Targeted cytokine delivery to neuroblastoma

The aim of this study was to construct a fusion protein from the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) and a single-chain Fv fragment (scFv D29) and to investigate its potential to activate cells of the immune system against neuroblastoma cells expressing neural cell adhesion molecule (NCAM). Mammalian cell expression of the scFv D29-GM-CSF fusion protein was compared using a number of vectors, including retroviral and adenoviral vectors. The resultant fusion protein, expressed by HeLa cells, was found by ELISA to bind immobilized recombinant NCAM. Moreover, FACS analysis confirmed binding to the human neuroblastoma cell line SKNBE and a murine neuroblastoma cell line engineered to express the glycosylphosphatidylinositol form of human NCAM (N2A-rKNIE). The fusion protein was also found to stimulate the proliferation of the FDC-P1 haemopoietic cell line, which is dependent on GM-CSF (or interleukin 3) for continued growth. In vitro clonogenic assays indicated that scFv-GM-CSF could selectively induce growth inhibition of SKNBE cells by murine lymphoid cells

Targeted cytokine delivery to neuroblastoma

The aim of this study was to construct a fusion protein from the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) and a single-chain Fv fragment (scFv D29) and to investigate its potential to activate cells of the immune system against neuroblastoma cells expressing neural cell adhesion molecule (NCAM). Mammalian cell expression of the scFv D29-GM-CSF fusion protein was compared using a number of vectors, including retroviral and adenoviral vectors. The resultant fusion protein, expressed by HeLa cells, was found by ELISA to bind immobilized recombinant NCAM. Moreover, FACS analysis confirmed binding to the human neuroblastoma cell line SKNBE and a murine neuroblastoma cell line engineered to express the glycosylphosphatidylinositol form of human NCAM (N2A-rKNIE). The fusion protein was also found to stimulate the proliferation of the FDC-P1 haemopoietic cell line, which is dependent on GM-CSF (or interleukin 3) for continued growth. In vitro clonogenic assays indicated that scFv-GM-CSF could selectively induce growth inhibition of SKNBE cells by murine lymphoid cells

A DISCORDANCE BETWEEN THE HUMAN MUSCLE NCAM SEQUENCE AND THOSE SEEN IN OTHER NCAM CDNA CLONES

A cDNA clone, designated NC7, has been isolated from human foetal kidney that partially codes for the 140 kDa isoform of human NCAM. This clone contains a 6 bp insert that is not present in the human muscle cDNA clone lambda-4.4. This same sequence has also been found in both a cDNA clone obtained from a human Small Cell Lung Carcinoma (SCLC) line and in human genomic DNA. Furthermore, an equivalent sequence to the 6 bp region identified in the above samples is present in mouse, rat and chicken NCAM. The 6 bp insertion does not lie at a predicted intron/exon boundary as extrapolated by homology studies with the chicken and, therefore, the mechanism by which the sequence is deleted from the human muscle clone lambda-4.4 remains unclear