Assessment of fusion cells from patient-derived ovarian carcinoma cells and dendritic cells as a vaccine for clinical use

Abstract

Purpose.

To evaluate a protocol that allowed the successful generation of DC and OVCA cells, fusion of these two cell types and assessment of stimulatory ability of the fusion cells for clinical use.

Patients and methods.

Ovarian cancer (OVCA) cells and dendritic cells (DC) were isolated or generated from 22 patients with OVCA and subsequently fused with PEG. The stimulatory ability of fusion cells including T cell proliferation and induction of cytotocic T lymphocytes (CTL) was assessed. In addition, the impact of radiation, freezing and thawing of the fusion cells was evaluated.

Results.

OVCA cells derived from 22 patients were successfully fused with autologous DC. The created heterokaryons expressed tumor-associated antigens, such as MUC1 and CA-125, and DC-derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, CD4 and CD8 T cells derived from patients with ovarian cancer were stimulated by fusion cells and produced IFN-γ as demonstrated with intracellular staining. Significantly, T cells primed by fusion cells produced MHC class I-dependent lysis of autologous ovarian tumor cells. One cycle of fusion-cell stimulation can maintain the CTL activity up to 25 days.

Conclusions.

The fusion of human OVCA cells and DC created immunogenic cells capable of stimulating CD4 and CD8 T cells. The effects of the processes required for preparing a vaccine for clinical use, including freezing and thawing and irradiation, do not interfere with the immunogenic properties of the fusion cells.

Introduction

Despite recent advances in post-surgical chemotherapy, ovarian cancer (OVCA) remains the leading cause of death for patients with gynecologic malignancies [1]. The majority of patients with OVCA are diagnosed at an advanced stage of the disease and are treated with surgical debulking followed by platinum-based chemotherapy. Most patients who respond to such therapy ultimately die of relapse from residual and/or drug-resistant disease. A novel therapy that reduces the risk of relapse is urgently needed. In this context, targeted immunotherapy may represent a useful anti-OVCA strategy since such therapy may be effective against chemotherapy-resistant disease with minimal toxicity.

One of the requirements for this approach is the identification of antigens capable of mediating tumor rejection. Certain antigen targets for immunotherapy of ovarian cancer, such as MUC1 [2], [3], Her-2/neu [4], [5] and CA-125 [6], [7], are already known, and others, although they remain unidentified, are likely to exist. The majority of tumor antigens, however, are nonmutated self-antigens to which the immune system is tolerant. It is well documented that tumor cells do not express costimulatory molecules and seldom express MHC class II molecules both of which are essential for the induction of cellular immunity. As a result, tumor antigen may not be effectively presented to the immune system [8], [9], or worse, T cells responding to tumor antigens may be anergized [10], [11], [12]. Therefore, a strategy is needed to overcome these barriers that prevent the induction of effective antitumor immunity against antigens selective for, or overexpressed in, ovarian carcinomas.

One strategy in development is the fusion of dendritic cells (DC) and tumor cells. DC are bone-marrow-derived leukocytes that are critical in the initiation of T-cell-mediated immunity [13], [14]. These cells derive their potency from constitutive and inducible expression of essential costimulatory ligands on the cell surface, including CD80, CD86, CD56 and CD40 [15], [16]. DC have been fused to patient-derived ovarian carcinoma (OVCA) cells [17], [18]. The fusion cells have induced cytotoxic T cells against autologous OVCA cells. In the present study, using samples collected from 22 patients with OVCA, we have adopted a systematic approach to address the following issues: (i) development of a protocol that allowed the successful generation of DC and OVCA cells and subsequent fusion of these two cell types (DC/OVCA); (ii) assessment of the anti-OVCA immune responses induced by DC/OVCA fusion cells; (iii) conversion of such fused DC/OVCA cells into a vaccine that can be frozen, thawed, irradiated and administered to patients.