title: 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.
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 microg/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 microg/10(7) cells) augments both T-cell helper 1 (Th1) and Th2 cytokine mRNA expression (IL-2, IL-4, GM-CSF, IFN-gamma, and tumor necrosis factor-alpha). Comparison of various immune manipulation strategies in parallel, bulk PBMCs treated with FP (0.34 microg/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.
authors.affiliation: Department of Urology, UCLA Kidney Cancer Program, University of California Los Angeles, Los Angeles, California 90095, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Tso
authors.forename: A
authors.initials: A
authors.lastname: Zisman
authors.forename: A
authors.initials: A
authors.lastname: Pantuck
authors.forename: R
authors.initials: R
authors.lastname: Calilliw
authors.forename: J M
authors.initials: JM
authors.lastname: Hernandez
authors.forename: S
authors.initials: S
authors.lastname: Paik
authors.forename: D
authors.initials: D
authors.lastname: Nguyen
authors.forename: B
authors.initials: B
authors.lastname: Gitlitz
authors.forename: P I
authors.initials: PI
authors.lastname: Shintaku
authors.forename: J
authors.initials: J
authors.lastname: de Kernion
authors.forename: R
authors.initials: R
authors.lastname: Figlin
authors.forename: A
authors.initials: A
authors.lastname: Belldegrun
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000276:immunology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D019496:Cancer Vaccines / Q000235:genetics / Q000276:immunology; D002292:Carcinoma, Renal Cell / Q000097:blood / Q000276:immunology* / Q000628:therapy; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology; D015972:Gene Expression Regulation, Neoplastic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D006801:Humans; D007680:Kidney Neoplasms / Q000097:blood / Q000276:immunology* / Q000628:therapy; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D018411:Spodoptera / Q000821:virology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D016207:Cytokines; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C400566:antigen G250; D016178:Granulocyte-Macrophage Colony-Stimulating Factor