Gene Therapy-Mediated Reprogramming Tumor Infiltrating T Cells Using IL-2 and Inhibiting NF-κB Signaling Improves the Efficacy of Immunotherapy in a Brain Cancer Model

Abstract

Immune-mediated gene therapy using adenovirus expressing Flt3 ligand and thymidine kinase followed by ganciclovir administration (Flt3/TK) effectively elicits tumor regression in preclinical glioma models. Herein, we assessed new strategies to optimize Flt3L/TK therapeutic efficacy in a refractory RG2 orthotopic glioblastoma model. Specifically, we aimed to optimize the therapeutic efficacy of Flt3L/TK treatment in the RG2 model by overexpressing the following genes within the brain tumor microenvironment: 1) a TK mutant with enhanced cytotoxicity (SR39 mutant TK), 2) Flt3L-IgG fusion protein that has a longer half-life, 3) CD40L to stimulate DC maturation, 4) T helper cell type 1 polarizing dendritic cell cytokines interleukin-12 or C-X-C motif ligand 10 chemokine (CXCL)-10, 5) C-C motif ligand 2 chemokine (CCL2) or C-C motif ligand 3 chemokine (CCL3) to enhance dendritic cell recruitment into the tumor microenvironment, 6) T helper cell type 1 cytokines interferon-γ or interleukin-2 to enhance effector T-cell functions, and 7) IκBα or p65RHD (nuclear factor kappa-B [NF-κB] Neurotherapeutics (2012) 9:827-843 DOI 10.1007 immunity and tumor specific effector T-cell functions were assessed by cytotoxic T lymphocyte assay and intracellular IFN-γ staining. Our data showed that overexpression of interferon-γ or interleukin-2, or inhibition of the nuclear factor kappa-B within the tumor microenvironment, enhanced cytotoxic T lymphocyte-mediated immune responses and successfully extended the median survival of rats bearing intracranial RG2 when combined with Flt3L/TK. These findings indicate that enhancement of T-cell functions constitutes a critical therapeutic target to overcome immune evasion and enhance therapeutic efficacy for brain cancer. In addition, our study provides novel targets to be used in combination with immunetherapeutic strategies for glioblastoma, which are currently being tested in the clinic.

Blockade of mTOR Signaling via Rapamycin Combined with Immunotherapy Augments Antiglioma Cytotoxic and Memory T-Cell Functions

“…Addition of Ad-IL-2 to the Ad-Flt3L + Ad-TK/GCV immunotherapy enhanced the ratio of cytotoxic T lymphocytes/Tregs resulting in augmented anti-tumor CD8 + T cell responses. Similar effects were obtained by the inhibition of NF-κB signaling that resulted in reduced Foxp3 expression in CD4 + T cells and enhanced IFN-γ release by CD8 + T cells (17). …”

Immunosuppressive Myeloid Cells’ Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy

“…[17][18][19][20] The gene therapy approach entails using adenoviruses (Ads) encoding herpes simplex virus type-I thymidine kinase (Ad-TK) and Fms-like tyrosine kinase 3 ligand (Flt3L), which are injected into the tumor followed by ganciclovir (GCV) administration. Thymidine kinase converts GCV to its active metabolite, which leads to tumor cell lysis, with concomitant release of tumor antigens and damage-associated molecular pattern molecules, i.e., HMGB1.…”

“…17,19,64,69 All cell suspensions obtained from tissues were resuspended in PBS containing 2% fetal calf serum (FCS) (flow buffer) for antibody staining for flow cytometry. All flow data were acquired on a FACSAria flow cytometer (BD Biosciences) and analyzed using Flow Jo version 10 (Treestar).…”

Induction of antigen-specific cytotoxic T-cell response by dendritic cells generated from ecto-mesenchymal stem cells infected with an adenovirus containing the MAGE-D4a gene

“…In pediatric patients with glioma, DC-based immunotherapies may exert certain clinical benefits, particularly in individuals with minimal residual disease; however, additional investigation of such treatment is necessary (7)(8)(9). Unconventional treatment options, including gene therapy combined with immunocyte-based immunotherapy, also offer potential therapeutic approaches to reduce glioma-associated mortality (10). Gene therapies have been implicated in the treatment of brain tumors, with animal models demonstrating preclinical efficacy, in addition to encouraging safety profiles observed in phase I clinical trials; however, in phase III clinical trials, such therapies have thus far failed to demonstrate significant therapeutic efficacy (11,12).…”