Production and characterization of a novel site-specific-modifiable anti-OX40-receptor single-chain variable fragment for targeted drug delivery

Biochem Biophys Res Commun. 2018 Feb 5;496(2):614-620. doi: 10.1016/j.bbrc.2018.01.051. Epub 2018 Jan 9.

Abstract

OX40 receptor (tumor necrosis factor receptor superfamily, member 4; CD134) is a T-cell co-stimulatory molecule that plays an important role in T-cell activation and survival. OX40 receptor is activated by its ligand, OX40L; and modulation of the OX40-OX40L interaction is a promising target for the treatment of autoimmune diseases and cancers. Here, we generated a high-affinity anti-OX40 single-chain variable fragment carrying a C-terminal cysteine residue (scFvC). Physicochemical and functional analyses revealed that the scFvC bound to OX40-expressing cells and was internalized via OX40-mediated endocytosis without inducing phosphorylation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), an important complex in the classical NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway. In addition, mutation of the 36th cysteine residue in variable region of light chain enabled site-specific chemical modification to carboxy terminal cysteine and improved the thermal stability of the scFvC. These results suggest that this novel high-affinity anti-OX40 scFvC may be useful as a transporter for targeted delivery of small compounds, proteins, peptides, liposomes, and nanoparticles, into OX40-expressing cells for the treatment of autoimmune diseases and cancers.

Keywords: Antibody drug conjugate; Drug delivery; Internalization; OX40; Single chain antibody(scFv); Thermal stability of antibody.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Drug Delivery Systems
  • Escherichia coli / genetics
  • Gene Expression
  • Humans
  • Immunoconjugates / chemistry
  • Immunoconjugates / genetics
  • Immunoconjugates / immunology*
  • Jurkat Cells
  • Models, Molecular
  • Point Mutation
  • Protein Stability
  • Receptors, OX40 / immunology*
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology*

Substances

  • Immunoconjugates
  • Receptors, OX40
  • Single-Chain Antibodies