Researchers at the University of Delaware have developed a novel bio-functional hydrogel-coated membrane (HCM) platform that significantly reduces T-cell exhaustion during CAR-T cell manufacturing while maintaining robust activation and expansion capabilities. The study, published in Frontiers in Immunology, demonstrates how engineered biomaterials can address critical challenges in CAR-T production scalability and quality control.
Addressing Manufacturing Challenges in CAR-T Therapy
CAR-T cell therapy has emerged as a pivotal treatment for hematological cancers, achieving remission rates of 40-90%. However, high variability in CAR-T function between donors and overall scalability issues have hindered broader adoption of this treatment paradigm. Current industry standards for T-cell activation, such as TransAct and Dynabeads, rely on synthetic particles that promote rapid activation but often lead to T-cell exhaustion, limiting therapeutic efficacy.
The research team engineered hydrogel-coated membranes using poly(ethylene glycol) diacrylate (PEGdiPDA) with physiologically-relevant mechanical properties and integrated biotin pendant groups for modular functionalization with co-stimulatory molecules. This platform enables precise control over the T-cell microenvironment during manufacturing.
Optimized Co-Stimulatory Molecule Combinations
The researchers investigated different combinations of co-stimulatory ligands: anti-CD3, anti-CD28, anti-4-1BB, and anti-OX40, maintaining a constant total concentration of 20 μg·mL⁻¹ across all conditions. The study focused on five specific combinations, with particular emphasis on the "All 3:1" formulation containing anti-CD3 (10 μg·mL⁻¹) and equal amounts of anti-CD28, anti-4-1BB, and anti-OX40 (3.33 μg·mL⁻¹ each).
Primary human CD3+ T cells from four donors were activated using the HCM platform and compared against TransAct controls over a 9-day manufacturing timeline. Flow cytometry analysis revealed that HCM-activated T cells exhibited significantly lower exhaustion markers, particularly Tim3 expression, on both day 3 and day 9 compared to TransAct-activated cells.
Enhanced T-Cell Phenotype and Function
The HCM platform promoted a gradual shift from naïve T cells to predominantly central memory (Tcm) phenotypes, which are associated with superior antitumor effects and proliferation compared to effector memory (Tem) phenotypes. On day 9, all HCM conditions showed trends toward increased Tcm and decreased Tem populations compared to TransAct controls.
Transduction experiments using both model GFP lentivirus and clinically relevant CD19-CAR lentivirus demonstrated enhanced gene transfer efficiency with HCM-activated T cells. The All 3:1 combination achieved 3-4-fold increases in transduction for multiple donors, attributed to the increased presence of naïve T cells and reduced exhaustion profiles.
Superior Killing Efficacy Against Cancer Cells
The functional superiority of HCM-produced CAR-T cells was validated through cytolysis assays using Raji-Luc2 leukemia cells as targets. At a 3:1 effector-to-target ratio, CAR-T cells activated with the All 3:1 HCM combination achieved 28% cytolysis at 72 hours compared to 19% for TransAct-activated cells, representing a statistically significant improvement in killing potential.
The enhanced performance correlated with the lower exhaustion profiles and improved memory phenotypes observed throughout the manufacturing process. CAR-T cells produced using HCMs also demonstrated greater consistency between donors, addressing variability concerns in current manufacturing approaches.
Scalable Manufacturing Platform
The hydrogel platform was designed for integration into tangential flow filtration devices, enabling scalable production while maintaining the benefits of controlled T-cell activation. The flat-sheet regenerated cellulose membrane base allows for future incorporation into flow-based manufacturing systems that could combine co-stimulation effects with enhanced viral concentration through flow dynamics.
Stability testing confirmed that the hydrogel-coated membranes maintained their functional properties over the 9-day manufacturing timeline under physiologically relevant conditions, supporting their practical application in industrial CAR-T production workflows.
Clinical Translation Potential
The modular nature of the HCM platform allows for personalized optimization of T-cell activation based on individual donor characteristics. The researchers noted that the approach could be particularly valuable for producing CAR-T therapies targeting solid tumors, where enhanced persistence and reduced exhaustion are critical for therapeutic success.
While the current study utilized cells from healthy donors, the researchers acknowledged that future investigations with cells from leukemia patients will be necessary to fully validate the platform's clinical potential, as significant differences in cell behavior have been reported between healthy and diseased populations.
The bio-inspired hydrogel platform represents a significant advancement in CAR-T manufacturing technology, offering improved control over T-cell quality while maintaining scalability requirements for clinical production. The combination of reduced exhaustion, enhanced memory phenotypes, and superior killing efficacy positions this approach as a promising solution for next-generation CAR-T manufacturing.
