Intracellular single chain Fv antibody inhibits Ras activity in T‐cell antigen receptor stimulated Jurkat cells

Abstract

A mammalian expression vector directing the synthesis of a cytoplasmic single chain Fv version of the Y13-259 anti-Ras antibody was constructed and co-transfected into the human lymphoid cell line Jurkat together with a reporter construct containing the bacterial gene for chloramphenicol acetyl transferase under the transcriptional control of several copies of the binding site for the transcription factor NF-AT. The Ras specific antibody interferes with NF-AT activation upon direct activation of the T-cell antigen receptor, whereas activation by direct protein kinase C stimulation is less sensitive to the anti-Ras antibody. Furthermore, the observed inhibition is dependent on the ratio of antibody to reporter plasmid utilized in the transfection experiments.

Regulation of growth and survival of activated T cells by cell‐transducing inhibitors of Ras

“…6C). The Y13-259 mAb has been shown to inhibit Ras signalling in Jurkat cells [14] however, in our study TAT-Y13 was the least effective among the TAT-coupled Ras inhibitors tested. A possible explanation may have to do with the different method of delivery employed.…”

“…Also, transduction of eoshinophils with TAT-dominant negative Ras, down-modulated IL5-mediated ERK activity and cell survival [25], and decreased cell adhesion mediated by beta-2 integrin binding to intracellular adhesion molecule-1 [26]. We chose inhibitors known for their high specificity; the neutralizing Y13-259 mAb [14] and the RBD of Raf-1. The RBD is the minimum region of Raf-1 that binds to active Ras with high affinity (Kd of 20 nM), whereas its affinity for RasGDP is 3 orders of magnitude lower [27].…”

The mode of action of Y13‐259 scFv fragment intracellularly expressed in mammalian cells

“…Evidence for the neutralization of p21ras activity in mammalian cells by the intracellular expression of Y13‐259 scFvs has also been presented [11, 12]. Moreover, as a step towards cancer gene therapy, intratumor transduction of HCT116 colon carcinoma cells with the Y13‐259 scFv using an adenoviral vector leads to tumor regression in nude mice [13].…”

The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco

“…Cytosolic expression of functional scFv antibodies in plants and other eukaryotes [2,3,41] is remarkable. The two intramolecular disulphide bridges (one in V H and one in VL) which are assumed to be necessary for folding into a stable and functional scFv [ 14] are expected not to be formed in the reducing environment of the cytosol because of the absence of the enzyme protein disulphide isomerase [13], which catalyses the formation of such bonds.…”