Synthesis of antibody conjugated polymeric nanoparticles for controlled activation, priming and tracking of T cells in the context of cancer immunotherapy

Cancer is one of the most fatal diseases affecting 40% of men and women during their life span. Conventional treatments such as surgery, radiation, and chemotherapy suffer from incomplete removal of the malignant tissue, thus reoccurrence of the tumor and metastasis are the main reason for cancer-related death causes. The development of cancer involves a complex interplay between genetic alterations and the evasion of the immune system, and in this regard, immunotherapy emerges as a promising approach by shaping the patients' immune system to detect and fight cancer again. Adoptive cell transfer (ACT) is a type of immunotherapy that involves the isolation, differentiation, expansion, and reinfusion of cancer-specific cytotoxic lymphocytes into the patient, and in recent years has shown promising results in pre-clinical and clinical studies. Improved antitumor responses have been linked to the differentiation state of the transferred T cells, highlighting the importance of the methods used to obtain large quantities of functional T cells with antigen specificity but also with a long life span in form of a young phenotype. In addition, after infusion into the patient, upon engagement with the tumor, T cells have to overcome the immunesuppressing tumor environment, which often impairs the therapeutic outcome. Ways to overcome this problem include the use of immune-checkpoint inhibitors, whose therapeutic effect can block anti-inflammatory ligands in the tumor microenvironment and untie the physiological break for T cells. This effect can further be enhanced with pro-inflammatory cytokines that enable full effector functions of the T cell. However, due to the cytokines’ low serum half-life, therapies require high dosing, which often comes with severe side effects due to their high potency. Lastly, the therapeutic evaluation of ACT often comes with weeks of delay after the initial treatment, putting pressure on critical patients awaiting a curative response. Therefore, it is essential to develop precise imagebased tools for monitoring the biodistribution of the transferred cells to predict ACT outcomes.
The use of nanoparticles for medicinal purposes can improve immunotherapy due to their ability to carry either conjugated or encapsulated biologically active molecules. Biodegradable polymeric systems are made of versatile biocompatible materials that allow precise engineering of the particle properties in regards to size, shape, charge, and conjugation of targeting ligands that can further improve the interaction with the biological environment. Particularly, antibodies show unique properties in guiding the carrier towards its target and some can further induce therapeutic effects. Importantly, orientation and density, which is dictated by the conjugation chemistry used, of the antibodies present in the particles' surface strongly influence their targeting and therapeutic outcome. Due to their flexibility, polymeric nanoparticles can encapsulate, protect, and sustainably release either hydrophobic or hydrophilic molecules, making them ideal candidates for the delivery of protein drugs. Nonetheless, particles usually suffer from an initial burst release caused by concentration gradients inside the particles, but to address this, several possibilities are available such as coformulation of stabilizing agents, thus reducing the initial high loss of the active pharmaceutical ingredient.
In this thesis, different efforts were made to improve ACT at their key hurdles of expansion, priming, and tracking of T cells. In Chapters 3 and 4, a poly-lactideglycolic-acid (PLGA) based particle system was developed to investigate the influence of antibody orientation and density on T cell activation, expansion and differentiation stage (phenotype). To do so, different fundamental parameters on the system were fine-tuned, such as antibody conjugation chemistry, polymer length, PEGylation degree, co-formulation of stabilizers. It was demonstrated that the particles required the anti-fouling polymer PEG to prevent spontaneous physical adsorption of the antibody during the coupling reaction. Control of the antibody orientation through conjugation chemistry used can be linked to the expansion levels and final phenotype of the expanded T cells. In addition, Ca²⁺ sensing has shown to be a meaningful tool to explore the functional state of the antibody conjugated to nanoparticles. We show that random immobilized antibodies require higher ligand densities to perform equally well to their oriented counterparts, and thus underline the importance of the antibody orientation on T cell activating nanoparticles. In chapter 5, knowledge gained during previous chapters was used to develop a polymeric particle system loaded with cytokines and an immune-checkpoint inhibitor to help T cells overcome the immune-suppressing tumor microenvironment. This was achieved by conjugating anti-PD-L1 antibodies to the surface of a particle system encapsulating Interleukin-2 (IL-2). We show that the particles were able to specifically bind PD-L1 receptors on CT26 and B16.OVA cancer cells in an expression-dependent manner. In addition, the initial burst release of the encapsulated IL-2 could be prolonged by 17 hours, showing sustained biological activity with expanding OVA-specific T cells in vitro over 5 days. Lastly, it was demonstrated that PD-L1 targeting and IL-2 encapsulating particles enhanced the killing properties of OVA-specific T cells in different phenotypic states in vitro, validating the system for future in vivo testing. Chapter 6 focuses on providing a solution for the in vivo T cell tracking dilemma. We present an approach for labeling T cells with giant shell quantum dots (GSQDs) for uptake and trafficking studies. In addition, an automated segmentation script was developed that allowed evaluation of the uptake efficiency of GSQDs into T cells in high cell content samples. We show that highly cationic particles were incorporated into Jurkat and primary T cells, however, also unveiling toxic side effects. These findings led to the design of antibody-conjugated GSQDs to target endocytotic receptors on the surface of T cells. While further experiments are needed to determine the influence of ligand density and particle size on T cell uptake, this approach highlights the potential labeling capacity of GSQDs for T cell tracking purposes.
Overall, the work presented in this Ph.D. thesis covers the impact of antibody-conjugated nanoparticles on their potential as artificial antigen-presenting cells, checkpoint inhibiting T cell boosters, and labeling agents for T cell tracking. The developed systems have shown promising results, setting the basis for future exploration using particles with well-oriented antibodies for T cell activation, priming, and tracking purposes in the context of cancer immunotherapy.