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Induction of G250-targeted and T-Cell-mediated Antitumor Activity against Renal Cell Carcinoma Using a Chimeric Fusion Protein Consisting of G250 and Granulocyte/Monocyte-Colony Stimulating Factor

Departments of Urology [C-L. T., A. Z., A. P., R. C., J. M. H., S. P., D. N., B. G., J. d. K., R. F., A. B.], Medicine [R. F.], and Pathology [P. I. S.] and Jonsson Comprehensive Cancer Center [C-L. T., A. Z., A. P., R. C., J. M. H., S. P., D. N., B. G., P. I. S., J. d. K., R. F., A. B.], UCLA Kidney Cancer Program, University of California Los Angeles, Los Angeles, California 90095

Immunotherapy targeting for the induction of a T-cell-mediated antitumor response in patients with renal cell carcinoma (RCC) appears to hold significant promise. Here we describe a novel RCC vaccine strategy that allows for the concomitant delivery of dual immune activators: G250, a widely expressed RCC associated antigen; and granulocyte/macrophage-colony stimulating factor (GM-CSF), an immunomodulatory factor for antigen-presenting cells. The G250-GM-CSF fusion gene was constructed and expressed in Sf9 cells using a baculovirus expression vector system. The M_r 66,000 fusion protein (FP) was subsequently purified through a 6xHis-Ni²⁺-NTA affinity column and SP Sepharose/fast protein liquid chromatography. The purified FP retains GM-CSF bioactivity, which is comparable, on a molar basis, to that of recombinant GM-CSF when tested in a GM-CSF-dependent cell line. When combined with interleukin 4 (IL-4; 1000 units/ml), FP (0.34 µg/ml) induces differentiation of monocytes (CD14⁺) into dendritic cells (DCs) expressing surface markers characteristic for antigen-presenting cells. Up-regulation of mature DCs (CD83⁺CD19^-; 17% versus 6%) with enhanced expression of HLA class I and class II antigens was detected in FP-cultured DCs as compared with DCs cultured with recombinant GM-CSF. Treatment of peripheral blood mononuclear cells (PBMCs) with FP alone (2.7 µg/10⁷ cells) augments both T-cell helper 1 (Th1) and Th2 cytokine mRNA expression (IL-2, IL-4, GM-CSF, IFN-, and tumor necrosis factor-). Comparison of various immune manipulation strategies in parallel, bulk PBMCs treated with FP (0.34 µg/ml) plus IL-4 (1000 units/ml) for 1 week and restimulated weekly with FP plus IL-2 (20 IU/ml) induced maximal growth expansion of active T cells expressing the T-cell receptor and specific anti-RCC cytotoxicity, which could be blocked by the addition of anti-HLA class I, anti-CD3, or anti-CD8 antibodies. In one tested patient, an augmented cytotoxicity against lymph node-derived RCC target was determined as compared with that against primary tumor targets, which corresponded to an 8-fold higher G250 mRNA expression in lymph node tumor as compared with primary tumor. The replacement of FP with recombinant GM-CSF as an immunostimulant completely abrogated the selection of RCC-specific killer cells in peripheral blood mononuclear cell cultures. All FP-modulated peripheral blood mononuclear cell cultures with antitumor activity showed an up-regulated CD3⁺CD4⁺ cell population. These results suggest that GM-CSF-G250 FP is a potent immunostimulant with the capacity for activating immunomodulatory DCs and inducing a T-helper cell-supported, G250-targeted, and CD8⁺-mediated antitumor response. These findings may have important implications for the use of GM-CSF-G250 FP as a tumor vaccine for the treatment of patients with advanced kidney cancer.

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