CHIMERIC RECEPTORS PROVIDING BOTH PRIMARY AND COSTIMULATORY SIGNALINGIN T-CELLS FROM A SINGLE-GENE PRODUCT

Single chain Fv chimeric receptors, or T-bodies, are described with in tracellular sequences comprising the costimulatory signaling domain of CD28 in series with the zeta-chain from the TCR complex. Using an eng ineered human single chain Fv derived from P67, an mAb with specificit y for human CD33, and a spacer comprising an Ab hinge region with eith er Fc gamma or part of the CD28 extracellular region, fusion molecules were constructed to test the ability of single chain designs to media te both primary signaling and costimulation from one extracellular bin ding event. Constructs with the CD28 signaling domain proximal and the zeta-chain distal to the membrane were found to express more efficien tly in Jurkat than constructs with the opposite orientation and were c apable of mediating up to 20 times more IL-2 production on stimulation with solid phase Ag when compared with transfectants expressing chime ric receptors with zeta-chain intracellular signaling domains only, IL -2 production was specific to Ag challenge and was completely inhibite d by incubation with free Ab of the same specificity as the extracellu lar binding site of the construct, but not by an isotype-matched contr ol Ab, The CD28 intracellular domain of these fusion proteins was show n to be capable of binding the p85 subunit of phosphatidylinositol 3'- kinase, These constructs represent the first of a new generation of si ngle gene multidomain chimeric receptors capable of mediating both pri mary and costimulatory signaling specifically from a single extracellu lar recognition event.