764. Attenuation of Treg Cells by GITR Ligand Led to Synergistic Enhancement of Treatment Efficacy with Immune-Stimulatory Therapies in Mice Bearing Metastatic 4T1 Breast Carcinoma

Abstract

CD25+CD4+ regulatory T cells (Treg) are capable of suppressing cytolytic activity of CD8+ T cells which may hamper the development of effective immune therapies against cancer. The glucocorticoid-induced TNF receptor family-related gene (GITR) is a membrane receptor is predominantly expressed on these Treg cells, and the immune-suppressive activity of Treg can be inhibited by its interaction with GITR Ligand (GITRL) expressed on antigen presenting cells. Depletion of Treg using anti-CD25 or blocking GITR signaling by GITRL alone, however, was not capable of eradicating pre-established tumors in laboratory animals. Implantation of poorly immunogenic but highly tumorigenic 4T1 breast carcinoma cells in the mammary glands of syngeneic Balb/c mice led to the development of orthotopic lesions that are spontaneously metastatic. We showed that an immune enhancement therapy by intratumoral delivery of a recombinant adenovirus vector expressing murine GMCSF and IL12 (Ad.mGMCSF-IRES-mIL12), in combination with intraperitoneal administration of a fusion protein of murine immunoglobulin with the ecto-domain of murine 4-1BB ligand (mIg-m4-1BBLs), in mice bearing 4T1 breast carcinoma led to significantly prolonged survival, although the treated animals eventually succumbed to tumor relapse. The Treg population was significantly elevated in the splenocytes of the treated animals and depletion of CD4+CD25+ cell population led to a significant increase in splenic T cell proliferation activity in vitro. We hypothesized that addition of a fusion protein made of murine immunoglobulin and the ecto-domain of GITR ligand (mIg-mGITRLs) to the treatment regimen would have a synergistic effect on anti-tumor immune responses through attenuation of Treg cell-mediated immune suppression, which would lead to much enhanced treatment efficacy. The data presented here show that mIg-mGITRLs has the ability to attenuate the immune suppressive activity of CD4+CD25+ Treg cells in vitro. Intraperitoneal administration of mIg-mGITRLs with Ad.mGMCSF-IRES-mIL12 + mIg-m4-1BBLs treatment in 4T1-bearing mice resulted in tumor-free and long-term survival in >60% of the animals. Furthermore, this combination therapy induced robust tumor-specific CTL activities and the long-term surviving animals were able to reject a challenge of live parental tumor cells. There were no apparent toxic effects and taken together, the results suggest this novel combination therapy induced a systemic and effective anti-tumor immunity that is persistent, which may be translated into an effective and safe treatment modality for metastatic breast cancer in humans.