ARTICLE ABSTRACTImmunotherapy using OX40 agonist antibodies shows great preclinical efficacy in mouse tumor models. But in a clinical setting, OX40 agonist antibody alone or in combination with checkpoint blockade exhibits only modest efficacy due to lack of sufficient activation. We hypothesized that the limited antitumor activity in patients may due to insufficient clustering of OX40 antibody in the tumor. To test this hypothesis, we generated a tetravalent programmed death ligand-1 (PD-L1)/OX40 BsAb by fusing two PD-L1 VHH fragments to the C-terminus of a nonblocking agonistic anti-OX40 antibody. The resulting BsAb had intact function of each parental antibody, including efficiently blocking PD1/PD-L1 interaction and inducing OX40 activation. In addition, this BsAb showed significantly enhanced potency in activation of OX40-expressing T cells when PD-L1–expressing tumor cells or dendrite cells were present, through PD-L1–mediated cross-linking of OX40. Moreover, the BsAb exhibited superior antitumor activities over the parental monospecific antibodies alone or in combination in multiple in vivo tumor models. These results demonstrated a great potential for further clinical development of the potent immunostimulatory PD-L1/OX40 bispecific antibody.