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

Cho Lea Tso, Amnon Zisman, Allan Pantuck, Randy Calilliw, Jose M. Hernandez, Sun Paik, David Nguyen, Barbara Gitlitz, Peter I. Shintaku, Jean De Kernion, Robert Figlin, Arie Belldegrun, Robert Figlin, Peter I. Shintaku, Cho Lea Tso, Amnon Zisman, Allan Pantuck, Randy Calilliw, Jose M. Hernandez, Sun PaikDavid Nguyen, Barbara Gitlitz, Peter I. Shintaku, Jean De Kernion, Robert Figlin, Arie Belldegrun

Research output: Contribution to journalArticlepeer-review

Abstract

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 Mr 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-γ, and tumor necrosis factor-α). 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.

Original languageEnglish
Pages (from-to)7925-7933
Number of pages9
JournalCancer Research
Volume61
Issue number21
StatePublished - 1 Nov 2001
Externally publishedYes

Fingerprint

Dive into the research topics of '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'. Together they form a unique fingerprint.

Cite this