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CAR-T cells and other engineered T cells are designed to recognize and attack specific targets, such as proteins found on cancer cells. Finding T cells that respond strongly to the right target is an important part of developing effective cell therapies, but laboratory screening methods do not always reproduce the complex way T cells interact with real cells.
Researchers at the California Institute of Technology (Caltech), working with collaborators at UCLA, investigated whether engineered vesicles could make these screening systems more realistic.
Using nanovials to screen T cells
The researchers focused on nanovials, tiny hydrogel particles containing microscopic cavities that can capture individual cells. Nanovials can be coated with antigens, molecules recognized by T cells, allowing researchers to screen large numbers of cells and measure how they respond.
Traditionally, these surfaces can be coated with purified proteins or antigen complexes. While useful, isolated proteins do not necessarily behave the same way as proteins embedded within the membrane of a living cell. T cells normally recognize targets through a complex contact point called the immunological synapse.
The researchers developed a way to make the surface of nanovials more closely resemble this natural environment.
Engineered extracellular vesicles improve single-cell capture and T cell activation of hydrogel nanovials
(A) Top: schematic of a plasmid construct containing a membrane protein fused to a Gly3Ser spacer, and an EABR vesicle-forming sequence. Bottom: schematic of membrane-bound cell-surface protein with EABR appended on the cytoplasmic tails induce budding of a vesicle composed of a lipid bilayer with embedded membrane proteins that contain the EABR sequence. (B) Schematic showing production and purification of the engineered EABR-mediated extracellular vesicles. (C) Postfabrication biotinylation of hydrogel nanovials via gelatin are then functionalized with streptavidin, followed by biotinylated capture antibodies. Capture antibodies can be targeted to any protein present on the vesicle surface, including the EABR- tagged membrane protein that induces vesicle formation. Cells, such as CAR-T cells, bind to the captured extracellular vesicles within the nanovial cavity, and cytokines released by activated cells are captured using the biotinylated capture antibodies covering the nanovial surface. (D) Fluorescent sandwich antibodies detect cytokines released by activated cells that are captured within the nanovial to enable single-cell measurements of both cell capture and cell activation by flow cytometry. Measurement of cell activation is gated based on the loaded nanovials containing Calcein AM+ cells.
Using vesicles to mimic cell surfaces
The team generated antigen-presenting vesicles and cancer-mimicking exosomes using cultured human cells. An engineered sequence called an ESCRT- and ALIX-binding region, or EABR, encouraged cells to release vesicles displaying selected proteins.
Importantly, the proteins remained embedded in a lipid membrane, similar to how they would be presented on the surface of a real antigen-presenting or cancer cell. The researchers then attached these vesicles to the cavities of hydrogel nanovials.
This approach allowed the nanovials to present full-length membrane proteins in a more natural cellular environment instead of relying solely on purified proteins attached directly to the hydrogel.
Improving T cell capture and activation
When the researchers tested the vesicle-coated nanovials, they found that they captured 1G4 T cells more effectively than nanovials coated with conventional recombinant proteins.
They also tested HER2 CAR-T cells, which are engineered to recognize the HER2 protein found at high levels in some cancers. Nanovials coated with cancer-mimicking exosomes produced stronger T cell activation, measured through increased secretion, than surfaces containing soluble recombinant proteins.
These results suggest that the physical environment in which an antigen is presented can influence how effectively therapeutic T cells recognize and respond to it.
A more realistic approach to T cell screening
High-throughput screening is valuable for identifying promising T cell receptors and CAR-T cell designs from large numbers of candidates. Making these screening systems more closely resemble interactions with actual cells could help researchers identify T cells based on functional responses rather than antigen binding alone.
Cancer-mimicking exosomes and antigen-presenting vesicles provide a way to bring natural membrane structures into nanovial-based screening. By combining engineered extracellular vesicles with high-throughput cell analysis, the approach could support the development and evaluation of more effective T cell therapies.
