• Authors: Enderle L. et al.
  • Year: 2021
  • Journal: MAbs 13 1933690
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi


IgGs, DATEs, and d-DATEs were produced using the human Expi293 expression system. Expi293F cells were maintained in Expi293 expression medium on orbital shaking platforms (25 mm, 125 rpm) at 37°C and 8% CO2. For protein production, Expi293 cells were grown to a density of approximately 2.5 × 106 cells/mL and heavy- and light-chain DNA was transfected at 1:1 ratios using FectoPro transfection reagent (Polyplus Transfection #116-010). After 5 d the cell supernatant was harvested by centrifugation, incubated with rProteinA Sepharose FastFlow overnight, and collected in Polyprep columns for purification.


In order to direct T cells to specific features of solid cancer cells, we engineered a bispecific antibody format, named Dual Antigen T cell Engager (DATE), by fusing a single-chain variable fragment targeting CD3 to a tumor-targeting antigen-binding fragment. In this format, multiple novel paratopes against different tumor antigens were able to recruit T-cell cytotoxicity to tumor cells in vitro and in an in vivo pancreatic ductal adenocarcinoma xenograft model. Since unique surface antigens in solid tumors are limited, in order to enhance selectivity, we further engineered "double-DATEs" targeting two tumor antigens simultaneously. The double-DATE contains an additional autonomous variable heavy-chain domain, which binds a second tumor antigen without itself eliciting a cytotoxic response. This novel modality provides a strategy to enhance the selectivity of immune redirection through binary targeting of native tumor antigens. The modularity and use of a common, stable human framework for all components enables a pipeline approach to rapidly develop a broad repertoire of tailored DATEs and double-DATEs with favorable biophysical properties and high potencies and selectivities.