Concurrent induction of T-cell activation and apoptosis of osteosarcoma cells by adenovirus-mediated B7-1/Fas chimeric gene transfer

Abstract

To establish an effective B7-based gene therapy against osteosarcoma, we transferred B7-1/Fas chimeric gene adenovirally into poorly immunogenic osteosarcoma cells. We found that adenovirus-mediated rat B7-1/Fas gene transfer induced (i) expression of rat B7-1/Fas chimeric molecules in osteosarcoma cells, (ii) activation of murine T cells, (iii) apoptosis of murine osteosarcoma cells in the presence of anti-rat B7-1 mAb in vitro, and (iv) therapeutic effects more prominently than B7-1 gene transfer on the development of pulmonary metastasis and survival of mice. These findings collectively support the therapeutic value of adenovirusmediated B7-1/Fas gene transfer on poorly immunogenic osteosarcoma, which is resistant to a treatment protocol using transduction of B7-1 alone. Cancer Gene Therapy (2003) 10, 717-725. doi:10.1038/sj.cgt.7700624Keywords: B7-1; Fas; chimeric gene; adenovirus; osteosarcoma; apoptosis T reatment of patients with osteosarcoma, especially those resistant to current chemotherapy protocols, has been a major challenge. Active immunotherapy is a potential clue for overcoming such therapeutic difficulties. Using a rat syngeneic model system, we demonstrated that B7-1-transfected tumor vaccine as well as adenovirus-mediated in vivo B7-1 gene transfer induces antitumor immunity against pre-established pulmonary metastasis of osteosarcoma. 1,2 On the other hand, we also found that B7-1-transfected vaccine has limited efficacy on a poorly immunogenic osteosarcoma cell line. 3 As seen in other studies, 4,5 the effect of B7-1 costimulation on T-cell-mediated tumor immunity varies depending on the inherent immunogenecity of tumors. To obtain more potent antitumor effects of B7-expressing osteosarcoma vaccine, we constructed a chimeric gene consisting of the extracellular domain of B7-1 and the transmembrane and intracellular domains of Fas, a member of the tumor necrosis factor family acting as an inducer of apoptosis. Materials and methods Animals, cell lines and reagentsMale C3H/He and BALB/c mice and LEW rats, 6-week old, were purchased from Japan SLC Inc. (Hamamatsu, Japan), and housed in a specific pathogen-free animal facility in our Institute.A murine osteosarcoma cell line, LM8, derived from Dunn osteosarcoma was kindly provided by the Department of Orthopaedics, Osaka University Graduate School of Medicine. 6 Chinese hamster ovary (CHO) cells expressing murine B7-1 (mB7-1-CHO) were prepared as described previously. 7 LM8 cells were maintained in DMEM (GIBCO, Grand Island, NY) containing 10% fetal calf serum (GIBCO), vitamin solution, sodium pyruvate, nonessential amino acids and l-glutamate at 371C in 5% CO 2 , and mB7-1-CHO cells in aMEM (GIBCO) containing 10% fetal calf serum.Monoclonal antibodies (mAbs) used were mouse antirat B7-1 mAb (3H5), 8 rat anti-mouse B7-1 mAb (1G10; PharMingen, San Diego, CA), 9 rat anti-mouse MHCclass I (H-2D k ) mAb (15-5-5; PharMingen), rat antimouse MHC-class II mAb (1E4; a kind gift from Dr K Ogasawara, Hokkaido University), 10 rat anti-mouse Mac-1 mAb (M1/70...

A quest for therapeutic antigens in bone and soft tissue sarcoma

“…In addition, the utilization of tumor-draining lymph node as a source of antigen-specific lymphocytes and the manipulation of co-stimulatory pathways such as B7/CD28 and 4-1BB/4-1BBL have enhanced the success rate of CTL induction [28,29]. The efficacy in manipulation of costimulatory pathways to elicit immunological responses in vivo has been applied to animal models with osteosarcoma [30-34] and used in clinical trials on patients with malignant melanoma [35,36]. …”

Nanocarriers in Veterinary Medicine: A Challenge for Improving Osteosarcoma Conventional Treatments

“…Although the mechanisms of IL-12 anti-tumor activity are not fully understood, they may be linked to both its ability to inhibit angiogenesis and stimulate T-cells and NK cells. Adenovirus-mediated B7-1/Fas chimeric gene transfer has also been reported to activate T-cell activation and induce apoptosis of osteosarcoma cells [87].…”