358 Systemic Combination Virotherapy for Malignant Melanoma with Vesicular Stomatitis Virus and Adoptive T Cell Transfer Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American So[.]
CANCER-ONCOLYTIC VIRUSES 356 CD8:Class I MHC Interactions Influence T Cell Activity Mediated by Chimeric Antigen Receptors David H Aggen,1 Jennifer D Stone,1 Adam S Chervin,1 Andrea Schietinger,2 Karin Schreiber,2 Hans Schreiber,2 David M Kranz.1 Department of Biochemistry, University of Illinois UrbanaChampaign, Urbana, IL; 2Department of Pathology, Committee on Cancer Biology, University of Chicago, Chicago, IL One approach to adoptive T cell therapy uses chimeric antigen receptors (CARs) that contain an antibody (scFv) specific for a tumor-associated antigen CARs derived from antibodies were originally conceived to target non-MHC restricted antigens, and unlike most full-length T cell receptors, should in principle be able to redirect T cell activity independent of the coreceptors CD4 or CD8 To examine the ability of a single-chain fragment variable (scFv) CAR to redirect T cells to tumor antigen, we used a model murine scFv, called 237, to redirect T cell activity against a tumorspecific antigen The 237 antibody recognizes a mutated glycopeptide expressed by an aggressive murine fibrosarcoma tumor cell line called Ag104a The tumor-specific glycopeptide is generated as a result of a mutated chaperone protein Cosmc that when repaired, ablates the 237 neo-epitope Using a CAR that consisted of the 237 scFv fused to the intracellular signaling domains of CD28,CD3ζ, and Lck, we demonstrate here that T cell specific activation occurred in the presence of the Ag104a tumor line, but not in the presence of a cell line with a repaired chaperone protein that lacks the tumor antigen (ACosmc) To assess whether CD8 plays any role in the activation, we generated a T cell hybridoma line that expressed the 237 CAR, with and without CD8 The CD8- 237 and CD8+ 237 expressing cells mediated similar activation with Ag104a tumor cells However, when the 237 CAR lines were incubated with plate-bound glycopeptide antigen, the CD8+ line was completely inactive, whereas the CD8line was stimulated effectively Thus, CD8 expression inhibited antigen-specific activity when antigen was targeted that lacked MHC Class I We propose that Lck sequestration by the co-receptors away from the immunological synapse reduced signaling mediated by interactions with non-MHC ligands Additional lines of evidence included the inhibition of activity of the 237 CAR/CD8 line, in the presence of the Ag104a APC, with an antibody to CD8 In the normal activation of CD8+ T cells, CD8 with its associated Lck colocalizes at the immunologic synapse with T cell receptors, facilitating efficient T cell responses CD8 contributed to CAR mediated activity, presumably through interactions with class I MHC molecules that cluster near the scFv antigen epitopes This observation that class I MHC can facilitate the activity of a CAR through a CD8-dependent mechanism has important implications for CARs directed against non-MHC antigens in tumor immunotherapy For example, reduced class I MHC levels on a patient’s tumor cells may reduce the efficacy of CAR-mediated T cell activity by allowing Lck to be sequestered away form the synapse Support from NIH Program Project # P01 CA097296 357 Genetically Engineered T-Cells Targeting Cancer Associated Fibroblasts for Cancer Immunotherapy Sunitha Kakarla,1,3 Lisa Wang,1 David Rowley,1 Klaus Pfizenmaier,2 Stephen Gottschalk.1,3 Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX; 2Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany; 3Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Introduction: Adoptive T-cell therapy has had considerable success in effectuating antitumor responses, however complete eradication of bulky disease is rarely observed This limited efficacy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy is most likely due to the tumor stroma, which is not targeted by tumor-specific T cells Cancer associated fibroblasts (CAFs), the central component of the tumor stroma, secrete inhibitory factors and nutrient depleting enzymes that are detrimental to effector T-cell function In addition, CAFs promote angiogenesis and secrete extracelluar matrix components, which act as a physical barrier CAFs express fibroblast activation protein (FAP); a membrane bound serine protease, which is an attractive immunotherapeutic target The aim of this project was to generate FAP-specific T cells and determine if targeting the tumor stroma with FAP-specific T cells has antitumor effects Methods: To generate FAP-specific T cells we took advantage of chimeric antigen receptors (CARs), which consists of antigen-specific single chain variable fragments (scFv) linked to T-cell receptor signaling domains Using this approach we generated CARs, which are specific for human FAP (hFAP) or human and murine FAP (mhFAP) T cells expressing hFAP-CARs or mhFAP-CARs were generated by retroviral transduction (hFAPor mhFAP-T cells) Ex vivo, efficacy of hFAP- and mhFAP-T cells was determined by their ability to 1) secrete cytokines in coculture experiments with FAP-positive tumor or stroma cells, and 2) kill FAP-positive targets in cytotoxicity assays To test in vivo, if selective targeting of FAP on tumor stroma prevents the development of human tumors in a xenograft model, we took advantage of lymphoblastoid cell lines (LCL), which are FAP-negative Results: hFAP-CARs or mhFAP-CARs were successfully expressed on T cells as judged by FACS analysis hFAP- T cells recognized FAP-positive human tumor and stroma cells as judged by cytokine production and cytotoxicity assays In addition to recognizing human FAP positive targets, the mhFAP-T cells also recognized and killed murine FAP targets To evaluate if targeting the tumor stroma, prevents the development of tumors, FAP-negative, luciferase-expressing LCLs, were mixed with hFAP, mhFAP-, or nontransduced T cells prior to the s.c injection into flanks of SCID mice While LCLs tumor readily established in mice injected with LCL/hFAP-T cells or LCL/NT-T cells; LCL tumor growths was 10 to 100 fold slower in mice injected with LCL/mhFAP-T cells as judged by serial bioluminescence imaging Conclusions: Our results indicate that targeting FAP-positive stroma with T cells delays the development of tumors in human xenograft models Thus, targeting the tumor stroma in addition to cancer cells with FAP-specific T cells has the potential to improve current immunotherapy approaches for cancer Cancer-Oncolytic Viruses 358 Systemic Combination Virotherapy for Malignant Melanoma with Vesicular Stomatitis Virus and Adoptive T Cell Transfer Diana M Rommelfanger,1,2 Phonphimon Wongthida,1 Karen K Kaluza,1,3 Rosa M Diaz,1,3 Jill M Thompson,1 Timothy J Kottke,1 Richard G Vile.1,3 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 2Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN; 3Department of Immunology, Mayo Clinic, Rochester, MN Our goal is to develop truly systemic virotherapy treatment regimens for metastatic cancer that not necessitate direct intratumoral injection We have shown in the B16ova melanoma model that combining adoptively transferred OT-I T cells (1x10^6 naïve cells), which are specific for the surrogate tumor-associated antigen (TAA) OVA, with intratumoral VSV (5x10^8 PFU) encoding the OVA antigen (VSV-ova) results in regression of established tumors and an enhancement of survival compared to either treatment alone (p