4 Discussion

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Effective amplification of immune responses induced by a cancer vaccine is an important strategy to achieve clinical responses in cancer patients receiving immunotherapeutic treatment. The important role of effective immune response amplifiers, called adjuvants, has captured the imagination of immunologists for decades and has led to important developments, such as the use of killed Bordetella pertussis for diphtheria and tetanus toxoids (21), muramyl dipeptide extracted form the mycobacterial cell wall (22) and immune stimulatory complexes, called ISCOMs, which simultaneously serve as antigen carriers and adjuvants (23). Additional adjuvants are cytokines and more recently also chemokines, such as IL-2 (24) or fractalkine, the former also used successfully as an adjuvant in a clinical trial of melanoma patients with a respectable response of 42% in patients receiving a gp-100 peptide vaccine. Preclinical data using fractalkine in cancer immunotherapy are limited (25) and results from clinical applications using this chemokine are not available at this time.

Here I demonstrate for the first time the efficacy of a fractalkine gene therapy approach in combination with targeted IL-2 in a neuroblastoma model using a genetically engineered fractalkine protein, constructed by PCR cloning. Expression of FKN and bioactivitity in vitro and in vivo was demonstrated (Fig. 3-6) [4]. The efficacy for this type of combination gene therapy was established in an immunocompetent syngeneic model for murine neuroblastoma using NXS2 cells that naturally express the disialoganglioside GD2. This model also features experimental metastases to liver, expresses the neuroblastoma tumor marker tyrosine hydroxylase, and represents many pathophysiological similarities to human neuroblastoma (26).

The immune response induced by genetically engineered NXS2 cells which produce fractalkine is partially effective as indicated by a reduction of primary tumor growth by 30%. The effect was mediated by CD8+ T cells as indicated by a strong infiltrate of primary tumors with CD8+ T cells (Fig. 6). This finding is in contrast to NK-cell-mediated immune mechanism observed in the same animal model induced by a recombinant anti-disialoganglioside GD2-interleukin-2 fusion protein monotherapy that directs interleukin-2 (IL-2) into the tumor microenvironment (26) where infiltrating CD8+ T cells were absent. Since such recombinant IL-2 fusion proteins were demonstrated to induce T cell mediated immune responses in other animal models, e.g. melanoma (27) and colon carcinoma (28), I concluded that poor immunogenicity and immunosuppressive factors secreted by NXS2 cells, e.g. IL-10 and TGF-β, could account for T-cell anergy in this model (26). However, this T-cell anergy is apparently overcome by the use of fractalkine gene therapy in combination with targeted IL-2.

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One of the major advantages of fractalkine over other chemokines is its proinflammatory TH1 potential capable of potentiating T- and NK- cell mediated cytotoxic responses. It is also a strong stimulator of TH1 CD4+ T cells. These cells elicit a helper function and are involved in the maturation of CD8+ T cells, the main effector cells observed in this experimental system. In fact, it was possible to demonstrate the presence of CD4+ T-cells in cellular infiltrates observed in primary tumors of mice previously vaccinated with fractalkine producing NXS2 cells (Fig. 6). Interestingly, this is again in contrast to previous studies with a recombinant antibody IL-2 fusion protein in this same tumor model, where such CD4+ T-cells cells were completely absent (26).

Despite the presence of CD8+ and CD4+ T cells in the tumor microenvironment, fractalkine gene therapy used as a monotherapy showed limited efficacy in this neuroblastoma model as indicated by a reduction of primary tumor growth by only 30% (Fig. 7). This finding is in contrast to results reported in the literature on C26 colon carcinoma and B16F10 melanoma models (25) with a reduction in primary tumor growth by almost 90% in both models. These differences are most likely a result of the tumor models used. A similar observation was made with exactly the same tumor models using tumor specific recombinant IL-2 fusion proteins (targeted IL-2). The induction of a T cell mediated memory immune response was demonstrated in melanoma (27) and colon carcinoma (28;29), but not in the neurobastoma model (20). The absence of an effective T cell response with targeted IL-2 or fractalkine gene therapy used as a monotherapy is a result of the poor immunogenicity of the NXS2 neuroblastoma model, thus reflecting immunologically the situation in man.

Based on these considerations, the efficacy of a novel approach was tested in this experiment, which specifically amplify a T-cell-mediated immune response initially induced with a cellular tumor vaccine genetically engineered to secrete fractalkine. Thus, tumor-specific targeting of the T cell growth factor IL-2 into the tumor microenvironment was shown to effectively boost a CD8+ T-cell-mediated immune response, as indicated by increased CD8+ T cell activation (Fig 10) and MHC class I restricted tumor cell killing (Fig. 9) in mice receiving both the fractalkine vaccine and tumor-specific IL-2 boost. The absence of such a response with a non-specific fusion protein clearly proves the concept of tumor-specific boost with ch14.18-IL-2 fusion protein specific for ganglioside GD2, which is highly expressed in the neuroblastoma tumor microenvironment (30). The primary effector cells involved in the anti-tumor immune response were CD8+ T cells, since depletion of this T cell subpopulation abrogated the effect of this combination therapy (Fig. 11-12). Interestingly, depletion of CD4+ T cells also abrogated the therapeutic effect indicating their important helper function.

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The increase in effective concentrations of IL-2 in the tumor microenvironment appears to be the crucial step in effective CD8+ T cell re-activation, which can be achieved with systemic injections of tumor-specific antibody-IL-2 fusion proteins (27;28;31-33). Thus, the presence of both tumor-associated T cell antigens and adequate co-stimulation in the tumor microenvironment provided by targeting of the tumor cell surface with IL-2, fulfills the necessary requirement for amplification of the CD8+ T-cell-mediated immune response.

This principle of amplifying an immune response initially induced by a cancer vaccine with a tumor-specific antibody-cytokine fusion protein could find broad application in immunotherapeutic cancer treatments. Indeed, additional treatments with tumor-specific antibody-IL-2 fusion protein could benefit many clinical trials with cancer vaccines based on cytokine and chemokine gene therapy approaches, dendritic cells pulsed with immunogenic tumor-associated peptides or DNA vaccines encoding for tumor-associated peptide antigens. Such effective adjuvants are of major interest based on the clinical response rates observed thus far in vaccine trials (34).

In summary, I demonstrate that targeting of IL-2 into the tumor microenvironment with a ch14.18-IL-2 fusion protein effectively amplifies a CD8+ T cell immune response induced by fractalkine gene therapy. This effect was specific, since non-specific ch225-IL-2 were ineffective in this regard. A mechanism for re-activated CD8+ memory T cells was provided by increased CD8+ T cell activation and MHC class I restricted tumor cell killing only in mice that received both the vaccine and the tumor-specific boost. Taken together, these data suggest that tumor targeted IL-2 may overcome weak immune responses induced by cancer vaccines and therefore lead to further improvement in the adjuvant treatment of patients with minimal residual disease.

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