Integrated src kinase and costimulatory activity enhances signal transduction through single-chain chimeric receptors...

Adoptive immunotherapy using receptor-modified T lymphocytes has shown promise in preclinical studies for the treatment of infectious and malignant diseases. These modified T cells express chimeric receptors that link ligand recognition and signal transduction domains in a single gene product. Typically, a single chain Fv fragment is genetically attached to the cytoplasmic domain of the T-cell receptor (TCR) zeta chain. Modulating the signaling characteristics of chimeric receptors will be important for their application to human immunotherapy. It was hypothesized that linking coreceptor and costimulatory signaling motifs together with the zeta signaling domain will enhance receptor function. The present study compares signaling characteristics of 9 single-chain receptors consisting of the H-2K(b) extracellular and transmembrane domains and various combinations of T cell signal transduction domains. Signal transduction regions studied include the TCR zeta chain, the CD4 coreceptor, the lck protein tyrosine kinase, and the CD28 costimulatory receptor. Biochemical characteristics of the receptors, analyzed using calcium flux, receptor, and ZAP-70 phosphorylation, and lck association may be predicted from the known functions of receptor constituents. The combination of zeta together with coreceptor and costimulatory function in a single receptor maximizes chimeric receptor sensitivity and potency. Combining zeta with either the costimulatory or coreceptor function independently also enhances receptor function, though to a lesser extent. It is therefore possible to link TCR, coreceptor, and costimulatory activities in a single functional entity using modular domains. Such receptors demonstrate distinct signaling properties and should prove useful in the development of chimeric receptors for therapeutic purposes.