A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells.
CAR-T
CD69
EGFRvIII
Jurkat
T cell
high-throughput
live imaging
plasmid
pre-clinical
screening
Journal
Molecular therapy. Methods & clinical development
ISSN: 2329-0501
Titre abrégé: Mol Ther Methods Clin Dev
Pays: United States
ID NLM: 101624857
Informations de publication
Date de publication:
13 Mar 2020
13 Mar 2020
Historique:
received:
06
12
2019
accepted:
27
01
2020
entrez:
22
2
2020
pubmed:
23
2
2020
medline:
23
2
2020
Statut:
epublish
Résumé
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
Identifiants
pubmed: 32083149
doi: 10.1016/j.omtm.2020.01.012
pii: S2329-0501(20)30023-1
pmc: PMC7021643
doi:
Types de publication
Journal Article
Langues
eng
Pagination
238-254Informations de copyright
Crown Copyright © 2020.
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