Depot Formulation of Interleukin-2 as an Immunotheraphy for B-Cell Lymphoma.

Relapses occurring in most lymphoma patients after treatment highlight the need for effective immunotheraphy approaches. Induction of tumor-specific adaptive immunity in cancer patients is an ideal approach because its selectivity and memory for the same tumors eventually prevent the relapse after conventional therapies. Vaccination with cytokines and tumor cells represents an attractive therapeutic approach for patients with B-cell lymphoma. Interleukin-2 (IL-2) has a wide range of immunologic effects, including the activation of cytotoxic T cells and natural killer cells (NK). The therapeutic use of IL-2 has generated substantial interest based on its ability to induce the regression of metastatic renal cell carcinomas and malignant melanomas tumors in humans. However, toxicity associated with systemic administration of large doses of cytokine is a major drawback for clinical application. In this work, we investigated whether in vivo vaccination with a cytokine-based immunotherapy using IL-2 adsorbed in alum, as a depot system, with lysed lymphoma cells could stimulate lymphoma-specific immunity and improve survival. We developed a reproducible syngenic model of B-cell lymphoma in Balb/c mouse based upon the A20 cell line. In this model animals died between day 35 and 40 after tumor cells inoculation. Mice were first injected subcutaneously with 1 x 10⁶ A20 cell in right flank. Four groups of mice received at days 3 and 7 after tumor cell inoculation by subcutaneous injections one of the following: lysed A20 cells with IL-2 in alumn (A20-IL-2); Lysed A20 cells in alumn (A20); IL-2 in alumn (IL-2); and phosphate-buffered saline (PBS) as control. Mice were followed for survival and immune response was evaluated. At day 22, 17% of A20-IL-2 group had systemic disseminated disease in comparison with 33% A20 group, 83% for IL-2 group and 100% of PBS group (p=0.009). Mice vaccinated with A20-IL-2 had longer survival compared with mice vaccinated with A20 or PBS (p=0.0019). Prolonged survival was related with a marked increase in the number of intratumoral CD4+ T cells (p=0.0001), CD8+ T cells (p=0.001) and NK cells (p=0.001). Aditionally, at day 22 groups of mice vaccinated with A20 showed a significantly lower percentages of intratumoral CD4+ CD25+ CD127- T_reg cells in the tumor as compared with PBS group (p=0.0001). At day 39 the percentage of intratumoral T_reg cells increased in the groups vaccinated with IL-2 (A20-IL-2 and IL-2 groups). Intratumoral CD4+, CD8+, NK and T_reg cells were evaluated by flow cytometry. Interestingly, intracellular cytokine analysis showed a greater number of intratumoral INF-γ producing CD4+ T cells in mice vaccinated with A20-IL-2. Proliferation assays by flow cytometry (using carboxy-fluorescein diacetate, succinimidyl ester and propidium iodide) showed enhanced proliferation upon stimulation with irradiated A20 cells in splenocytes from A20-IL-2 vaccinated mice (p=0.004). In conclusion, the results of this study indicate that vaccination with A20 antigens combined with a depot formulation of IL-2 elicits strong anti-tumor specific immunity and extended survival. This approach may be an interesting strategy to promote systemic immunity against B-cell lymphoma with therapeutic value.