Shao-An Xue

Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines

BACKGROUND & AIMSnVirus-specific T cells capable of controlling HBV and eliminating hepatocellular carcinoma (HCC) expressing HBV antigens are deleted or dysfunctional in patients with chronic HBV or HBV-related HCC. The goal of this study was to determine if T cell receptor (TCR) gene transfer can reconstitute HBV-specific T cell immunity in lymphocytes of chronic HBV patients and investigate whether HCC cells with natural HBV-DNA integration can be recognized by genetically modified T cells.nnnMETHODSnWe used vector-mediated gene transfer to introduce HLA-A2-restricted, HBV-specific TCRs into T cells of chronic HBV as well as HBV-related HCC patients.nnnRESULTSnThe introduced TCRs were expressed on the cell surface, evidenced by Vβ and pentamer staining. TCR transduced T cells produced IFN-γ, TNF-α, IL-2, and lysed HBV infected hepatocyte-like cell lines. Furthermore, HCC cell lines with natural HBV-DNA integration could be recognized by HBV-specific TCR-re-directed T cells.nnnCONCLUSIONSnTCR re-directed HBV-specific T cells generated from PBMC of chronic HBV and HBV-related HCC patients were multifunctional and capable of recognizing HBV-infected cells and HCC tumor cells expressing viral antigens from naturally integrated HBV DNA. These genetically modified T cells could be used to reconstitute virus-specific T cell immunity in chronic HBV patients and target tumors in HBV-related HCC.

Antibody targeted gene transfer to endothelium

One of the drawbacks of the currently available vectors for gene therapy is the lack of selectivity in gene delivery. We have therefore investigated a strategy to generate immunoliposomes to target non‐viral vectors to cell surface receptors on endothelium.

Virus-Associated RNA I-Deleted Adenovirus, a Potential Oncolytic Agent Targeting EBV-Associated Tumors

Given the growing number of tumor types recognizably associated with EBV infection, it is critically important that therapeutic strategies are developed to treat such tumors. Replication-selective oncolytic adenoviruses represent a promising new platform for anticancer therapy. Virus-associated I (VAI) RNAs of adenoviruses are required for efficient translation of viral mRNAs. When the VAI gene is deleted, adenovirus replication is impeded in most cells (including HEK 293 cells). EBV-encoded small RNA1 is uniformly expressed in most EBV-associated human tumors and can functionally substitute for the VAI RNAs of adenovirus. It enables replication to proceed through complementation of VAI-deletion mutants. We hypothesized that VAI-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to the presence of EBV-encoded small RNA1 with no (or poor) replication in normal or EBV-negative tumor cells. In this report, we show that high levels of replication occurred in the VAI-deleted mutant in the EBV-positive tumor cells compared with low (or negligible) levels in EBV-negative and normal human primary cells. Correspondingly, high toxicity levels were observed in EBV-positive tumor cells but not in EBV-negative tumor or normal human primary cells. In vivo, VAI-deleted adenovirus showed superior antitumoral efficacy to wild-type adenovirus in EBV-positive tumor xenografts, with lower hepatotoxicity than wild-type adenovirus. Our data suggest that VAI-deleted adenovirus is a promising replication-selective oncolytic virus with targeting specificity for EBV-associated tumors.

Effect of vectors on human endothelial cell signal transduction: implications for cardiovascular gene therapy.

Objective—Endothelium is an important target for gene therapy. We have investigated the effect of viral and nonviral vectors on the phenotype and function of endothelial cells (ECs) and developed methods to block any activation caused by these vectors. Methods and Results—Transduction of ECs with viral vectors, including adenovirus, lentiviruses, and Moloney murine leukemia virus, can induce a pro-inflammatory phenotype. This activation was reduced when nonviral vectors were used. We demonstrate that after transduction there is upregulation of dsRNA-triggered antiviral and PI3K/Akt signaling pathway. Blockade of the NF&kgr;B, PI3-K, or PKR signaling pathways all operated to inhibit partially virally induced activation, and inhibition of both PKR and PI3-K pathways totally blocked EC activation. Furthermore, inhibition of IFN-α/β in addition to PI3-K was effective at preventing EC activation. Conclusions—Viral vectors, although efficient at transducing ECs, result in their activation. Blockade of the signaling pathways involved in viral activation may be used to prevent such activation.

Adiponectin Receptor Signaling on Dendritic Cells Blunts Antitumor Immunity

Immune escape is a fundamental trait of cancer. Dendritic cells (DC) that interact with T cells represent a crucial site for the development of tolerance to tumor antigens, but there remains incomplete knowledge about how DC-tolerizing signals evolve during tumorigenesis. In this study, we show that DCs isolated from patients with metastatic or locally advanced breast cancer express high levels of the adiponectin receptors AdipoR1 and AdipoR2, which are sufficient to blunt antitumor immunity. Mechanistic investigations of ligand-receptor interactions on DCs revealed novel signaling pathways for each receptor. AdipoR1 stimulated IL10 production by activating the AMPK and MAPKp38 pathways, whereas AdipoR2 modified inflammatory processes by activating the COX-2 and PPARγ pathways. Stimulation of these pathways was sufficient to block activation of NF-κB in DC, thereby attenuating their ability to stimulate antigen-specific T-cell responses. Together, our findings reveal novel insights into how DC-tolerizing signals evolve in cancer to promote immune escape. Furthermore, by defining a critical role for adiponectin signaling in this process, our work suggests new and broadly applicable strategies for immunometabolic therapy in patients with cancer.

796. In Vivo Elimination of Human Leukaemia Cells by WT1 Specific TCR Transduced T Cells

Previously, we have shown that to circumvent T cell tolerance to the Wilms tumor antiegn-1 (WT1), cytotoxic T lymphocytes (CTL) specific for an HLA-A2-presented peptide epitope of the WT1 have been generated from allogeneic donors, and these CTL can selectively kill immature human leukemia progenitor and stem cells in vitro. In this study we have isolated the T cell receptor (TCR) genes of an allorestricted CTL line for retroviral gene transfer into T lymphocytes obtained from both leukemia patients and healthy donors. Here we show that TCR transduced CTL kill T2 target cells coated with the WT1 peptide but not with irrelevant peptide, and these T cells also kill leukemia cells in vitro and display WT1-specific cytokine production. Intravenous injection of TCR transduced T cells into NOD/SCID mice harbouring human leukemia cells resulted in leukaemia elimination, whilst transfer of control T cells transduced with an irrelevant TCR was ineffective. These data suggest that adoptive immunotherapy with WT1-TCR gene modified patient T cells should be considered for the treatment of human leukemia.

CREATION OF TOLEROGENIC ANTIGEN PRESENTING CELLS VIA INTRACELLULAR CTLA4: A NOVEL STRATEGY WITH POTENTIAL CLINICAL UTILITY IN TRANSPLANTATION.

Aims: Activation of T lymphocytes requires the recognition of peptide–MHC complexes and co-stimulatory signals provided by antigen-presenting cells (APCs). It has been shown that T cell activation without co-stimulation can lead to T cell anergy, a state of T cell hyporesponsiveness. In this study we developed a novel strategy to inhibit expression of B7 molecules by transfecting APCs with a gene construct encoding a modified CTLA4 molecule (CTLA4-KDEL) that is targeted to the endoplasmic reticulum (ER). Our approach is to express within the cells CTLA4-KDEL. This will bind to the newly synthesised B7 molecules in the ER and prevent them from reaching the cell surface. Methods: The CTLA4-KDEL gene was constructed using standard technology. It was then transfected into a range of APCs. In the case of DCs transfection was performed using a new non-viral gene therapy system. The APCs were then tested in their ability to activate peptide specific T cell clones and primary allospecific T cells. OVA TG model was used to assess in vivo tolerance. Results: Our in vitro and in vivo data using CTLA4-KDEL in various APCs show that this strategy does indeed block B7 expression by APC and that antigen-reactive (both allospecific and peptide-specific) T cells not only are hyporesponsive to antigen presented by these cells but also are rendered anergic. Conclusions: This gene-based strategy to inhibit expression of key surface receptors in order to generate ‘B7deficient’ APCs has significant advantages over the currently described approaches for treatment of transplant rejection. Transplant 6328