Established B16 tumors are rejected following treatment with GM-CSF-secreting tumor cell immunotherapy in combination with anti-4-1BB mAb

Abstract

Immunization with irradiated tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates potent, specific and long lasting anti-tumor immunity in clinical and preclinical settings. Efforts to further increase immunotherapy efficacy with immune-modulatory agents are under evaluation. Based on the immune-modulatory properties of 4-1BB (CD137), it has been postulated that agonistic 4-1BB antibodies may add additional anti-tumor efficacy to GM-CSF-secreting tumor cell immunotherapy. The combination of GM-CSF-secreting tumor cell immunotherapy and anti-4-1BB monoclonal antibody (mAb) treatment resulted in rejection of established tumors in the B16 melanoma model. These anti-tumor effects correlated with persistent tumor-specific CD8⁺ T cell responses. In addition, early tumor infiltration of functional CD8⁺ T cells and a greater expansion of antigen-specific memory T cells were found in mice treated with the combination therapy. In summary, an agonistic anti-4-1BB mAb combined with GM-CSF-secreting tumor cell immunotherapy may provide a novel and potent treatment strategy for patients with cancer.

Introduction

4-1BB (CD137) is a type I membrane protein of the TNFR family which has been shown to augment CD4 and CD8 T cell responses [1]. Its expression is upregulated on activated CD4⁺ and CD8⁺ T cells, NK cells and monocytes. It binds to the 4-1BB ligand (4-1BBL) expressed on antigen-presenting cells (APC) such as macrophages, dendritic cells (DC) and activated B cells. Binding of 4-1BB to 4-1BBL delivers a signal to T cells leading to their activation, survival and growth [2], [3], [4]. Ligation of 4-1BB by antibodies is known to enhance anti-tumor immunity in tumor-bearing mice [5], [6]. Anti-tumor immune responses include enhancement of proliferation and CTL activity of tumor-specific T cells [2], [7], prevention of activation induced cell death (AICD) [2], [3], [8], enhancement of T cell infiltration into the tumor [9] and generation and maintenance of antigen-specific CD8 memory cells [10], [11]. Considered together, these findings suggest that 4-1BB ligation may be an effective way to further enhance an on-going immune response.

Immunization with irradiated tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates potent, specific and long lasting anti-tumor immunity in clinical and preclinical settings [12], [13], [14], [15], [16], [17]. Anti-tumor protection requires both CD4 and CD8 T cells [16], [18], [19]. The mechanism underlying the stimulation of anti-tumor immunity involves improved tumor antigen presentation by DCs recruited to the site of immunization [20]. DCs are highly activated at the immunotherapy injection site by the GM-CSF-secreting tumor cell immunotherapy and traffic readily to the draining lymph nodes (DLN) to activate a tumor-specific immune response [20]. Tumor-specific T cells infiltrate the tumor, resulting in tumor destruction.

The studies reported here evaluated the combination of GM-CSF-secreting tumor cell immunotherapy with anti-4-1BB monoclonal antibody (mAb) in preclinical models. We demonstrate in the poorly immunogenic and highly aggressive B16 melanoma model that mice treated with the combination therapy rejected established tumors when therapy was initiated as late as 11 days after tumor inoculation. In addition, long-lived cytolytic activity and enhanced IFNγ secretion by CD8⁺ T cells were observed in the combination therapy-treated animals compared to animals treated with either monotherapy alone. Furthermore, higher numbers of activated effector CD8 T cells were detected in lymphoid tissues and the tumor microenvironment and a greater expansion of memory T cells was found in animals treated with the combination therapy. These data show that anti-4-1BB mAb combined with a GM-CSF-secreting tumor cell immunotherapy increases the level of anti-tumor efficacy in preclinical models.