PURPOSE: The B7 homolog 4 (B7-H4, VTCN1) is an immune checkpoint molecule that negatively regulates immune responses and is known to be overexpressed in many human cancers. Previously, we generated a mouse anti-human B7-H4 monoclonal antibody that did not have a significant antitumor effect in vivo probably because of molecule instability. In this study, we designed a B7-H4/CD3 bispecific antibody (BsAb) and investigated its antitumor activity in vitro and in vivo using a humanized mouse model. EXPERIMENTAL DESIGN: Complementary DNAs of the antibody-binding fragment (Fab)-single-chain variable fragment (scFv) and scFv-scFv of the anti-B7-H4/CD3 BsAb were synthesized, and the BsAb antibodies were produced in HEK293 cells. The anti-tumor activity against human breast cancer cells by human peripheral blood mononuclear cells (hPBMC) with BsAb was measured by lactate dehydrogenase (LDH) release in vitro, and in vivo using hPBMC transplanted major histocompatibility (MHC) class I and class II-deficient NOG mice. RESULTS: hPBMCs with anti-B7-H4/CD3 BsAbs successfully lysed the human breast cancer cell line MDA-MB-468 (EC50: 0.2 ng/ml) and other B7-H4-positive cell lines in vitro When BsAb was injected in a humanized mouse model, there was an immediate and strong antitumor activity against MDA-MB-468, HCC-1954 and HCC-1569 tumors and CD8(+) and granzyme B(+) CTL infiltration into the tumor, and there were no adverse effects after long-term observation. CD8(+) T cell depletion by an anti-CD8 antibody mostly reduced the antitumor effect of BsAb in vivo Conclusions:An anti-B7-H4/CD3 bispecific antibody may be a good therapeutic tool for patients with B7-H4-positive breast cancers.