Combokines: Platform for Cytokine Delivery in aAPC Engineering 2252933

Abstract

CAR-T therapy is promising, but poor T cell expansion causes up to 13% of patients to drop out. Traditional bead-based activation fails to present physiological signals, whereas artificial antigen presenting cells (aAPCs) provide a superior alternative to present membrane-bound proteins for antigen recognition, costimulation, and cytokine stimulation. aAPC engineering is constrained by lentiviral packaging limits and reduced viability after repeated transduction, which restricts the number of introduced signals. To overcome this, a membrane-bound cytokine complex, termed “Combokine”, was developed to present two cytokines simultaneously. Combinations of membrane-bound IL—7/IL-21, and IL-18/IL-21 constructs were tested. K562 cell lines were transduced with lentivirus, single-cell sorted, and validated with flow cytometry. IL-7 and IL-21 activity was confirmed by STAT3/5 phosphorylation using intracellular staining followed by flow cytometry. IL-18 activity was confirmed via NF-kB signaling detected with HEK-Blue IL-18 Reporter cells. IL-7/IL-21 Combokines induced STAT3/5 phosphorylation. IL-18/IL-21 Combokines induced STAT3 phosphorylation and activated NF-kB signaling. Combokines with IL-21 in the second position produced greater STAT3 phosphorylation. Similarly, IL-18/IL-21 Combokines with IL-18 in the second position produced stronger NF-kB signaling. Combokine-expressing cell lines induced STAT3/5 phosphorylation and NF-kB signaling comparable to cell lines dual-transduced with IL-7/IL-21 and IL-18/IL-21. These findings establish Combokines as a platform to enhance T cell proliferation by integrating multiple cytokine stimulation pathways into a single construct. Future work will evaluate consequences on CAR-T cell proliferation and function. The Roy and Diana Vagelos Program in the Molecular Life Sciences Cytokines and Chemokines and their Receptors (CCR)