Induction of G250-targeted and T-cell-mediated antitumor activity against renal cell carcinoma using a chimeric fusion protein consisting of G250 andGranulocyte/Monocyte-Colony stimulating factor

Immunotherapy targeting for the induction of a T-cell-mediated antitumor re sponse in patients with renal cell carcinoma (RCC) appears to hold signific ant promise. Here we describe a novel RCC vaccine strategy that allows for the concomitant delivery of dual immune activators: G250, a widely expresse d RCC associated antigen; and granulocyte/macrophage-colony stimulating fac tor (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) wa s subsequently purified through a 6xHis-Ni2+-NTA affinity column and SP Sep harose/fast protein liquid chromatography. The purified Fl? 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 inte rleukin 4 (IL-4; 1000 units/ml), FP (0.34 mug/ml) induces differentiation o f 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 cult ured with recombinant GM-CSF. Treatment of peripheral blood mononuclear cel ls (PBMCs) with Fl? alone (2.7 mug/10(7) cells) augments both T-cell helper 1 (Th1) and Th2 cytokine mRNA expression (IL-2, IL-4, GM-CSF, IFN-gamma, a nd tumor necrosis factor-alpha). Comparison of various immune manipulation strategies in parallel, bulk PBMCs treated with FP (0.34 mug/ml) plus IL-4 (1000 units/ml) for I week and restimulated weekly with Fl? plus IL-2 (20 I U/ml) induced maximal growth expansion of active T cells expressing the T-c ell 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 ta rget was determined as compared with that against primary tumor targets, wh ich corresponded to an 8-fold higher G250 mRNA expression in lymph node tum or as compared with primary tumor. The replacement of Fl? with recombinant GM-CSF as an immunostimulant completely abrogated the selection of RCC-spec ific killer cells in peripheral blood mononuclear cell cultures. All FP-mod ulated peripheral blood mononuclear cell cultures with antitumor activity s howed an up-regulated CD3(+)CD4(+) cell population. These results suggest t hat GM-CSF-G250 FP is a potent immunostimulant with the capacity for activa ting immunomodulatory DCs and inducing a T-helper cell-supported, G250-targ eted, and CD8(+)-mediated antitumor response. These findings may have impor tant implications for the use of GM-CSF-G250 Fl? as a tumor vaccine for the treatment of patients with advanced kidney cancer.