Engineering single-vector logic-gated CAR T cells with transgene sizes beyond current limitations.

Area of Science:

• Immunotherapy
• Molecular Engineering
• Cellular Engineering

Background:

• Chimeric antigen receptor (CAR) T cells engineered with synthetic Notch (synNotch) receptors enhance specificity and reduce toxicity.
• Conventional dual-vector synNotch-CAR systems limit clinical translation due to dual transduction and cell sorting requirements.
• Manufacturing challenges with large transgenes in single-vector systems hinder CAR T cell therapy development.

Purpose of the Study:

• To engineer a single-vector synNotch (svsNotch) system integrating all components into one lentiviral vector.
• To develop an optimized CAR T cell production workflow for large lentiviral transgenes.
• To overcome limitations in CAR T cell manufacturing for clinical translation.

Main Methods:

• Engineered a single-vector synNotch (svsNotch) system integrating synNotch receptor and inducible CAR.
• Established an optimized CAR T cell production workflow to enhance transduction efficiency for large transgenes.
• Constructed HER2-MSLN svsNotch and HER2-MSLN-CBG svsNotch constructs for ovarian cancer targeting and in vivo monitoring.

Main Results:

• The optimized workflow increased T cell transduction rates by up to 14.8-fold, enabling production of CAR T cells with transgenes exceeding 9.2 kb.
• HER2-MSLN svsNotch T cells showed superior specificity, selectively killing HER2+MSLN+ tumor cells.
• In vivo studies demonstrated superior efficacy of HER2-MSLN-CBG svsNotch T cells against HER2lowMSLNhigh and HER2highMSLNhigh tumors.

Conclusions:

• Established a framework for single-vector logic-gated immunotherapies.
• Provided an optimized workflow for generating CAR T cells with transgenes exceeding current size limitations.
• Demonstrated the potential of svsNotch CAR T cells for targeted cancer therapy.