Abstract
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 mAb 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. cDNAs 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 antitumor activity against human breast cancer cells by human peripheral blood mononuclear cells (hPBMC) with BsAb was measured by lactate dehydrogenase release in vitro, and in vivo using hPBMC-transplanted MHC class I- and class II-deficient NOG mice. 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+ 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. An anti-B7-H4/CD3 BsAb may be a good therapeutic tool for patients with B7-H4+ breast cancers.
Highlights
Because of the recent success of immune checkpoint–blocking antibodies, clinical trials are underway to evaluate their efficacy in various cancers [1,2,3,4]
We found that the anti-B7-H4/CD3 Fab-scFv antibody had potent cytotoxic activity against a B7-H4þ breast cancer cell line in vitro, and in vivo using a MHC-double knockout NOG mouse model [18]
We found that the B7-H4/CD3 bispecific antibodies (BsAb) had potent cytotoxic activity against B7-H4þ breast cancer cell lines in vitro, and in vivo using a MHC-double knockout NOG mouse model
Summary
Because of the recent success of immune checkpoint–blocking antibodies, clinical trials are underway to evaluate their efficacy in various cancers [1,2,3,4]. A majority of patients with cancer are unlikely to benefit from anti- programmed death-1 (PD-1)/ PD-ligand 1 (PD-L1) antibody treatment because the response rate is approximately 20%–40% even in PD-L1þ cancers. In addition to these immunomodulatory receptor blockade therapies, other modulating technologies have been developed [5, 6]. Improvements in protein engineering technology have enabled the creation of various types of artificial antibodies with greater flexibility in design, size, specificity, half-life, and distribution, and dozens of BsAb formats have been proposed [6, 14]
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