A functional role for CD28 costimulation in tumor recognition by single-chain receptor-modified T cells

T cells engineered to express single-chain antibody receptors that incorporate TCR-ζ and cluster designation (CD)28 signaling domains (scFv-α-erbB2-CD28-ζ) can be redirected in vivo to cancer cells that lack triggering costimulatory molecules. To assess the contribution of CD28 signaling to the function of the scFv-CD28-ζ receptor, we expressed a series of mutated scFv-CD28-ζ receptors directed against erbB2. Residues known to be critical for CD28 signaling were mutated from tyrosine to phenylalanine at position 170 or proline to alanine at positions 187 and 190. Primary mouse T cells expressing either of the mutant receptors demonstrated impaired cytokine (IFN-γ and GM-CSF) production and decreased proliferation after antigen ligation in vitro and decreased antitumor efficacy in vivo compared with T cells expressing the wild-type scFv-CD28-ζ receptor, suggesting a key signaling role for the CD28 component of the scFv-CD28-ζ receptor. Importantly, cell surface expression, binding capacity and cytolytic activity mediated by the scFv-CD28-ζ receptor were not diminished by either mutation. Overall, this study has definitively demonstrated a functional role for the CD28 component of the scFv-CD28-ζ receptor and has shown that incorporation of costimulatory activity in chimeric scFv receptors is a powerful approach for improving adoptive cancer immunotherapy.