Functional expression of chimeric receptor genes in human T cells

Tumor immunotherapy has been limited to date by the poor antigenicity of mo st tumors, the immunocompromised state of many cancer patients, and the slo w tumor penetration and short half-life of exogenously-introduced anti-tumo r antibodies. Our group has developed a model immunotherapy system using a chimeric construct containing an antibody V region fused to a T cell activa tion molecule (T body) introduced by transfection into cytotoxic T cell lin es, or populations of activated primary T or natural killer (NK) cells, In this study we have optimized the conditions needed for efficient transducti on of human peripheral lymphocytes (PBL) using retroviral vectors pseudotyp ed with the gibbon ape leukemia virus (GaLV) envelope. Selection of packagi ng cells producing high virus titers was performed following transfection w ith constructs containing the green fluorescent protein (GFP), and FAGS sor ting. As a model chimeric receptor gene we used a tripartite construct cons isting of a single-chain anti-TNP antibody variable region linked to part o f the extracellular domain and the membrane spanning regions of the CD28 co receptor molecule and joined at its 5' end to a gene fragment encoding the intracellular moiety of the gamma activation molecule common to the Fc epsi lon and Fc gamma receptors. Enriched preparations of retrovectors containin g this chimeric receptor and the GFP gene could stably and efficiently tran sduce human PBL co-activated by anti-CD3 and anti-CD28 antibodies. In routi ne experiments, the transgene was expressed in 35-70% of the human T cells. Such lymphocytes express the chimeric receptors on their surface and upon stimulation with hapten immobilized on plastic they can produce IL-2. Trans fectomas activated in this manner also undergo specific proliferation in th e absence of exogenous IL-2. Moreover, the transduced lymphocytes could eff ectively lyse target cells expressing the TNP hapten on their surface. Thes e studies establish the conditions for the optimal transfection of effector lymphocytes to redirect them against a variety of tumor targets. (C) 2001 Elsevier Science B.V. All rights reserved.