1 Introduction

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The effective treatment of stage 4 neuroblastoma is one of the major challenges in pediatric oncology, since its outcome remains poor, even after high dose chemotherapy and autologous bone marrow or stem cell transplantation. The development of an effective adjuvant immunotherapeutic strategy appears to be an important option to further improve the outcome of this neoplasm. New approaches are under investigation including passive immunotherapy with monoclonal anti-GD2 antibody ch14.18 [1]. Active immunotherapy is also a promising approach, such as DNA vaccination using tyrosine hydroxylase or MHC class I peptide ligands derived from tyrosine hydroxylase as a tumor antigen to induce an active host-anti-tumor immune response (1), [2], [3].

Based on the consideration that tumor-associated leukocytes are residual evidence of the host’s ineffective antitumor immune response, a major goal of immunotherapy may be further accumulation and activation of such immune cells in the tumor microenvironment. Given the chemoattractive and stimulative properties of chemokines on different leukocyte subpopulations, therapeutic manipulation of the chemokine environment constitutes one strategy to stimulate protective responses by delivering chemokines to the tumor microenvironment at more relevant concentrations than could be given systemically.

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There are approximately 40 chemokines identified to date, which can be classified into four groups according to the number of NH2-terminal cysteine motif: C, CC, CXC, and CX3C. Furthermore, chemokines can be distinguished between ‘inflammatory’ (alternatively called inducible) chemokines, such as SLC (secondary lymphoid tissue chemokine), and ‘homeostatic’ (alternatively called constitutive, housekeeping or lymphoid) chemokines, such as IP10 (interferon-inducible protein 10), based on the pathophysiological condition and the location of chemokine production as well as the cellular distribution of chemokine receptors.

Fractalkine (FKN), which is also called neurotactin, is the sole member of CX3C chemokine subfamily consisting of a CX3C motif with three amino acids between the two terminal cysteines. It is expressed predominantly by endothelial cells and its expression is both constitutive and inducible upon stimulation with TPA, LPS, TNF-α or IL-1 (2) (3).

FKN is different from other chemokines, since it exists in both a soluble and a membrane-anchored form. Following the predicted signal peptide (Fig 1, blue), FKN contains an N-terminal chemokine domain (Fig. 1, red, residues 1 to 76) with the unique 3-residue insertion between cysteines. Its structure is also characterized by a mucin like stalk (Fig. 1, purple, residues 77 to 317) with predicted O-glycosylated serine and threonine residues, providing for a distance between the membrane anchor and the chemokine domain. The transmembrane domain (Fig. 1, green, residue 318 to 336) and the intracellular domain (Fig. 1, pink, residue 337 to 373) constitute the anchor in the cell membrane.

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Fig 1. Schematic structure of FKN (4)

Soluble FKN is released from the membrane bound version by proteolytic cleavage at a membrane-proximal region by the TNF-α-converting enzyme and exhibits the chemotactic activity to CX3C receptor positive cells in a way similar to other chemokines (5). In contrast to other chemokines, the membrane-bound FKN induces firm adhesion directly, rather than indirectly through selectins and integrins. In general,the transmigration of leukocytes from the blood vessels into the surrounding tissue comprises several steps. It starts with the selectin-mediated interactions between leukocytes and the endothelium, which is followed by the activation of integrins on the leukocytes surface induced by chemokines, resulting in firm adhesion between leukocytes and the endothelium. Finally, leukocytes transmigrate through the endothelial layer in response to a chemokine gradient (6). As a result of the peculiar structure of FKN, it has a dual function of mediating both adhesion and migration and might directly mediate cell-cell interaction without involving selectins and integrins, which may represent a parsimonious solution to this complicated molecular event of leukocyte transmigration (7), (8). To date, FKN and the newly described CXCL16 are the only chemokines identified that have this kind of structure (9). This unique dual function may provide superior efficacy in therapeutic application of this chemokine in cancer immunotherapy.

Furthermore, FKN plays an exceptional role in polarized TH1 type immune responses not only because of its uniquestructure but also on the basis of the following characteristics. First, it executes its multiple functions through the CX3CR1 receptor which is a seven-transmembrane protein containing several motifs conserved among the chemokine receptor superfamily. Importantly, CX3CR1 is expressed mainly on CD16+ NK cells, CD8+/CD3+ T cells, CD4+/CD3+ T cells and CD14+ monocytes (10). These cell populations are identical with cells migrating towards soluble FKN. Noteworthily, expression of CX3CR1 in both CD4+ and CD8+ T cells was strongly up-regulated by IL-2. Second, CX3CR1 is selectively expressed on various lineages of lymphocytes characterized by a high content of intracellular perforin and granzyme B including NK cells, γδT cells, and terminally differentiated CD8+ T cells (11). These cytotoxic lymphocytes are the major effector cells against tumor cells. Third, TH1 type cytokines and TH2 type cytokines have divergent effects on FKN expression. The FKN mRNA and protein expression can be induced by IL-1, TNF-α and INF-γ in human endothelial cells. Furthermore, INF-γ and TNF-α showed a synergistic effect in inducing FKN expression. In contrast, IL-4 and IL-13 do not induce FKN expression and even suppressed its induction by INF-γ and TNF-α (12). Moreover, the membrane-bound form of FKN can induce INF-γ produced by NK cells (13). In rheumatoid arthritis patients, peripheral CX3CR1+/CD4+ cells expressed INF-γ and TNF-α to a greater extend than CX3CR1-/CD4+ , CX3CR+/CD8+ cells expressed INF-γ to a greater extend than CX3CR1-/CD8+ cells (14).

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Based on these effects of FKN on providing for a TH1 milieu, and the unique dual function of this chemokine, I selected this chemokine for immunogenetherapy of neuroblastoma.

However, the application of cytokine gene therapy used as a monotherapeutic approach has failed to translate the induction of tumor specific T-cells into objective clinical responses (15). Therefore, I expanded the efforts to combine FKN-gene therapy with a second immunotherapeutic strategy involving targeted IL-2, which was demonstrated to effectively amplify a suboptimal immune response following gene therapy with IL-12 (16).

The strategy of targeted IL-2 uses an antibody-cytokine fusion protein consisting of an anti-ganglioside GD2 antibody (ch14.18) [1] fused with interleukin-2 (ch14.18-IL-2), constructed by fusion of the synthetic sequence encoding for human IL-2 to the carboxy terminus of each IgG heavy chain of ch14.18. This construct can direct IL-2 specifically to ganglioside GD2, which is extensively expressed in neuroblastoma and melanoma. It was demonstrated in previous experiments that this immunocytokine, ch14.18-IL-2, is an effective agent in the treatment of murine melanoma through activating and expanding CD8+ T cell (17). Furthermore, a long-lived protective immunity was demonstrated by a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2) in a murine carcinoma model (18). In addition, many investigators have clearly shown that IL-2 is of key importance for boosting the efficacy of anti-tumor immunity (19).

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Here, the hypothesis was tested that the anti-tumor immune response induced by the increased production of FKN through transduction of the FKN gene, which may influence the trafficking of resting and activated T cells, is amplified by ch14.18-IL-2 and subsequently, the development of tumor growth. I report a novel immunotheraputic strategy combining chemokine gene therapy with targeted IL-2 as a promising approach to neuroblastoma treatment. For this purpose, the murine FKN was cloned and expressed in the neuroblastoma cell line NXS2. Its chemotactic activity was determined both in vitro and in vivo. The antitumor effect of FKN combined with targeted IL-2 was demonstrated both for primary tumor growth and metastasis in syngeneic A/J mice. The main effector cells involved in induction of systemic immunity were indicated by the strongest T cell activation following the combination treatment. This was also demonstrated by up-regulation of T cell activation markers, TH1 cytokines and CTL activity only in the combination group over all controls. In vivo depletion of CD4+ and CD8+ T cells abrogated the therapeutic effect, further supporting the pivotal role of these T-cell subpopulations in this antitumor immunity.

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