Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

MAbs. 2017 Apr;9(3):438-454. doi: 10.1080/19420862.2016.1277301. Epub 2017 Jan 5.

Abstract

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.

Keywords: Antibody engineering; bispecific monovalent antibodies; dual targeting; single-chain antibody; tethered single-chain antibodies.

MeSH terms

  • Animals
  • Antibodies, Bispecific / biosynthesis*
  • Antibodies, Bispecific / isolation & purification
  • ErbB Receptors / immunology
  • Humans
  • Immunoglobulin G / immunology*
  • Protein Engineering / methods*
  • Proteolysis
  • Receptor, IGF Type 1 / immunology
  • Single-Chain Antibodies / biosynthesis*
  • Single-Chain Antibodies / isolation & purification

Substances

  • Antibodies, Bispecific
  • Immunoglobulin G
  • Single-Chain Antibodies
  • EGFR protein, human
  • ErbB Receptors
  • Receptor, IGF Type 1