Targeting the cytotoxicity of fusogenic membrane glycoproteins in gliomas through protease–substrate interaction

Abstract

Fusogenic membrane glycoproteins (FMG) are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. FMG expression constructs caused massive syncytia formation followed by cytotoxic cell death in glioma cell lines, and antitumor activity has been shown in glioma xenografts. FMG-induced fusion in glioma cells can involve heterologous cell lines including normal astrocytes and fibroblasts, therefore making targeting important. Here we report on the use of matrix metalloproteinase (MMP) cleavable linkers to target cytotoxicity of FMGs against gliomas. Expression constructs were made expressing the hyperfusogenic version of the Gibbon Ape Leukemia Virus envelope glycoprotein (GALV) linked to a blocking ligand (the C-terminal extracellular domain of CD40 ligand) via either an MMP cleavable linker (GALV M40), a factor Xa protease cleavable linker (GALV X40), or a noncleavable linker (GALV N40). Unmodified GALV expressing constructs were used as positive controls. The glioma cell lines U87, U118, and U251 previously characterized by zymography and MMP-2 activity assay as high, medium, and low MMP expressors, respectively; normal human astrocytes and the MMP-poor cell line TE671 were transfected with the GALV, GALV N40, GALV X40, and GALV M40 constructs. In contrast to unmodified GALV constructs, transfection with GALV X40 and GALV N40 constructs blocked fusion and cytotoxic cell death. Fusion occurred, however, after transfection with constructs containing MMP cleavable linkers to an extent dependent on MMP expression in the specific cell line. Use of the broad-spectrum MMP inhibitors, 1,10-phenanthroline and N-hydroxy-piperazine-carboxamide completely abolished the ability of MMP constructs to induce fusion. In cell mixing experiments, mixing of MMP-poor cell lines transfected with GALV M40 constructs with the MMP overexpressing untransfected U87 glioma cells led to partial restoration of fusion. Use of U87 supernatant did result in a similar effect. Establishment of stable tranfectants expressing the membrane-type MMPs, MT-1 MMP and MT-2 MMP did restore fusion in the MMP-poor cell line TE671 after transfection with GALV M40, thus indicating that both membrane-type MMPs and soluble MMPs activate the MMP cleavable constructs. In addition, the GALV M40 construct retained its cytotoxic activity against U87 cells in vivo, although less effectively as compared to unmodified GALV. Our data indicate that GALV-induced cytotoxicity in glioma cell lines can be blocked by display of the CD40 ligand. Incorporation of an MMP cleavable linker can selectively restore cytotoxicity in MMP expressing glioma cell lines both in vitro and in vivo, while sparing normal human astrocytes. Given the high frequency of MMP overexpression in gliomas, this represents a promising targeting strategy. Gene Therapy (2003) 10, 725-732.

Transcriptionally targeted gene therapy to detect and treat cancer

“…For example, the attenuated adenovirus dl1520 (ONXY-015), lacking viral E1B 55k protein, was reported to selectively replicate and kill p53 deficient tumor cells and not normal cells ( [2] and references within). In another example, the cytotoxic activity of a fusogenic glycoprotein (GALV) was engineered to be activated by matrix metalloproteinase (MMP) cleavage of a blocking domain [6]. This modulated GALV exhibited selective cytotoxicity to MMP-expressing glioma cells, while sparing normal human astrocytes.…”

Death by fusion for acute leukemia cells

“…17 The potential of this mutant gene for cancer therapy has been explored in a variety of settings. [17][18][19][20][21] In this context, the current report by Tan et al further adds to the growing literature that supports the use of SIN as an oncolytic agent. 6 The authors show that AML cell lines as well as primary cells taken from patients with the disease overexpress the laminin receptor, making them excellent targets for therapy with SIN.…”

Generation of optimized and urokinase-targeted oncolytic Sendai virus vectors applicable for various human malignancies

“…8,12,19,20 Their data show that those vectors can target the cytotoxicity of the fusogenic membrane glycoprotein in MMP-overexpressing tumor lines and xenografts, and maintain significant antitumor activity both in vitro and in vivo. MMPtargeted vectors, irrespective of the vectors' backbone, may be confronted with a similar difficulty in clinical settings.…”