Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies

Bioconjug Chem. 2018 Nov 21;29(11):3586-3594. doi: 10.1021/acs.bioconjchem.8b00545. Epub 2018 Oct 17.

Abstract

Antibody-based diagnostic and therapeutic reagents armed with effector molecules such as dyes and drugs offer hope in the battle against cancer. Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled, and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a single-pot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye700 and Alexa-Fluor647. This dual-labeled antibody bound to EGFR+ ovarian cancer cell lines and tissue samples with high specificity, and its phototherapeutic efficacy was confirmed by the selective killing of EGFR+ cells in vitro.

MeSH terms

  • Cell Line, Tumor
  • Coloring Agents / chemistry
  • ErbB Receptors / immunology
  • ErbB Receptors / metabolism
  • Female
  • Flow Cytometry
  • Humans
  • Immunoconjugates / chemistry
  • Microscopy, Confocal
  • Ovarian Neoplasms / pathology
  • Protein Binding
  • Protein Conformation
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / immunology
  • Single-Chain Antibodies / chemistry*
  • Single-Chain Antibodies / immunology

Substances

  • Coloring Agents
  • Immunoconjugates
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • scFv 425
  • ErbB Receptors