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Development of CAR Constructs for Human Solid Tumors

This study aims to design and optimize the expression of second-generation CAR constructs targeting solid tumors. Antibody sequences recognizing representative solid tumor antigens such as EGFRvIII and IL13Rα2 were identified and used to generate scFvs. These scFvs were incorporated into CAR constructs consisting of the human CD8 hinge and transmembrane regions, along with the intracellular signaling domains of 4-1BB and CD3ζ. The resulting CAR proteins are introduced into immune cells through various delivery approaches to generate solid tumor–specific cellular therapeutics. As these engineered cells exhibit antigen-specific cytotoxicity, the second-generation CAR constructs developed in this study are intended to serve as candidate cellular therapeutics for the treatment of solid tumors.

Kim Byung-Soo

Seoul National University

Department of Chemical Engineering

South Korea

1 Gwanak-ro

Gwanak-gu

Seoul

8826

Unpublished

No

No

human CD8, 41BB, CD3 zeta

EGFR epidermal growth factor receptor [ Homo sapiens (human) ]

EGFRvIII-41BBz-T2A-LNGFR

lentiviral expression plasmid for anti-EGFRvIII-CAR in human cells

We designed a second-generation CAR construct for a project aimed at developing CAR-T cells targeting the human EGFRvIII protein. The extracellular domain was constructed by fusing an EGFRvIII-specific scFv with the human CD8 hinge and CD8 transmembrane regions. For the intracellular signaling domains, the human 4-1BB and CD3ζ sequences were sequentially incorporated. To facilitate detection of CAR protein expression, a reporter protein (LNGFR) was linked to the CAR construct via a T2A cleavage sequence, enabling the design of a single ORF. Each component was amplified by PCR, and the sequences were assembled into a continuous ORF using the Gibson assembly system. The assembled ORF was subsequently amplified by PCR and cloned into the pELPs lentiviral expression vector to generate the final plasmid.

None

8954

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