Improvement in the function of self-activating chimeric antigen receptor by replacing the linker sequence

Area of Science:

• Immunology
• Cancer Therapy
• Molecular Engineering

Background:

• Chimeric antigen receptor (CAR)-T cell therapy shows promise for hematological cancers but faces challenges in solid tumors, particularly controlling CAR-T cell exhaustion.
• Tonic signaling from CAR self-activation is linked to CAR-T cell exhaustion, with the extracellular CAR structure influencing signal magnitude.
• The specific role of the single-chain variable region (scFv) linker sequence in CAR tonic signaling and T cell function remains unclear.

Purpose of the Study:

• To investigate the impact of different scFv linker sequences (G4S vs. Whitlow/218) on the function of self-activating SKM-CAR T cells targeting malignant mesothelioma.
• To determine if linker variations affect tonic signaling, cytokine production, and anti-tumor activity.

Main Methods:

• Comparison of two scFv linkers (G4S and Whitlow/218) within a self-activating SKM-CAR construct targeting a mesothelioma-specific antigen (HEG1).
• Analysis of cell surface phenotype, NFAT/NFκB signaling, gene expression, cytokine production post-antigen stimulation, and in vitro/in vivo tumor cell killing.
• Utilized CAR-T cells engineered with the CD28 co-stimulatory domain.

Main Results:

• No significant differences were observed in cell surface phenotype, NFAT/NFκB signaling intensity, or gene expression profiles between SKM-CAR T cells with G4S and Whitlow/218 linkers.
• Switching to the Whitlow/218 linker significantly altered cytokine expression upon antigen stimulation compared to the G4S linker.
• The Whitlow/218 linker improved in vitro tumor cell killing activity but did not enhance in vivo tumor control.

Conclusions:

• The scFv linker sequence plays a role in modulating CAR-T cell function, specifically cytokine production and in vitro cytotoxicity.
• The Whitlow/218 linker demonstrates advantages over the G4S linker for certain CAR-T cell functions in the context of solid tumor targeting.
• Further research is needed to optimize linker design for improved in vivo efficacy in solid tumor CAR-T cell therapy.