre Vantourout*w, Hardev Pandhaz, Adrian C. Hayday*w. *Peter Gorer Department of Immunobiology, King’s College London; w London Research Institute, Cancer Research UK, London; zPostgraduate Medical School, University of Surrey, Guildford, UK. Dysregulated cells including solid tumours express stress molecules that can be recognised by cell surface receptors on gd T cells, including the Vg9Vd2 T cell receptor (which recognises pyrophosphate molecules such as IPP that may be expressed on tumour cell surfaces) and the killer activatory receptor NKG2D. Because gd T cells exhibit marked functional pleiotropy upon activation, we employed extensive microarrays to demonstrate that the use of low molecular weight pyrophosphate antigens in combination with low-dose IL-2 could induce the expression of cytokines and cytolytic agents (eg, Granzymes, Perforin and TRAIL) that are known to be efficacious against tumours. Consequently, in a Phase I trial in which two cohorts of nine stage IV hormone-refractory prostate cancer patients received the amino-bisphosphonate zoledronate (which blocks IPP metabolism) with or without IL-2, we observed profound activation of gd effector responses in vivo, as well as an indication of therapeutic efficacy in the cohort receiving zoledronate with IL-2. However, repeated administration of gd-activating agents can lead to cell exhaustion in vivo; an indication of this is provided by the failure of the bisphosphonate-induced acute phase response to recur after the initial dose. Moreover, a combination of the tumour milieu and concurrent therapies could limit the capacity of some individuals to generate an adequate population of activated gd T cells able to target the tumour. It is therefore logical to complement bisphosphonate treatment by the activation of autologous gd T cells ex vivo and the subsequent infusion of them. This approach has been validated in mouse models of prostate cancer, and more recently in nude mice bearing human glioblastoma tumours that were treated by adoptive transfer of expanded human gd T cells. By extension, we are aiming to generate reproducible protocols for the ex vivo expansion of human Vg9Vd2 T cells, with the objective of adapting the system to GMP for intended treatment of patients with solid tumours. This approach is critically dependent upon the optimisation of key factors, such as the use of cytokines that, in addition to low-dose IL-2, might serve to augment survival and effector potential of such cells in long-term cultures. Furthermore, ongoing work also comprises the development of appropriate functional assays, in order that we can begin to predict in vitro the likely success of subsequent cell engraftment in vivo. Dissection of Therapy-Induced Melanoma-Reactive Cytotoxic T Cell Responses Ton Schumacher. Department of Immunology, Netherlands Cancer Institute, Amsterdam, Netherlands. There is strong evidence that melanoma-reactive T cell responses induced by immunotherapeutic interventions such as anti-CTLA4 treatment or TIL therapy can exert clinically meaningful effects. However, at present we do not know which cytotoxic T cell reactivities mediate cancer regression. Furthermore, as the number of melanoma-associated antigens to which these responses can be directed is very high, classical strategies to map cytotoxic T cell reactivity do not suffice. Knowledge of such reactivities would be useful to design more targeted strategies that selectively aim to induce immune reactivity against these antigens. In the past years we have aimed to address this issue by designing MHC class I molecules occupied with UV-sensitive ‘‘conditional’’ peptide ligands, thereby allowing the production of very large collections of pMHC complexes for T cell detection. Secondly, we have developed a ‘‘combinatorial coding’’ strategy that allows the parallel detection of dozens of different T cell populations within a single sample. The combined use of MHC ligand exchange and combinatorial coding allows the high-throughput dissection of disease- and therapy-induced CTL immunity, and we have now used this platform to monitor immune reactivity against a panel of over 200 melanoma-associated epitopes. First data on the composition and engraftment of TIL products used for adoptive cellular therapy will be presented. A Data Mining Architecture for Studying Cytokine Production and T Cell Repertoire of Tumor-Specific T Cells Generated from Tumor-Infiltrating Lymphocytes Janet C. Siebert*, Sachin Puriw, Tarsem Moudgilw, William Millerw, Edwin B. Walkerw, Carlo Bifulcow, Brendan D. Curtiw, Walter J. Urbaw, Bernard A. Foxw. *CytoAnalytics, Denver, CO; w Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Cancer Center, Portland, OR. 864 | www.immunotherapy-journal.com r 2010 Lippincott Williams & Wilkins
J Immunother Volume 33, Number 8, October 2010 Abstracts Generating T cells with specific reactivity against tumor-associated antigens is a key component of adoptive immunotherapy. In our work, we have established lymphocyte cultures from tumorinfiltrating lymphocytes. After digestion, cells from a specific tumor are grown under different culture conditions such as IL-2, anti-OX40 mAb, NOS inhibitor+Arginase inhibitor, NOS inhibitor+Arginase inhibitor+OX40, IL-15+Low Dose IL-2, or IL- 15+High Dose IL-2. We refer to the mixed cell population from each of these tumor-culture conditions as CLOIDs. If a particular CLOID was successfully expanded over a 3 to 8 week period, that CLOID was in turn aliquoted and subjected to multiple stimulation conditions such as activation by autologous tumor, allogenic tumor, or anti-CD3. Each of these aliquots were inspected by ELISA for the release of cytokines such as IFN-g, IL-5, and IL-17. Additionally, the T cell repertoire of each CLOID was examined by multiparameter flow cytometry for subphenotypes as defined by CD4, CD8, CD107a, CCR7, CD45RA, CD27 and CD28 mAb staining. Making sense of this complex data for multiple tumors, multiple culture conditions and multiple stimulation conditions is the challenge addressed here. We created a Rich Analytical Environment by integrating both the ELISA data and the flow cytometry data in a relational database. We also included relevant descriptive data such as source tumor and culture conditions. This approach gave us fast and reliable access to all data from the study. Then, we addressed relevant questions using a combination of visual and statistical techniques. We were able to show that: For 8 of 9 tumors, there were no statistically significant differences between the number of cells produced by tumor reactive CLOIDs (n = 156 total) digested with enzyme and those digested mechanically. For some tumors, CLOIDs digested with enzyme produced significantly more IFN-g than those digested mechanically. In tumors digested with enzyme, the autologous tumor-specific central memory (CCR7+CD45RA CD27+CD28+) and central memory-like (CCR7+CD27+CD28+) CD4 and CD8 T cell populations were significantly higher when cultured with IL-15 and low dose IL-2 than with high dose IL-2 alone (0.001< P
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