Surface Engineering of T Cells for Improved Adoptive Cell Therapy

Abstract

T cells, one of the most abundant immune cell types in the human body, play a central role in diverse pathophysiologies and currently dominate the clinical landscape of cell therapies. Their inherent target specificity and ability to induce durable therapeutic responses position them as a powerful modality for precision therapy. Today, T-cell therapies represent a leading frontier in cellular medicine, with extensive ongoing efforts to fully harness their therapeutic potential. Their applications span a broad spectrum of indications, from cancer to autoimmune diseases. In my dissertation, I begin with a detailed analysis of this clinical landscape of adoptive T-cell therapies. The historical milestones that have shaped the evolution of T cells into transformative therapies are outlined, followed by a comprehensive analysis of their clinical translation. This work offers a quantitative and contextualized view of the progress made in T-cell therapy to date. The analysis highlights that the majority of T-cell therapeutic applications seek to leverage their cytotoxic capabilities to eliminate malignant cells, particularly in the context of cancer treatment. Remarkable therapeutic responses observed in hematological malignancies following adoptive cell transfer (ACT) of cytotoxic T lymphocytes (CTLs), specifically CAR T cells, have been pivotal in driving the clinical translation of T-cell therapies. However, despite promising advancements over the past decade, their application to solid tumors has remained relatively limited due to a range of biologically unique challenges. Studies in newly established tumor models have shown that naturally occurring antitumor CTLs gradually lose control over tumor growth due to the development of immune evasion mechanisms within the solid tumor microenvironment (sTME). This sTME, predominantly immunosuppressive in nature, similarly hinders adoptively transferred CTLs. To enhance the efficacy of ACT in solid tumors, there is growing interest in developing strategies that can sustain and reinforce CTL functionality post-ACT. Under physiological conditions, CTLs maintain their fitness and survival through tonic stimulation, a form of low-level signaling during routine immunosurveillance. In vitro studies have shown that steric exclusion of CD45-phosphatase can effectively initiate this form of tonic stimulation in CTLs. Building on these insights, my dissertation research presents a biomaterial-based strategy for surface engineering of CTLs via phosphatase exclusion to deliver tonic stimulation that locally reinforces their functionality post-ACT. This cell surface engineering platform uses phosphatase-excluding micropatches to drive tonic stimulation and sustain CTL functionality after infusion. First, the design of Polymeric Micropatches for CD45-Phosphatase Exclusion (PMPEs) is described, featuring a core composed of a polylactic-co-glycolide (PLGA) biopolymer. PMPEs bind efficiently to CTLs and induce micron-scale exclusion of CD45-phosphatase at the cell contact interface. This exclusion, validated through probabilistic mathematical modeling and experimental analysis, offers insights into the design features of PMPEs as an effective means for CTL stimulation. A comprehensive set of in vitro assays, including bulk RNA sequencing, calcium flux analysis, cytokine secretion profiling, immunofluorescence, and cytotoxicity assays, alongside in vivo studies, such as biodistribution analysis, immunophenotyping, and pathology assessment, demonstrate that PMPE-modified CTLs exhibit enhanced persistence, robust type 1 immuno-permissive responses, and excellent tolerability. In aggressive B16F10 melanoma and EG7 solid lymphoma mouse models, PMPE-modified CTLs significantly enhance tumor control and extend survival. Notably, the combination of PMPE-CTLs with systemic IL-15 superagonist (IL15sa) therapy in B16F10 melanoma-bearing mice results in 46% survival beyond 35 days, with 15.4% of mice achieving complete tumor remission, compared to none in the IL15sa + CTL group alone. PMPEs represent a simple yet highly promising biomaterial-based approach for sustaining functionality post-transfer and enhancing the therapeutic efficacy of CTLs in solid tumors.

The probabilistic mathematical model from the PMPE study indicates micron-scale close contact formation as the primary driver of tonic stimulation. Supporting this, experimental studies show that PMPEs functionalized with either anti-CD45 or anti-CD44 antibodies induced robust stimulation, whereas soluble antibodies or polymeric nanoparticles failed to do so. These findings highlight the material-independent and purely contact-driven nature of this reinforcement strategy, emphasizing its strong translational potential. To further substantiate this principle, Hydrogel Micropatches for Phosphatase Exclusion (HMPEs) based surface engineering was developed, using a hyaluronic acid (HA) hydrogel core, a material platform markedly distinct from PLGA-based PMPEs. HMPEs exhibited significantly greater hydrophilicity (87° smaller contact angle with water) and ~470-fold lower stiffness compared to PMPEs. Despite these differences, HMPEs engineered to establish firm micron-scale contacts with CTLs successfully promoted CD45 exclusion and reinforced CTL fitness. Altogether, these findings highlight the versatility of the contact-driven reinforcement strategy, laying the foundation for broadly applicable biomaterial interventions. This approach offers future opportunities to integrate drug-loading strategies, tune mechanical properties, and incorporate additional immunomodulatory molecules to further improve adoptive cell therapies across diverse clinical settings.