Engineering Bifunctional Antibodies with Constant Region Fusion Architectures

J Am Chem Soc. 2017 Dec 27;139(51):18607-18615. doi: 10.1021/jacs.7b09641. Epub 2017 Dec 14.


We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent in vitro activity, physiochemical properties, and stability. Among these, a Her2 × CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 single-chain variable fragment (ScFv) into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2 × CD3 BsAb shows potent in vivo antitumor activity in mouse Her22+ and Her21+ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multifunctional antibodies for therapeutic applications.

MeSH terms

  • Animals
  • Antibodies, Bispecific / chemistry*
  • Antibodies, Bispecific / genetics*
  • Antibodies, Bispecific / immunology
  • CD3 Complex / immunology
  • Cattle
  • Female
  • Humans
  • Immunoglobulin Constant Regions / chemistry*
  • Immunoglobulin Constant Regions / genetics*
  • Immunoglobulin Constant Regions / immunology
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / genetics
  • Immunoglobulin Fab Fragments / immunology
  • Mice
  • Protein Engineering*
  • Protein Stability
  • Receptor, ErbB-2 / immunology
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology
  • Xenograft Model Antitumor Assays


  • Antibodies, Bispecific
  • CD3 Complex
  • Immunoglobulin Constant Regions
  • Immunoglobulin Fab Fragments
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • Erbb2 protein, mouse
  • Receptor, ErbB-2