Radiopharmaceuticals based on the highly potent FAP inhibitor (FAPi) UAMC-1110 have shown great potential in molecular imaging, but the short tumor retention time of the monomers do not match the physical half-lives of the important therapeutic radionuclides ¹⁷⁷Lu and ²²⁵Ac. This was improved with the dimer DOTAGA.(SA.FAPi)₂, but pharmacological and radiolabeling properties still need optimization. Therefore, the novel FAPi homodimers DO3A.Glu.(FAPi)₂ and DOTAGA.Glu.(FAPi)₂. were synthesized and quantitatively radiolabeled with ⁶⁸Ga, ⁹⁰Y, ¹⁷⁷Lu and ²²⁵Ac. The radiolabeled complexes showed high hydrophilicity and were generally stable in human serum (HS) and phosphate-buffered saline (PBS) at 37 °C over two half-lives, except for [²²⁵Ac]Ac-DOTAGA.Glu.(FAPi)₂ in PBS. In vitro affinity studies resulted in subnanomolar IC₅₀ values for FAP and high selectivity for FAP over the related proteases PREP and DPP4 for both compounds as well as for [^natLu]Lu-DOTAGA.Glu.(FAPi)₂. In a first proof-of-principle patient study (medullary thyroid cancer), [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ was compared to [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂. High uptake and long tumor retention was observed in both cases, but [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ significantly reduces uptake in non-target and critical organs (liver, colon). Overall, the novel FAPi homodimer DOTAGA.Glu.(FAPi)₂ showed improved radiolabeling in vitro and pharmacological properties in vivo compared to DOTAGA.(SA.FAPi)₂. [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ and [²²⁵Ac]Ac-DOTAGA.Glu.(FAPi)₂ appear promising for translational application in patients.

Crohn’s disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibrostenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate the mechanisms underlying fibrostenosis in CD, we analyzed the transcriptome of cells isolated from the transmural ileum of patients with CD, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from patients without CD. Our computational analysis revealed that profibrotic signals from a subset of monocyte-derived cells expressing CD150 induced a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) deposition. Genetic and pharmacological inhibition of TWIST1 prevents fibroblast activation, reducing ECM production and collagen deposition. Our findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibrostenosis in CD.

Cell surface proteases (also known as ectoproteases) are transmembrane and membrane-bound enzymes involved in various physiological and pathological processes. Several members, most notably dipeptidyl peptidase 4 (DPP4/CD26) and its related family member fibroblast activation protein (FAP), aminopeptidase N (APN/CD13), a disintegrin and metalloprotease 17 (ADAM17/TACE), and matrix metalloproteinases (MMPs) MMP2 and MMP9, are often overexpressed in cancers and have been associated with tumour dysfunction. With multifaceted actions, these ectoproteases have been validated as therapeutic targets for cancer. Numerous inhibitors have been developed to target these enzymes, attempting to control their enzymatic activity. Even though clinical trials with these compounds did not show the expected results in most cases, the field of ectoprotease inhibitors is growing. This review summarizes the current knowledge on this subject and highlights the recent development of more effective and selective drugs targeting ectoproteases among which small molecular weight inhibitors, peptide conjugates, prodrugs, or monoclonal antibodies (mAbs) and derivatives. These promising avenues have the potential to deliver novel therapeutic strategies in the treatment of cancers.

Human transmembrane serine protease 2 (TMPRSS2) has garnered substantial interest due to its clinical significance in various pathologies, notably its pivotal role in viral entry into host cells. The development of effective strategies to target TMPRSS2 is a current area of intense research and necessitates a consistent source of active TMPRSS2 with sufficient stability. Here, we comprehensively characterised human seminal‐fluid extracellular vesicles (SF‐EVs, also referred to as prostasomes), bearing a native source of surface‐exposed, enzymatically active TMPRSS2 as demonstrated by high‐sensitivity flow cytometry and a fluorometric activity assay. Additionally, we recombinantly produced human TMPRSS2 ectodomain in mammalian cells adopting a directed activation strategy. We observed comparable catalytic parameters and inhibition characteristics for both native SF‐EV‐associated and recombinant TMPRSS2 when exposed to serine protease inhibitor Nafamostat mesylate. Leveraging these findings, we developed a robust in vitro biochemical assay based on these SF‐EVs for the screening of TMPRSS2‐targeting compounds. Our results will accelerate the discovery and advancement of efficacious therapeutic approaches targeting TMPRSS2 and propel further exploration into the biological role of SF‐EV‐associated active TMPRSS2.

Fibroblast activation protein alpha is a postprolyl proteolytic enzyme highly expressed in the tumor microenvironment, particularly in cancer-associated fibroblasts. Although previously thought to be restricted to cancer-associated fibroblasts, malignant cells, and pathological fibroblasts, recent studies have identified fibroblast activation protein expression in natural killer cells. However, its expression and activity in natural killer cells remain poorly characterized. Here, we investigated fibroblast activation protein expression and activity in resting and cytokine-stimulated (interleukin-2 and interleukin-15) primary human natural killer cells and NK92 cells. Natural killer cell activation resulted in a significant decrease in fibroblast activation protein expression and enzymatic activity. Treatment with the fibroblast activation protein inhibitor UAMC-1110 altered the expression of activating and inhibitory natural killer cell receptors and reduced perforin expression, though it did not impact degranulation or cytotoxic function. Culturing natural killer cells in cancer-associated fibroblast–conditioned medium or direct co-culture with cancer-associated fibroblasts increased fibroblast activation protein expression and activity in NK92 cells, with donor-dependent effects observed in primary natural killer cells. These conditions also led to a reduction in natural killer activating and inhibitory receptor expression. Furthermore, hypoxia upregulated fibroblast activation protein in both NK92 and primary natural killer cells. Overall, our findings demonstrate that fibroblast activation protein is downregulated in/on natural killer cells upon activation with interleukin-2 and interleukin-15, whereas it is upregulated under tumor microenvironment–mimicking conditions. This may suggest that fibroblast activation protein contributes to the phenotype formation of natural killer cells, particularly within the tumor microenvironment, where we hypothesize that natural killer cells might prioritize invasive capacity over cytotoxic capacity, thus upregulating fibroblast activation protein.

Fibroblast activation protein α (FAP) is highly expressed on cancer-associated fibroblasts and has emerged as a promising target for tumor molecular imaging and therapy. Although many FAP-targeted ligands have entered clinical evaluation, important aspects of FAP biochemistry, enzyme kinetics, and target engagement are still not well understood, limiting rational ligand design and therapeutic optimization. The overarching aim of this thesis was to establish robust recombinant production of highly active FAP, and to develop an in vitro screening framework for FAP-targeted theranostic ligands that enables quantitative kinetic characterization of target engagement and residence time. First, this thesis reviews the historical development of FAP-targeting strategies in oncology (Chapter 3). Subsequently, recombinant production of human FAP (Chapter 4) and related proteases (Chapter 5) was optimized, focusing on yield, specific activity and stability. The enhanced quality of the recombinant human FAP facilitated a first structural characterization with a FAP-inhibitor, and the development of more elaborate enzymatic assay formats. Building on this foundation, medium-throughput kinetic assays were established to accurately determine association and dissociation rate constants of FAP-inhibitors, allowing target-residence time to be quantified as a key parameter for ligand selection (Chapter 6). Application of this framework revealed that structurally related FAP-inhibitors can display markedly distinct kinetic profiles despite similar equilibrium potency, highlighting the limitations of conventional affinity-based screening. In addition, this work also describes the development of a FAP-targeted gold nanoparticle conjugate as an exploratory translational tool for FAP research (Chapter 7). Multiple targeting strategies were evaluated, including monoclonal antibodies, small-molecule FAP-inhibitors, and single-domain antibodies, to assess their compatibility with gold nanoparticle conjugation and retention of functional FAP binding. The final conjugate was evaluated in a proof-of-principle anti-FAP lateral flow assay. Beyond this diagnostic application, the conjugate represents a versatile platform with broader potential, for example as a tracking reagent to study FAP localization and behavior in vitro, including live-cell imaging applications. Collectively, this work provides a mechanistic basis for the rational design and evaluation of FAP-targeted radioligands and diagnostic tools. The findings contribute to ongoing efforts to improve the specificity, efficacy, and clinical robustness of FAP-based theranostic strategies.