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J Vis Exp ; (200)2023 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-37955379

RESUMO

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.


Assuntos
Receptores de Antígenos Quiméricos , Anticorpos de Cadeia Única , Humanos , Receptores de Antígenos Quiméricos/genética , Leucócitos Mononucleares , Linhagem Celular Tumoral , Células Jurkat , Anticorpos de Cadeia Única/genética , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/genética
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