Bio-functional hydrogel coated membranes to decrease T-cell exhaustion in manufacturing of CAR T-cells

Aida López Ruiz¹Eric Slaughter¹Kartik Bomb¹Paige LeValley¹Zaining Yun¹Jacob McCoskey²Kara Levine²Jonathan Steen²Joseph Almasian²Aparajita Chatterjee²Christina Carbrello²Dustin Chang³Aida Fuseini³Yama Abassi³Abraham Lenhoff³Catherine Fromen¹April Kloxin^1*

• ¹University of Delaware, Newark, Delaware, United States
• ²Millipore Sigma, St. Louis, Missouri, United States
• ³Agilent Technologies (United States), Santa Clara, California, United States

Cell therapies have revolutionized cancer treatment, with chimeric antigen receptor (CAR) T-cell therapies at the forefront for the treatment of hematological cancers. However, current manufacturing protocols rely on rapid T-cell activation, which can induce exhaustion and undesirable phenotypes, ultimately reducing the efficacy and persistence of CAR T-cells. Given the importance of T-cell activation as a fundamental step to achieve proliferative phenotypes for cell engineering and expansion, approaches are needed to control activation and increase CAR T-cell quality. To address this need, in this work we utilized a bioinspired, scalable, tunable platform to direct T-cell activation and decrease exhaustion during CAR T production. Specifically, hydrogel-coated membranes (HCMs) were designed with different co-stimulatory ligands and a physiologically-relevant substrate modulus inspired by the native microenvironment in which s are programmed. With this controlled and well-defined system, we hypothesized that a combination of ligands inspired by antigen-presenting cells would promote desired T-cell phenotypes with reduced exhaustion and thereby improved killing efficacy. Phenotype, activation, and exhaustion markers were used to compare T cells cultured with HCMs or industry standard TransAct, finding memory phenotype, minimal exhaustion, and similar activation profiles with HCMs. Next, transduction with a CD19 CAR lentivirus was performed, where increased T-cell transduction and decreased exhaustion for the CAR T population were observed with HCMs. Further, the killing potential of the resulting CAR T product was evaluated, finding improved in vitro cytolysis of target cells with lower variability with HCMs. These results demonstrate the importance of lower T-cell exhaustion in CAR T manufacturing and present significant opportunities to modulate T-cell phenotypes for cell therapy applications using engineered bioinspired materials that display combinations of co-stimulatory molecules.