Balanced secretion of anti-CEA × anti-CD3 diabody chains using the 2A self-cleaving peptide maximizes diabody assembly and tumor-specific cytotoxicity

Gene Ther. 2017 Apr;24(4):208-214. doi: 10.1038/gt.2017.3. Epub 2017 Jan 11.


Adoptive transfer of genetically engineered human cells secreting bispecific T-cell engagers has shown encouraging therapeutic effects in preclinical models of cancer. However, reducing the toxicity and improving the effectiveness of this emerging immunotherapeutic strategy will be critical to its successful application. We have demonstrated that for gene-based bispecific antibody strategies, two-chain diabodies have a better safety profile than single-chain tandem scFvs (single-chain variable fragments), because their reduced tendency to form aggregates reduces the risk of inducing antigen-independent T-cell activation. Here, we demonstrate that the incorporation of a 2A self-processing peptide derived from foot-and-mouth disease virus conveying co-translational cleavage into a two-chain anti-CD3 × anti-CEA diabody gene enables near-equimolar expression of diabody chains 1 and 2, and thus increases the final amount of assembled diabody. This was found to maximize diabody-mediated T-cell activation and cytotoxicity against carcinoembryonic antigen-positive tumor cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Bispecific / genetics
  • Antibodies, Bispecific / immunology*
  • CD3 Complex / genetics*
  • CD3 Complex / immunology
  • Carcinoembryonic Antigen / immunology*
  • Cytotoxicity, Immunologic
  • Foot-and-Mouth Disease Virus / genetics
  • Foot-and-Mouth Disease Virus / immunology
  • Humans
  • Immunotherapy / methods
  • Jurkat Cells
  • Lymphocyte Activation / immunology
  • Neoplasms / immunology
  • Neoplasms / therapy*
  • Peptides / genetics
  • Peptides / immunology
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology
  • T-Lymphocytes / immunology
  • Tumor Necrosis Factor Receptor Superfamily, Member 7 / genetics*
  • Tumor Necrosis Factor Receptor Superfamily, Member 7 / immunology


  • Antibodies, Bispecific
  • CD3 Complex
  • Carcinoembryonic Antigen
  • Peptides
  • Single-Chain Antibodies
  • Tumor Necrosis Factor Receptor Superfamily, Member 7