Novel single-chain variant of antibody against mesothelin established by phage library

Cancer Sci. 2019 Sep;110(9):2722-2733. doi: 10.1111/cas.14150. Epub 2019 Aug 28.

Abstract

Mesothelin (MSLN) shows increased expression in various cancer cells. For clinical application of antibodies as a positron emission tomography (PET) imaging reagent, a human shortened antibody is essential both for avoiding redundant immune responses and for providing rapid imaging. Therefore, we cloned a single-chain fragment of variable regions (scFv) from a human-derived gene sequence. This was achieved through the construction of a naïve phage library derived from human tonsil lymphocytes. Using a column with human recombinant MSLN, we carried out bio-panning of phage-variants by colony formation. We first obtained 120 clones that were subjected to selection in an ELISA using human recombinant MSLN as a solid phase antigen, and 15 phage clones of scFv with a different sequence were selected and investigated by flow cytometry (FCM). Then, six variants were selected and the individual scFv gene was synthesized in the VL and VH domains and expressed in Chinese hamster ovary cells. Mammalian cell-derived human-origin scFv clones were analyzed by FCM again, and one MSLN highly specific scFv clone was established. PET imaging by 89 Zr-labeled scFv was done in mice bearing xenografts with MSLN-expressing cancer cells, and tumor legions were successfully visualized. The scFv variant established in the present study may be potentially useful for cancer diagnosis by PET imaging.

Keywords: 89Zr (zirconium-89); mesothelin; phage library; positron emission tomography (PET) imaging; single-chain fragment of variable region.

MeSH terms

  • Animals
  • CHO Cells
  • Cell Line, Tumor
  • Cloning, Molecular
  • Cricetulus
  • GPI-Linked Proteins / genetics
  • GPI-Linked Proteins / immunology*
  • GPI-Linked Proteins / isolation & purification
  • GPI-Linked Proteins / metabolism
  • Humans
  • Male
  • Mesothelin
  • Mice
  • Mice, Inbred BALB C
  • Mice, Nude
  • Molecular Imaging / methods
  • Neoplasms / diagnostic imaging*
  • Neoplasms / pathology
  • Peptide Library
  • Positron Emission Tomography Computed Tomography / methods
  • Radioisotopes
  • Radiopharmaceuticals / administration & dosage
  • Radiopharmaceuticals / immunology*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / isolation & purification
  • Single-Chain Antibodies / administration & dosage
  • Single-Chain Antibodies / immunology*
  • Xenograft Model Antitumor Assays
  • Zirconium

Substances

  • GPI-Linked Proteins
  • MSLN protein, human
  • Msln protein, mouse
  • Peptide Library
  • Radioisotopes
  • Radiopharmaceuticals
  • Recombinant Proteins
  • Single-Chain Antibodies
  • Zirconium
  • Mesothelin
  • Zirconium-89