The efficacy of a novel vaccine approach using tumor cells that ectopically express a codon-optimized murine GM-CSF in a murine tumor model

Abstract

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that is known to facilitate vaccine efficacy by promoting the development and prolongation of both humoral and cellular immunity. Here, we investigated a novel vaccine approach using a human papillomavirus (HPV)-16 E6/E7-transformed cell line, TC-1, that ectopically expresses a codon-optimized 26-11-2015 murine GM-CSF (cGM-CSF). Ectopically expressing cGM-CSF in TC-1 (TC-1/cGM) cells significantly increased expression of a GM-CSF that was functionally identical to wt GM-CSF by 9-fold compared with ectopically expressed wild type GM-CSF in TC-1 cells (TC-1/wt). Mice vaccinated with irradiated TC-1/cGM cells exhibited enhanced survival compared with mice vaccinated with TC-1/wt cells when both groups were subsequently injected with live TC-1. Consistently, mice vaccinated with irradiated TC-1/cGM cells exhibited stronger IFN-γ production in HPV E7-specific CD8⁺ T cells. More dendritic cells were recruited to the draining lymph nodes (dLNs) of mice vaccinated with TC-1/cGM cells than C-1/wt cells. Regarding dLN cell recall responses, both proliferation and IFN-γ production in the HPV E7-specific CD8⁺ T cells were enhanced in mice that were vaccinated with TC-1/cGM cells. Our results demonstrate that a novel practical molecular strategy utilizing a codon-optimized GM-CSF gene overcomes the limitation and improves the efficacy of tumor cell-based vaccines.

Introduction

Cervical cancer is the second most frequent gynecological malignancy worldwide and accounts for approximately 12% of all cancers in women [1]. Greater than 99% of cervical cancer patients are carriers of human papillomaviruses (HPV), which makes HPV a major risk factor for cervical cancer [2]. HPVs are double-strand DNA viruses with a genome size of approximately 8 kb that encode 8 or 9 open reading frames consisting of eight early (E1–E9) and two late (L1 and L2) gene products [3]. HPV integration into the genome and high-level E6 and E7 expression are often associated with the manifestation of high-grade cervical neoplasia [2], [4], [5]. Although virus-like particle (VLP)-based vaccines have been developed with prophylactic activities to prevent most HPV infections [6], [7], the therapeutic effect of VLP vaccines has yet to be demonstrated for those who were already infected [8]. Additionally, although prophylactic vaccines induce a humoral immune response against the viral capsid protein L1, a cellular immune response against virus early proteins, E6 and E7, is required to eliminate previously infected cells [9], [10]. The viral transforming proteins, E6 and E7, are consistently expressed in cervical cancer cell lines and HPV-associated neoplasms. Thus, E6 and E7 represent true tumor-specific antigens and serve as a target for the development of immunotherapeutic strategies to combat HPV-associated cancers.

Granulocyte macrophage-colony stimulating factor (GM-CSF) was initially characterized as a factor that can support the in vitro colony formation of granulocyte-macrophage progenitors [11]. GM-CSF also serves as a growth factor for erythroid, megakaryocyte, and eosinophil progenitors. GM-CSF is produced by various cell types, including T cells, B cells, macrophages, mast cells, endothelial cells, fibroblasts, and adipocytes, in response to cytokine or inflammatory stimuli. In mature hematopoietic cells, GM-CSF is a survival factor and activates the effector functions of granulocytes, monocytes/macrophages, and eosinophils. In recent years, both murine tumor models and human clinical trials revealed that GM-CSF-secreting tumors cells can serve as an option for immunotherapy in several tumor types, including non-small cell lung carcinoma [12], pancreatic [13], prostate [14], melanoma [15], leukemia [16], gliomas [17], cervical [18], and renal cell carcinoma [19]. However, GM-CSF gene expression is highly regulated at multiple levels [20], [21], [22]. Native GM-CSF protein is poorly expressed in a tissue-specific and activation-dependent manner. A clinical trial demonstrated that a threshold level of GM-CSF is required for the induction of effective immunity [23]. We previously described that the co-administration of plasmids encoding the codon-optimized murine GM-CSF (cGM) sequence with a DNA vaccine resulted in a strong and protective antibody and cytotoxic T lymphocyte (CTL) immune response against recombinant vaccinia virus challenge [24].

In the present study, we applied a lentiviral-delivered codon-optimized murine GM-CSF into a HPV-16 E6/E7-transformed cell line, TC-1, as a vaccine adjuvant approach. The goal of the present study was to assess whether codon-optimized murine GM-CSF that is transduced into cancer cells as a vaccine would increase (dendritic cell) DC recruitment and result in enhanced antigen (Ag)-specific immune responses [28]. The lentiviral-modified TC-1/cGM cells showed significantly increased GM-CSF protein expression levels as demonstrated by Western blot and (enzyme-linked immunosorbent assay) ELISA assays. The GM-CSF secreted from the lentiviral-modified TC-1/cGM cell culture medium exhibited identical biological functions to the WT GM-CSF in its ability to support NFS-60 cell growth. The infected cells were continuously propagated for more than 30 passages, and stable lentiviral transgene expression was confirmed. We subcutaneously (s.c.) immunized C57BL/6 mice with a lethally irradiated TC-1/cGM cell-based vaccine and subsequently quantified CD11c⁺ DC and CD8⁺ by flow cytometry analysis. Vaccination with TC-1/cGM induced strong systemic CD8⁺ T cell immune responses against lethal TC-1 challenge and specific MHC class I HPV E7 epitopes, as demonstrated by an enzyme-linked immunospot (ELISPOT) assay. Peak DC recruitment was detected at 72 h post-inoculation in dLNs and was significantly increased (p < 0.05) in mice that were vaccinated with TC-1/cGM cells compared with control mice or mice inoculated with TC-1/wt cells. These results support the use of this novel codon-optimized GM-CSF for immunotherapeutic cancer vaccine strategies.