Transmembrane Domain Protein Effects on Chimeric Antigen Receptor T Cell Cytotoxic and Proliferation Capabilities

Abstract

This thesis investigated the role of the transmembrane domain in modulating chimeric antigen receptor (CAR) internalization kinetics and its downstream effects on CAR-T cell expansion, exhaustion, and differentiation when combined with CAR-Enhancer (CAR-E) therapy. While CAR optimization has traditionally focused on the single-chain variable fragment, hinge, and costimulatory domains, the transmembrane domain has been largely overlooked as a design parameter. Two panels of CAR constructs were generated, each identical in all domains except the transmembrane region, which was derived from nine endogenous T cell surface proteins (CD28, 4-1BB, CD45, CD43, CD62L, CD27, CD30, CD3ε, and OX40). In vitro internalization assays demonstrated that transmembrane domain identity alone dramatically altered CAR internalization rates, with CD28 internalizing the slowest and OX40 the fastest. These internalization rankings were consistent across both experiments despite the use of structurally distinct CAR backbones. In vivo studies in immunodeficient mice revealed that slower-internalizing constructs consistently expanded more robustly and, in the tumor-bearing model, the CD28-transmembrane construct demonstrated the strongest antitumor response, with near-complete tumor elimination across all treated mice. Notably, despite driving the greatest expansion, the CD28 construct also exhibited the lowest exhaustion marker expression, suggesting that CAR-E-mediated crosstalk between the 4-1BB costimulatory and IL-2 receptor endodomains operates through a signaling axis with kinetic requirements distinct from conventional CAR signaling. Transmembrane domain identity also influenced tissue distribution and memory subset differentiation. These findings establish the transmembrane domain as a functionally significant and tunable parameter in CAR design, with direct implications for optimizing CAR-E-based immunotherapies.