Development of antibody-targeted concepts for immunomodulation, biomarker imaging and enzymatic evaluation of bioresponsive pseudoglucosinolates

Abstract

Nature’s vast biodiversity and its complex biological systems have long been an inspirational source for drug development. A wealth of potent, life-saving substances has been found in nature and learning from nature’s design of functionality has revolutionized significant advancements in drug development. For instance, the evolution of antibodies from targeting specific antigens to recognizing precise biomarkers has refined diagnostic and therapeutic capabilities. Nature’s intermediates, like those in riboflavin biosynthesis activating MAIT cells, inspire the creation of artificial immunomodulators, enhancing our ability to regulate immune responses. Additionally, the transition from natural glucosinolates (GSL) releasing reactive isothiocyanates (ITCs) to synthetic pseudoglucosinolates (psGSLs) as ITC-prodrugs exemplifies the potential of nature-inspired innovations in creating potent treatments. By emulating these natural mechanisms, we can develop more effective and targeted medical therapies. Advancements in the detection and analysis of pathological biomarkers are pivotal for enhancing diagnostic accuracy and therapeutic development. This PhD thesis presents a comprehensive investigation into the synthesis and evaluation of novel antibody-targeted mass peptide conjugates as MALDI-MS-imaging (MALDI-MSI) probes, the development of immunomodulators targeting Mucosal Associated Invariant T (MAIT) cells, and the evaluation of enzyme-responsive compounds as potential prodrugs. The overarching aim is to improve the detection, visualization, and modulation of biomarkers in biological samples, thereby contributing to the fields of diagnostics and targeted therapeutics. Chapter 1 explores the development of a method for multiplex detection of pathological biomarkers in disease context. The study presents the development of antibody-targeted MALDI-MSI and fluorescence imaging probes for multiplexed, selective imaging of matrix metalloproteinases (MMPs), particularly MMP-7, in tissue samples. The probes were evaluated for their photocleavability and applied in fluorescence microscopy and MALDI-MSI of murine kidney slices, demonstrating enhanced detection and spatial distribution visualization of pathological biomarkers. Chapter 2 focuses on immunomodulation, the role of MAIT cells and the potential of artificial MAIT cell modulating ligands for clinical applications. The research introduces the design and synthesis of artificial MAIT cell activating and inhibiting ligands, aiming to bypass the instability of natural ligands, such as 5 OP RU. Biological evaluations of these synthetic ligands showed promising results in modulating MAIT cell activity. Additionally, the concept of Antibody-Immunomodulator-Conjugates (AIC) was developed, proposing a novel therapeutic strategy that combines an antibody-targeted approach to immunomodulation. Chapter 3 investigates enzyme-responsive pseudoglucosinolates (psGSLs) and the corresponding in situ formed isothiocyanates (ITCs) for their potential in targeted drug delivery as a prodrug. psGSLs were synthesized and evaluated for their responsiveness to enzymes, such as nitroreductase (NTR) and azoreductase (AzoR). The study developed enzymatic assays to assess ITC formation, confirming the efficacy of these compounds in releasing active payloads in response to specific enzymatic activity. The findings underscore the potential of enzyme-responsive systems in creating targeted and controlled therapeutic interventions. The thesis demonstrates the synthesis and application of novel antibody-peptide conjugates for enhanced imaging of pathological biomarkers, the development of synthetic immunomodulators targeting MAIT cells, and the development of enzymatic assays to investigate enzyme-responsive compounds for their controlled ITC release. Future research will focus on refining these techniques, scaling up for in vivo experiments, and exploring additional biomarkers and therapeutic targets. The integration of these innovations holds significant promise for advancing diagnostic precision and personalized medicine.