Site-specific PEGylation of an anti-CEA/CD3 bispecific antibody improves its antitumor efficacy

Haitao Pan,1,2 Jiayu Liu,1,2 Wentong Deng,1,2 Jieyu Xing,1,2 Qing Li,1,2 Zhong Wang1,2 1School of Pharmaceutical Sciences, 2Centre for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, People’s Republic of China Introduction: Bispecific antibodies that engage immune cells to kill cancer cells are actively pursued in cancer immunotherapy. Different types of bispecific antibodies, including single-chain fragments, Fab fragments, nanobodies, and immunoglobulin Gs (IgGs), have been studied. However, the low molecular weight of bispecific antibodies with single-chain or Fab fragments generally leads to their rapid clearance in vivo, which limits the therapeutic potential of these bispecific antibodies. Materials and methods: In this study, we used a site-specific PEGylation strategy to modify the bispecific single-domain antibody-linked Fab (S-Fab), which was designed by linking an anticarcinoembryonic antigen (anti-CEA) nanobody with an anti-CD3 Fab. Results: The half-life (t1/2) of PEGylated S-Fab (polyethylene glycol-S-Fab) was increased 12-fold in vivo with a slightly decreased tumor cell cytotoxicity in vitro as well as more potent tumor growth inhibition in vivo compared to S-Fab. Conclusion: This study demonstrated that PEGylation is an effective approach to enhance the antitumor efficacy of bispecific antibodies. Keywords: Fab, nanobody, PEGylation, bispecific antibody, half-life, CEA