REDIRECTED CYTOTOXIC EFFECTOR FUNCTION - REQUIREMENTS FOR EXPRESSION OF CHIMERIC SINGLE-CHAIN HIGH-AFFINITY IMMUNOGLOBULIN-E RECEPTORS

The aim of this study was to construct a single chain chimeric Fc epsi lon RI alpha receptor capable of effector function in leukocytes, incl uding cytotoxic lymphocytes. To determine the most effective single ch ain Fc epsilon RI alpha receptor with respect to IgE binding and signa ling function, a variety of chimeric gene constructs were transiently transfected into COS-7 cells. The most effective chimera consisted of four parts including: wild-type or mutated extracellular domains (Trp( 130) --> Ala(130), W130A) of Fc epsilon RI alpha, membrane proximal an d transmembrane regions of Fc gamma RIIa, and intracellular CD3 zeta ( epsilon(IIa)IIa zeta). Scatchard analysis indicated that these Fc epsi lon RI alpha chimeric receptor bound ligand with an affinity of 0.9 to 2.2 x 10(9) M(-1). Ligand binding capacity was dramatically reduced w ith the deletion of 11 membrane proximal amino acids of Fc epsilon RI alpha; however, function was restored by substitution with the equival ent region of Fc gamma RIIa, suggesting a crucial requirement for a '' spacer'' segment between the transmembrane and extracellular ligand bi nding domain. Chimeras that bound IgE effectively also mediated phagoc ytosis. Chimeric receptors that contained transmembrane zeta were expr essed as multimers and consequently did not bind IgE effectively; howe ver, cotransfection of these chimeras with gamma-chain largely reconst ituted IgE-mediated phagocytosis. The mouse cytotoxic T lymphocyte cel l line, CTLLR8 was stably transfected with epsilon(IIa)IIa zeta, and c loned transfectants were demonstrated to lyse target cells in an anti- Fc epsilon RI alpha or IgE antibody-dependent manlier. Therefore, func tional single chain chimeric Fe epsilon RI alpha receptors were expres sed in the absence or presence of associated zeta or gamma molecules a nd were used to redirect killer lymphocytes to target cells.