Citation

  • Authors: D'Onofrio A. et al.
  • Year: 2021
  • Journal: Eur J Pharm Biopharm 158 233-244
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: HEK-293 6E
    Description: Human embryonic kidney Fibroblast cell line genetically modified with a truncated version of EBNA1 which grows in suspension and chemically defined serum-free medium.

Method

- Recombinant proteins were produced using the mammalian HEK293-6E/pTT transient expression system. - HEK293-6E cells were grown in Freestyle F17 medium containing 4 mM GlutaMAX, 0.1% Pluronic® F-68 and 25 μg/mL G418 at 37 °C, 5% CO2 and 120 rpm. - For transfection, the vector DNA was mixed with FectoPRO (Polyplus) transfection reagent in F17 medium without supplements, according to the manufacturer’s instructions. - After five days of culture to allow protein expression, cultures were subjected to low speed centrifugation and the media collected. - Samples could be used immediately for direct capture and immobilization (dCI) experiments or snap-frozen and stored at −80 °C until required. - ScFv-Fc fusions were purified from clarified expression media using a HiTrap™ MabSelect column, followed by extensive dialysis against phosphate-buffered saline (PBS).

Abstract

The tumour endothelial marker 1 (TEM1/endosialin/CD248) is a receptor overexpressed in several human solid tumours and silenced in normal adult tissues, representing a suitable and potentially safe target for radioimmunotherapy of sarcoma. To develop new tools with improved TEM1 targeting properties, a new panel of antibody fragments was for the first time evaluated preclinically following 125I radiolabelling. The antibody fragment 1C1m-Fc, with the highest human/murine TEM1 binding affinity, was extensively characterized in vitro and in vivo in a Ewing's sarcoma human xenograft mouse model. In silico studies were also performed to elucidate the influence of a single amino acid mutation in the complementarity-determining region (CDR3) of the heavy chain, upon affinity maturation of the parental clone 1C1-Fc. From this study, 1C1m-Fc emerged as a promising candidate for the development of TEM1-targeted radioimmunoconjugates, namely to be further explored for theranostic applications with other suitable medical radionuclides.

Pubmed