Stephane Paul

Bypassing tumor-associated immune suppression with recombinant adenovirus constructs expressing membrane bound or secreted GITR-L

Recent evidence has resurrected the concept of specialized populations of T lymphocytes that are able to suppress an antigen-specific immune response. T-regulatory cells (T-reg) have been characterized as CD4+ CD25+ T cells. Previous reports describing differential gene expression analysis have shown that the glucocorticoid-induced tumor necrosis family receptor family-related gene (GITR) is upregulated in these cells. Furthermore, antibodies specific for GITR have been shown to inhibit the T-suppressor function of CD4+ CD25+ T-reg. The ligands for both mouse and human GITR have been cloned recently. We have inserted the sequences for natural, membrane-bound GITR-ligand (GITR-L) and a truncated secreted form of GITR-L (GITR-Lsol) into the adenovirus-5 genome. Coculture experiments show that cells infected with Ad-GITR-L and supernatants from cells infected with Ad-GITR-Lsol can increase the proliferation of both CD4+ CD25- and CD8+ T cells in response to anti-CD3 stimulation, in the presence, as well as in the absence, of CD4+ CD25+ T cells. The virus constructs were injected into growing B16 melanoma tumors. Ad-GITR-L was shown to attract infiltration with both CD4+ and CD8+ T cells. Both constructs were shown to inhibit tumor growth.

Improvement of Adoptive Cellular Immunotherapy of Human Cancer Using Ex-Vivo Gene Transfer

A variety of adoptive cellular strategies, aimed at boosting the immune system, have been tested in the management of metastatic diseases. Despite the drawbacks associated with ex vivo cell manipulation and upscaling, several such approaches have been assessed in the clinic. The use of lymphokine-activated killer (LAK) cells, auto-lymphocyte therapy (ALT) and tumor-infiltrating lymphocytes (TIL) have been the best studied and further trials are ongoing. Thus far, these approaches have not consistently shown benefit when compared to standard immune-based treatment with biologic response modifiers, notably, high-dose interleukin-2 (IL-2). More recently, it has been shown, in various animal models, that the ex vivo transfer of genes to cells of the immune system can have a dramatic impact on cancer immunotherapy. The application of gene transfer techniques to immunotherapy has animated the field of cell-based cancer therapy research. A wide variety of viral and non-viral gene transfer methods have been investigated in this context. Ex vivo strategies include gene delivery into tumor cells and into cellular components of the immune system, including cytotoxic T cells, NK, macrophages and dendritic cells (DC). Several of these approaches have already been translated into cancer therapy clinical trials. In this review, we focus on the rationale and types of ex vivo gene-based immunotherapy of cancer. Finally, the use of genetically modified DC for tumor vaccination and its prospects are discussed.

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

Tumor gene therapy by MVA-mediated expression of T-cell–stimulating antibodies

Immune responses to tumor-associated antigens are often dampened by a tumor-induced state of immune anergy. Previous work has attempted to overcome tumor-induced T-cell anergy by the direct injection of vectors carrying the genes encoding one of a variety of cytokines. We hypothesised that the polyclonal stimulation of T cells, preferably through the TCR complex, would result in a cascade of cytokines associated with T-cell activation and would be best able to overcome T-cell anergy. Here we use the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, either of three membrane-bound monoclonal antibodies specific for murine TCR complex. Using this system, we have expressed antibodies specific for the CD3ɛ chain (KT3), TCRα/β complex (H57-597), and Vβ7 chain (TR310). Tumor cells bristling with these antibodies are capable of inducing murine T-cell proliferation and cytokine production. When injected into growing tumors (P815, RenCa, and B16F10), these constructs induce the activation of immune effector cells and result in the rejection of the tumor. Histological and FACS analysis of tumor-infiltrating leukocytes reveal that the injection of recombinant virus-expressing antibodies specific for the TCR complex attracts and activates (CD25+, CD69+) CD4 and CD8 lymphocytes. This approach represents a novel strategy to overcome T-cell anergy in tumors and allow the stimulation of tumor-specific T cells.

The combination of a chemokine, cytokine and TCR-based T cell stimulus for effective gene therapy of cancer

Abstract.Cytotoxic T cells can recognize and kill tumor cells that present peptides derived from tumor-associated antigens (TAA) on their surface when associated with major histocompatibility complex (MHC) class I molecules. However, immune responses to tumor-associated antigens are often suppressed by a tumor-induced state of immune anergy. Previous work has attempted to overcome tumor-induced T cell anergy by the direct injection of vectors carrying genes encoding one of a variety of cytokines. Polyclonal stimulation of T cells, preferably via the TCR complex, results in a cascade of cytokines associated with T cell activation and thus may be better able to overcome T cell anergy. We have previously reported the use of the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, antibodies specific for the CD3ε chain (KT3). When injected into growing tumors, these constructs induce the activation of immune effector cells and result in rejection of the tumor. A variety of recombinant adenovirus (Ad) vectors expressing immunostimulatory and/or immunoattractant molecules have now been produced. With this collection of viruses, we have carried out in vivo analyses of combinations of vectors in tumor therapy experiments. For example, we have tested, in murine tumor models, the combination of MVA-KT3 with Ad expressing recently identified cytokines [for example interleukin-12 (IL-12), IL-18] as well as chemokines (e.g. RANTES, MIP1β). One combination, MVA-KT3/Ad-IL-12/Ad-MIP1β causes rejection of 100% of growing RENCA tumors. Much attention has been focused on cancer gene therapy using gene transfer of single agents. These data show that antigenic stimulation via the MHCI/TCR-CD3+cytokine+chemokine combination may provide a new and promising approach to cancer gene therapy which is more likely to bypass tumor immunosuppression mechanisms.

Lack of evidence for an immunosuppressive role for MUC1.

Abstract The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1- and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells.

Redirected cellular cytotoxicity by infection of effector cells with a recombinant vaccinia virus encoding a tumor-specific monoclonal antibody.

Cytotoxicity is an important function of the immune system that results in the destruction of cellular targets by humoral and/or cellular mechanisms. We wanted to assess the possibility of targeting the lytic function of immune cells toward cancer cells, which express the gene coding for a known tumor antigen (Ag) (GA733-2/epithelial cell adhesion molecule), using a viral vector encoding a monoclonal antibody (mAb) specific for said tumor Ag (CO17-1A). To this end, we have constructed recombinant vaccinia viruses expressing the sequences corresponding to mAb CO17-1A, which recognizes a specific Ag (GA733-2) that is present on the surface of most gastrointestinal carcinomas. The recombinant vectors encoding either a secreted or membrane-anchored form of CO17-1A mAb were used to infect effector cells, which were subsequently assessed for their cytotoxic activity. The recombinant viruses were able to infect both granulocyte-macrophage colony-stimulating factor-activated human macrophages and Ag-stimulated murine cytotoxic T lymphocytes. Infected granulocyte-macrophage colony-stimulating factor-activated macrophages were found to be able to kill GA733-2-expressing tumor cells. Likewise, infected cytotoxic T lymphocytes, although conserving their original alloreactivity, gained the capability of killing GA733-2-expressing cancer cells.

Secretomers as a new tool for the monitoring of CTL responses

Efforts to follow tumor-specific immune responses in patients are often thwarted by lack of knowledge of the appropriate tumor antigens and the CTL epitopes of those antigens. There is, therefore, a growing need for techniques to monitor tumor-specific immune responses in settings where tumor antigens, and antigenic epitopes, remain unidentified. Here we describe a novel system to follow tumor-specific CTL immune responses. A truncated, soluble murine class I MHC (H-2Db) molecule was fused with a rat IgG2a Fc, in order to allow secretion of the complex. Tumor-specific CTL could then be detected as a result of the complex fastening to specific T cell receptors (TCR). These constructs were inserted into the genome of a recombinant adenovirus vector. Infection of tumor cells with these adenovirus constructs results in the secretion of the complexes into the culture supernatant. These soluble divalent class I MHC molecules were used to detect and activate specific CTL populations.