Expression of 5T4 extracellular domain fusion protein and preparation of anti-5T4 monoclonal antibody with high affinity and internalization efficiency

Protein Expr Purif. 2019 Jun:158:51-58. doi: 10.1016/j.pep.2018.07.003. Epub 2018 Jul 6.

Abstract

5T4, a membrane protein, is overexpressed in many tumor tissues but rarely expressed in normal tissues. Here, CHO-5T4+ cells were generated and served as the antigen to immunize mice. Hybridoma techniques were employed to produce monoclonal antibodies (mAbs). The recombinant protein of human IgG Fc-fused extracellular domain of 5T4 (5T4 ECD-Fc) was obtained from transient expression in HEK293F cells. The fusion protein 5T4 ECD-Fc and CHO-5T4+ cells were respectively utilized to screen anti-5T4 antibodies that could bind to the native antigen. In preliminary screening, three hundred and fifty mAbs were obtained. Via surface plasmon resonance and flow cytometry screening, seven anti-5T4 mAbs stood out. Among them, H6 showed a high affinity (KD = 1.6 × 10-11 M) and internalization percentage (36% for 1 h and 80% for 4 h). The molecular weight and isoelectric point of H6 were determined by LC-MS and iCIEF. Moreover, the specific reactivity of H6 was demonstrated by western blotting, flow cytometry, and immunohistochemistry, respectively. In conclusion, we produced human recombinant protein of 5T4 extracellular domain and developed high-affinity internalizing monoclonal antibodies which may be applied in the 5T4-targeting ADC therapy and basic research.

Keywords: 5T4; Hybridoma; Mammalian expression; Monoclonal antibody.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Murine-Derived* / chemistry
  • Antibodies, Monoclonal, Murine-Derived* / immunology
  • Antibody Affinity*
  • Antineoplastic Agents, Immunological* / chemistry
  • Antineoplastic Agents, Immunological* / immunology
  • CHO Cells
  • Cricetulus
  • Female
  • Humans
  • Immunoglobulin Fc Fragments* / biosynthesis
  • Immunoglobulin Fc Fragments* / chemistry
  • Immunoglobulin Fc Fragments* / immunology
  • Immunoglobulin Fc Fragments* / isolation & purification
  • Membrane Glycoproteins* / biosynthesis
  • Membrane Glycoproteins* / chemistry
  • Membrane Glycoproteins* / immunology
  • Membrane Glycoproteins* / isolation & purification
  • Mice
  • Mice, Inbred BALB C
  • Protein Domains
  • Recombinant Fusion Proteins* / biosynthesis
  • Recombinant Fusion Proteins* / chemistry
  • Recombinant Fusion Proteins* / immunology
  • Recombinant Fusion Proteins* / isolation & purification

Substances

  • Antibodies, Monoclonal, Murine-Derived
  • Antineoplastic Agents, Immunological
  • Immunoglobulin Fc Fragments
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • trophoblastic glycoprotein 5T4, human