Multiplexed Aptamer-Lattice Nanodevice for One-Step Phenotyping and Functional Quality Control of CAR-T Cells

Area of Science:

• Biotechnology
• Immunotherapy
• Nanotechnology

Background:

• Chimeric antigen receptor T (CAR-T) cell therapy shows promise in cancer treatment.
• Cellular heterogeneity presents significant quality control challenges in CAR-T manufacturing.
• Existing methods for CAR-T cell analysis are often time-consuming and complex.

Purpose of the Study:

• To develop a novel nanodevice for rapid, multiplexed phenotypic and functional analysis of CAR-T cells.
• To establish a streamlined quality control method for engineered immune cells.
• To assess CAR-T cell activation, cytokine secretion, cytotoxic function, and donor variability.

Main Methods:

• Integration of aptamers targeting CD3, CD25, and CD122 into a DNA lattice scaffold (ALPHA).
• Development of a wash-free, orthogonal fluorescence activation system.
• Real-time monitoring of target cell lysis via detection of extracellular ATP.
• Correlation of the ALPHA-generated Activation Index with cytokine secretion and cytotoxic assays.

Main Results:

• ALPHA achieved one-step phenotypic and functional analysis of CAR-T cells within 30 minutes.
• The nanodevice accurately distinguished resting from activated T cells, generating a reliable Activation Index.
• The Activation Index strongly correlated with key functional parameters like cytokine secretion and cytotoxicity.
• ALPHA successfully captured donor-dependent functional variability in CAR-T cell samples.
• Real-time cytotoxicity monitoring was achieved through ATP detection from lysed target cells.

Conclusions:

• The Aptamer Lattice-Powered Hybrid Architecture (ALPHA) is a versatile and scalable tool for comprehensive CAR-T cell quality control.
• ALPHA provides rapid, accurate, and wash-free analysis, addressing key challenges in CAR-T manufacturing.
• This nanodevice facilitates efficient assessment of engineered immune cell function and consistency.