A spacer region between the single chain antibody- and the CD3 zeta-chain domain of chimeric T cell receptor components is required for efficient ligand binding and signaling activity

Abstract

The elimination of tumor cells by cytotoxic T lymphocytes (CTLs) could become the basis of a biological cancer therapy. The recognition specificity of cytotoxic T lymphocytes (CTLs) can be genetically modified by stable introduction of chimeric T cell receptor genes and thus be directed towards tumor cells. We designed a recombinant T cell receptor (TCR) component consisting of a single chain Fv derivative of a monoclonal antibody (scFv) serving as the extracellular antigen-binding domain and the zeta-chain of the TCR/CD3 complex serving as a signal transducing domain. Three chimeric receptor constructs differing in their molecular structure were derived and their functions in transduced T cells compared. A construct in which the scFv domain, specific for the ErbB-2 receptor, was fused directly to the zeta-chain, and two constructs containing different hinge regions between the functional domains, were made. The hinge regions serve as spacers which increase the distance of the scFv moiety from the plasma membrane. Only the two scFv-zeta chimeras containing a hinge region showed ErbB-2 binding activity, when expressed in T cells. The scFv-zeta construct which lacks a spacer segment did not. Consistently, only the spacer-containing chimeras transduced T cell receptor signals following ErbB-2 mediated crosslinking. An increase in intracellular Ca2+ concentrations and cytokine secretion was observed. ErbB-2 expressing tumor cells were efficiently lysed by CTLs which expressed the spacer-containing scFv-zeta chimeras. Our results will help to optimize the design of recombinant T cell receptor components useful in the grafting of a specificity of recognition on to cytotoxic T cells and possibly the gene therapy of cancer.