The Suicide Gene Therapy Challenge: How to Improve a Successful Gene Therapy Approach

The transfer of a suicide gene into donor lymphocytes to control alloreactivity in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the widest clinical application of T-cell based gene transfer, as shown by more than 100 patients treated worldwide to date, several phase I–II studies completed, and a registrative phase III study, sponsored by a biotech firm, about to begin. In this mini-review, we will summarize the clinical results obtained to date, and attempt to identify the steps envisaged to optimize the suicide gene therapy approach

Intratumor Heterogeneity of Cancer/Testis Antigens Expression in Human Cutaneous Melanoma Is Methylation-Regulated and Functionally Reverted by 5-Aza-2′-deoxycytidine

Abstract Cancer/testis antigens (CTA) are suitable targets for immunotherapy of human malignancies, and clinical trials are mainly focusing on MAGE-A3. However, the heterogeneous intratumor expression of CTA may hamper the effectiveness of CTA-directed vaccination through the emergence of CTA-negative neoplastic clones. We investigated the intratumor heterogeneity of CTA in human melanoma and the underlying molecular mechanism(s) at clonal level using 14 single cell clones generated from the melanoma lesion Mel 313. Reverse transcription-PCR revealed a highly heterogeneous expression of MAGE-A1, -A2, -A3, -A4, -A6, GAGE 1–6, SSX 1–5, and PRAME among melanoma clones. Only nine clones expressed MAGE-A3 and competitive reverse transcription-PCR identified relative differences in the number of mRNA molecules of up to 130-fold between clones 5 and 14. This clonal heterogeneity of MAGE-A3 expression correlated with the methylation status of specific CpG dinucleotides in MAGE-A3 promoter: i.e., hypomethylated CpG dinucleotides at positions −321, −151, −19, −16, −5, −2, +21, and +42 were found in clones expressing high but not low levels of MAGE-A3. Supporting the role of DNA methylation in generating the intratumor heterogeneity of CTA, the DNA hypomethylating agent 5-aza-2′-deoxycytidine (5-AZA-dCyd) invariably induced their expression in all CTA-negative clones. Furthermore, 5-AZA-dCyd–treatment reduced to 6 folds the differential expression of MAGE-A3 between clones 5 and 14, which became recognized to a similar extent by T cells specific for a MAGE-A–encoded peptide. These findings identify promoter methylation as directly responsible for the intratumoral heterogeneity of therapeutic CTA in melanoma and foresee the use of 5-AZA-dCyd to overcome the limitations set by their intratumor heterogeneous expression to CTA-based vaccine therapy

An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

The effects of a chimeric monoclonal antibody (chA6 mAb) that recognizes both the RO and RB isoforms of the transmembrane protein tyrosine phosphatase CD45 on human T cells were investigated. Chimeric A6 (chA6) mAb potently inhibited antigen-specific and polyclonal T cell responses. ChA6 mAb induced activation-independent apoptosis in CD4+CD45RO/RBhigh T cells but not in CD8+ T cells. In addition, CD4+ T cell lines specific for tetanus toxoid (TT) generated in the presence of chA6 mAb were anergic and suppressed the proliferation and interferon (IFN)-γ production by TT-specific effector T cells by an interleukin-10–dependent mechanism, indicating that these cells were equivalent to type 1 regulatory T cells. Similarly, CD8+ T cell lines specific for the influenza A matrix protein-derived peptide (MP.58-66) generated in the presence of chA6 mAb were anergic and suppressed IFN-γ production by MP.58-66–specific effector CD8+ T cells. Furthermore, chA6 mAb significantly prolonged human pancreatic islet allograft survival in nonobese diabetic/severe combined immunodeficiency mice injected with human peripheral blood lymphocytes (hu-PBL-NOD/SCID). Together, these results demonstrate that the chA6 mAb is a new immunomodulatory agent with multiple modes of action, including deletion of preexisting memory and recently activated T cells and induction of anergic CD4+ and CD8+ regulatory T cells

IL-7 and IL-15 allow the generation of suicide gene–modified alloreactive self-renewing central memory human T lymphocytes

Abstract Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (TEM) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (TCM) phenotype. To this, we generated suicide gene–modified TCM lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene–modified TCM cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene–modified T cells cultured with IL-7 and IL-15 persisted, differentiated in TEM cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene–modified TCM cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer

Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients

An Efficient Strategy to Induce and Maintain In Vitro Human T Cells Specific for Autologous Non-Small Cell Lung Carcinoma

BACKGROUND: The efficient expansion in vitro of cytolytic CD8+ T cells (CTLs) specific for autologous tumors is crucial both for basic and translational aspects of tumor immunology. We investigated strategies to generate CTLs specific for autologous Non-Small Cell Lung Carcinoma (NSCLC), the most frequent tumor in mankind, using circulating lymphocytes. PRINCIPAL FINDINGS: Classic Mixed Lymphocyte Tumor Cultures with NSCLC cells consistently failed to induce tumor-specific CTLs. Cross-presentation in vitro of irradiated NSCLC cells by autologous dendritic cells, by contrast, induced specific CTL lines from which we obtained a high number of tumor-specific T cell clones (TCCs). The TCCs displayed a limited TCR diversity, suggesting an origin from few tumor-specific T cell precursors, while their TCR molecular fingerprints were detected in the patient's tumor infiltrating lymphocytes, implying a role in the spontaneous anti-tumor response. Grafting NSCLC-specific TCR into primary allogeneic T cells by lentiviral vectors expressing human V-mouse C chimeric TCRalpha/beta chains overcame the growth limits of these TCCs. The resulting, rapidly expanding CD4+ and CD8+ T cell lines stably expressed the grafted chimeric TCR and specifically recognized the original NSCLC. CONCLUSIONS: This study defines a strategy to efficiently induce and propagate in vitro T cells specific for NSCLC starting from autologous peripheral blood lymphocytes

Rexpression of HLA class I antigens and restoration of antigen-specific CTL response in melanoma cells following 5-aza-2?-deoxycytidine treatment

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