Molecular Therapy: Methods & Clinical Development (Mar 2020)

A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells

  • Darin Bloemberg,
  • Tina Nguyen,
  • Susanne MacLean,
  • Ahmed Zafer,
  • Christine Gadoury,
  • Komal Gurnani,
  • Anindita Chattopadhyay,
  • Josée Ash,
  • Julie Lippens,
  • Doreen Harcus,
  • Martine Pagé,
  • Annie Fortin,
  • Robert A. Pon,
  • Rénald Gilbert,
  • Anne Marcil,
  • Risini D. Weeratna,
  • Scott McComb

Journal volume & issue
Vol. 16
pp. 238 – 254

Abstract

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Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, short-term co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced target-independent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization. Keywords: CAR-T, screening, pre-clinical, plasmid, EGFRvIII, high-throughput, CD69, live imaging, Jurkat, T cell