VHH characterization.Recombinant VHHs: Production, characterization and affinity

Anal Biochem. 2020 Jan 15:589:113491. doi: 10.1016/j.ab.2019.113491. Epub 2019 Oct 30.

Abstract

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology.

Keywords: Affinity; CD; Characterization; Differential scanning fluorimetry; Expression; Hydrogen/deuterium exchange; Ion-mobility; Mass spectrometry; Structure; Surface plasmon resonance; VHH.

MeSH terms

  • Animals
  • Antigens / immunology
  • Camelids, New World / immunology*
  • Cloning, Molecular
  • ErbB Receptors / immunology
  • Escherichia coli / genetics
  • Humans
  • Immunoglobulin Heavy Chains* / chemistry
  • Immunoglobulin Heavy Chains* / isolation & purification
  • Receptor, ErbB-2 / immunology*
  • Recombinant Proteins / immunology
  • Serum Albumin, Human / immunology*
  • Single-Domain Antibodies* / chemistry
  • Single-Domain Antibodies* / isolation & purification

Substances

  • Antigens
  • Immunoglobulin Heavy Chains
  • Recombinant Proteins
  • Single-Domain Antibodies
  • EGFR protein, human
  • ERBB2 protein, human
  • ErbB Receptors
  • Receptor, ErbB-2
  • Serum Albumin, Human