• Authors: Fierle JK. et al.
  • Year: 2021
  • Journal: Cell Rep Med 2 100362
  • 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.


Recombinant protein was produced using the HEK293-6E/pTT expression system and FectoPRO transfection protocol. ScFv-Fc fusions were purified from clarified expression media using a HiTrap MabSelect column, followed by extensive dialysis against PBS. TriloBiTE constructs were purified from clarified expression media by immobilized metal ion affinity chromatography (IMAC) using a HisTrap excel column.


Tumor endothelial marker 1 (TEM1) is an emerging cancer target with a unique dual expression profile. First, TEM1 is expressed in the stroma and neo-vasculature of many human carcinomas but is largely absent from healthy adult tissues. Second, TEM1 is expressed by tumor cells of mesenchymal origin, notably sarcoma. Here, we present two fully human anti-TEM1 single-chain variable fragment (scFv) reagents, namely, 1C1m and 7G22, that recognize distinct regions of the extracellular domain and possess substantially different affinities. In contrast to other, well-described anti-TEM1 binders, these fragments confer cytolytic activity when expressed as 2nd generation chimeric antigen receptors (CARs). Moreover, both molecules selectively redirect human T cell effector functions toward TEM1+ tumor cells when incorporated into experimental soluble bispecific trivalent engagers that we term TriloBiTEs (tBs). Furthermore, systemic delivery of 1C1m-tB prevents the establishment of Ewing sarcoma tumors in a xenograft model. Our observations confirm TEM1 as a promising target for cancer immunotherapy and illustrate the prospective translational potential of certain scFv-based reagents.