Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial

Abstract

Background aims

Dendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2⁺ patients) or with autologous/allogeneic tumor lysate (HLA-A2⁻ patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-α2b.

Results

Of 46 patients who initiated treatment, 10 stopped treatment within 1–4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2⁺ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25^high CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD.

Conclusions

Vaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.

Introduction

The incidence of malignant melanoma (MM) is increasing worldwide and the prognosis for patients with inoperable metastatic disease is poor. Conventional chemotherapy treatments have only very limited, if any, impact on survival, while high dose interleukin (IL)-2 has a response rate of 15–20% with 5–7% long-time survivors (1,2). During the past decade, numerous types of dendritic cell (DC) vaccines have been tested with more than 600 melanoma patients (3); in some of the studies achievement of a clinical response has been observed in a subset of patients, while more frequently induction of an antigen-specific immune response is found.

DC are present in peripheral blood but comprise less than 1% of mononuclear cells (MNC) (4), therefore isolation and in vitro differentiation of DC is required in order to generate DC-based vaccines. The optimal method for maturation of DC is a subject of debate. Typically the precursor cells (monocytes) are incubated with granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-4 for approximately 7 days (5,6). The most commonly used factors for DC maturation are IL-6, prostaglandin E₂ (PGE₂), IL-1β and tumor necrosis factor (TNF)-α (7). Used in combination, these are referred to as the standard maturation cocktail, and have been applied in several clinical trials including the present (8., 9., 10.). Mature DC (mDC) can be characterized both phenotypically and functionally. Phenotypically mDC is described using maturation markers such as C83, CD40, CD80 (B7.2), CD86 (B7.1), CCR-7 and HLA-DR, while the functional criteria for a matured DC are defined by IL-12 production, migration capacity and the ability to induce antigen-specific T-cell responses (11., 12., 13.).

Activated DC are pulsed with tumor-associated antigens, for example oncopeptides and tumor lysate. The tumor antigens survivin and telomerase are uniformly expressed in the majority of malignancies, while the expression in normal host cells is much less pronounced and in most cases only temporary (14,15). They are necessary for survival of tumor cells and therefore less likely to be down-regulated, making them suitable vaccine target candidates. Survivin belongs to the inhibitor of apoptosis protein family, while telomerase maintains chromosomal integrity by protecting telomeric DNA that otherwise would be lost with each cell division. In vitro studies have revealed that some cancer patients mount spontaneous immune responses against survivin and telomerase (16., 17., 18.). HLA class I-binding peptide epitopes have been identified for survivin and telomerase, and these peptides are capable of activating human T cells that are cytotoxic to cancer cells (16,19). Both survivin and telomerase have previously been targeted in DC vaccination trials, including our recently published trial in renal cell carcinoma patients (20., 21., 22.).

p53 is another obvious target for cancer vaccination therapy (23,24). Mutations that inactivate the p53 protein or members of the p53 pathways are the most common genetic alterations found in human cancers, and mutations in the p53 gene are found in approximately 40% of human tumors, including MM (25). Cytotoxic T cells (CTL) with HLA class I-restricted specificity for wild-type p53-derived peptides have been observed in the peripheral blood of cancer patients, suggesting immune surveillance of tumors expressing these peptides (26,27). We have previously targeted p53 in a DC vaccination trial in metastatic breast cancer patients employing autologous DC pulsed with six HLA-A2-binding p53 peptides (28,29).

Because of the diversity of tumor cells, the strategy in the present study was to administer DC loaded with multiple immunogenic epitopes for treatment of patients with metastatic MM. Therefore, a broad panel of HLA class I-binding peptides coding for survivin, telomerase and p53 epitopes was used for loading of DC from HLA-A2⁺ patients. For the HLA-A2⁻ patients, tumor antigen preparations were produced from either autologous tumor cells or allogeneic MM cell lines. This approach aimed to target a broad spectrum of antigens with induction of a polyclonal immune response against multiple targets on cancer cells, thereby theoretically reducing the possibility of tumor escape (30).