A recombinant bispecific single‐chain antibody induces targeted, supra‐agonistic CD28‐stimulation and tumor cell killing

Abstract Endowing tumor cells with costimulatory signals for T cell activation has emerged as a promising strategy for tumor immunotherapy. Costimulatory molecules were either transfected into tumor cells to generate vaccines or were fused, e.g. to antibodies against tumor‐associated antigens, to achieve targeted T cell costimulation in vivo . Here we report the production and purification of rM28, a recombinant bispecific single‐chain antibody directed to a melanoma‐associated proteoglycan and to the costimulatory CD28 molecule on human T cells. We found that a dimer of the recombinant molecule, bound to tumor target cells, induced pronounced T cell activation in peripheral blood mononuclear cell preparations without additional TCR/CD3 stimulation being required. Thelytic activity generated after 3 days of stimulation effectively prevented tumor cell growth. However, it was unspecific and predominantly mediated by non T cells. Our findings demonstrate that presentation of a CD28 antibody within a suitable recombinant, bispecific format may result in a "targeted supra‐agonistic stimulation" of the CD28 molecule, which leads to effective tumor cell killing after induction of unspecifically lytic cells.