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.

Recently, the first squaramide-(SA) containing FAP inhibitor-derived radiotracers were introduced. DATA^5m.SA.FAPi and DOTA.SA.FAPi with their non-radioactive complexes showed high affinity and selectivity for FAP. After a successful preclinical study with [⁶⁸Ga]Ga-DOTA.SA.FAPi, the first patient studies were realized for both compounds. Here, we present a new squaramide-containing compound targeting FAP, based on the AAZTA⁵ chelator 1,4-bis-(carboxylmethyl)-6-[bis-(carboxymethyl)-amino-6-pentanoic-acid]-perhydro-1,4-diazepine. For this molecule (AAZTA⁵.SA.FAPi), complexation with radionuclides such as gallium-68, scandium-44, and lutetium-177 was investigated, and the in vitro properties of the complexes were characterized and compared with those of DOTA.SA.FAPi. AAZTA⁵.SA.FAPi and its derivatives labelled with non-radioactive isotopes demonstrated similar excellent inhibitory potencies compared to the previously published SA.FAPi ligands, i.e., sub-nanomolar IC₅₀ values for FAP and high selectivity indices over the serine proteases PREP and DPPs. Labeling with all three radiometals was easier and faster with AAZTA⁵.SA.FAPi compared to the corresponding DOTA analogue at ambient temperature. Especially, scandium-44 labeling with the AAZTA derivative resulted in higher specific activities. Both DOTA.SA.FAPi and AAZTA⁵.SA.FAPi showed sufficiently high stability in different media. Therefore, these FAP inhibitor agents could be promising for theranostic approaches targeting FAP.

Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) ⁶⁸Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel ¹⁸F-labeled FAPI derivative. [¹⁸F]6 was labeled using a synthon approach based on the tetrazine ligation. It showed subnanomolar affinity for the FAP protein and a good selectivity profile against known off-target proteases. Small animal PET studies revealed high tumor uptake and good target-to-background ratios. [¹⁸F]6 was excreted via the liver. Overall, [¹⁸F]6 showed promising characteristics to be used as a PET tracer and could serve as a lead for further development of halogen-based theranostic FAPI radiopharmaceuticals.

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 (FAP) is a post-prolyl proteolytic enzyme that is prominently expressed in cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) and in activated fibroblasts that contribute to tissue remodeling in chronic inflammatory diseases, such as Crohn’s disease (CD). Interestingly, FAP exhibits low expression and activity in healthy tissues, making it a promising diagnostic and therapeutic target for cancer and chronic inflammatory diseases. Unfortunately, FAP’s functional role in these pathological conditions remains poorly understood, and current tools and methods for studying FAP are often characterized by a lack of selectivity. Additionally, research has primarily focused on FAP mRNA and protein expression, often overlooking its enzymatic activity, which is critical for understanding its biological functions. These limitations highlight the urgent need for innovative, highly selective tools and methods to detect FAP and elucidate its role in disease mechanisms. Hence, the goal of this doctoral research was to develop and validate novel FAP-selective tools and methods to enhance our understanding of FAP’s biological function in cancer and Crohn’s disease. To achieve this, we established an in-house recombinant human FAP (rhFAP) production system to ensure a robust supply of high-quality active enzyme for downstream evaluation and validation of novel FAP-targeting tools and compounds. We produced and purified a highly specific antibody against FAP and developed fluorescent FAP-targeting activity-based probes (ABPs) that enable selective, high-affinity visualization of FAP’s enzymatic activity. Furthermore, we evaluated novel [18F]-labeled FAP inhibitors ([18F]-FAPIs) for PET/CT imaging as a non-invasive diagnostic approach for tumors and fibrotic diseases in which FAP plays a role. With these tools in hand, we gained new insights into the expression and function of FAP in the TME, with a particular focus on its activity and role in natural killer (NK) cells and its interaction with CAFs. Our findings suggest that FAP is involved in NK cell activity and the activation status within the TME. In addition, we used these FAP-specific tools to investigate its function in CD, revealing its involvement in chronic inflammation and intestinal fibrosis. Overall, this research not only offers new insights into FAP’s role in the TME and CD, but also provides the scientific community with a comprehensive toolkit for the selective detection and functional analysis of FAP. By filling critical knowledge gaps in FAP’s biological function, our findings pave the way for novel diagnostic and therapeutic strategies targeting FAP in oncology, Crohn’s disease and beyond.