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

• Published in: Molecular therapy. Methods & clinical development Vol. 16; pp. 238 - 254
• Main Authors: Bloemberg, Darin, Nguyen, Tina, MacLean, Susanne, Zafer, Ahmed, Gadoury, Christine, Gurnani, Komal, Chattopadhyay, Anindita, Ash, Josée, Lippens, Julie, Harcus, Doreen, Pagé, Martine, Fortin, Annie, Pon, Robert A., Gilbert, Rénald, Marcil, Anne, Weeratna, Risini D., McComb, Scott
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.03.2020; American Society of Gene & Cell Therapy; Elsevier
• Subjects: CAR-T; CD69; EGFRvIII; high-throughput; Jurkat; live imaging; plasmid; pre-clinical; screening; T cell; live imaging; screening; CAR-T; T cell; high-throughput; plasmid; EGFRvIII; CD69; Jurkat; pre-clinical
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2020.01.012

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. [Display omitted]