Production of cell therapies: Integration of co-stimulatory ligands with hydrogel coated membranes to activate and transduce T cells

Introduction: Cell therapies have emerged as a critical tool to treat cancer. In particular chimeric antigen receptor (CAR) T cell therapies have become a fundamental treatment for hematological cancers with a remission rate of 60%-90%. However, the cost of the therapy ($350k up to 450k per injection) limits accessibility for patient. The cost of CAR T is directly related to the complex manufacturing process, which is a time-consuming, multi-step procedure that requires isolation from the patient, activation, transduction, expansion, and autologous infusion. Consequently, there is a need to develop efficient and scalable processes to engineer T-cells in a cost-effective manner. Hydrogel coated membranes (HCM) are tunable biomaterials that can be functionalized with a wide variety of ligands to achieve T-cell activation. Additionally, the use of tangential flow filtration (TFF) can improve transduction efficiency with a CAR. In this work, a combination of HCM and TFF was studied to improve T-cell activation and transduction (Fig 1 A,B).

Materials and

Methods: HCMs were functionalized using an avidin-biotin chemistry. First, surface of HCMs were blocked with 1% BSA solution for 1h at room temperature. After blocking, BSA solution was removed and 300L of 50 g·mL-1 Neutravidin solution was added (NA; ThermoScientific, Waltham, MA) letting it incubate for 4 h at room temperature. Afterwards, HCMs were washed with PBS twice for 15 min. The biotinylated cell activating antibodies were added to the HCM surface and incubated at 4 °C overnight at different concentrations. Activation of CD3+ T-cells was studied after 1, 3 and 9 days of incubation with HCMs or TransAct. Transduction experiments were performed after 3 days activation on a 96-well plate with HCMs or TransAct. Flow cytometry was used to determine presence of key markers for activation and transduction.

Results, Conclusions, and Discussions: Activation of T cells is a fundamental step to achieve proliferative phenotypes on T cells. Hence, in this in this work, four different co-stimulatory ligands have been functionalized on the surface of HCMs to study their activation potential. Phenotype, activation, and exhaustion markers were used to compare with industry standard TransActTM, finding memory phenotype, minimal exhaustion, and similar activation profiles with HCMs (Fig 1 C, E, F). Due to the increase in memory phenotypes, transduction experiments with a model lentivirus were performed with the same combinations of co-stimulatory molecules, where an increase in T cell transduction was observed (Fig 1 D). Lastly, the use of the TFF device was examined with primary CD3+ T-cells and two different combinations of co-stimulatory molecules to enhance transduction.This work presents significant opportunities to modulate T-cells phenotypes for cell therapy applications by using engineered soft materials furthermore the combination of HCMs and TFF has the potential to streamline the CAR-T manufacturing process.

Acknowledgements (Optional): This project was developed with an award from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) and financial assistance from the U.S. Department of Commerce, National Institute of Standards and Technology (70NANB21H086).