title: Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors.
abstract: BACKGROUND
  Adoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain.
  
  
  METHODS
  We prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity.
  
  
  RESULTS
  The DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs.
  
  
  CONCLUSIONS
  These findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.
authors.affiliation: Department Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON Canada.
authors.forename: Joanne A
authors.initials: JA
authors.lastname: Hammill
authors.affiliation: Department Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON Canada.
authors.forename: Heather
authors.initials: H
authors.lastname: VanSeggelen
authors.affiliation: Department Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON Canada.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Helsen
authors.affiliation: Department Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON Canada.
authors.forename: Galina F
authors.initials: GF
authors.lastname: Denisova
authors.affiliation: Department Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON Canada.
authors.forename: Carole
authors.initials: C
authors.lastname: Evelegh
authors.affiliation: Department Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON Canada.
authors.forename: Daniela G M
authors.initials: DG
authors.lastname: Tantalo
authors.affiliation: Department Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON Canada.
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Bassett
authors.affiliation: Department Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON Canada.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Bramson
keywords: CAR; Cancer; Chimeric antigen receptor; DARPin; Designed ankyrin repeat protein; Immunotherapy