HARNESSING SYK FAMILY TYROSINE KINASES AS SIGNALING DOMAINS FOR CHIMERIC SINGLE-CHAIN OF THE VARIABLE DOMAIN RECEPTORS - OPTIMAL-DESIGN FORT-CELL ACTIVATION

T cells of tumor bearers often show defective TCR-mediated signaling e vents and, therefore, exhibit impaired immune responses. As such, pati ents with heavy tumor burden are often not amenable to adoptive T cell therapy, To overcome this limitation, we have developed a chimeric re ceptor that joins an extracellular single chain Fv (scFv) of a specifi c Ab for Ag recognition to an intracellular protein tyrosine kinase (P TK) for signal propagation. Stimulation through the scFv-PTK receptor should bypass defective TCR-proximal events and directly access the T cell's effector mechanisms, In this study we describe the optimization of a scFv-PTK configuration, leading to complete T cell activation. T he cytosolic PTK Syk is superior to its family member, Zap-70, for int racellular signaling, As a transmembrane (TM) domain, CD4 performs bet ter than CD8 when plastic-immobilized Ag serves as a stimulator. Howev er, when APC are used to trigger chimeric receptors, the need for a fl exible spacer between the scFv and TM domains becomes apparent, The CD 8 alpha-derived hinge successfully performs this task in chimeric scFv -Syk receptors regardless of its cysteine content. A cytotoxic T cell hybridoma expressing chimeric receptor genes composed of scFv-CD8(hing e)-CD8(TM)-Syk or scFv-CD8(image)-CD4(TM)-Syk is efficiently stimulate d to produce IL-2 upon interaction with APC and specifically lyses app ropriate target cells in a non-MHC-restricted manner.