Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody.

Regulatory T cells (Tregs) are known to facilitate tumor progression by suppressing CD8+ T cells within the tumor microenvironment (TME), thereby also hampering the effectiveness of immune checkpoint inhibitors (ICIs). While systemic depletion of Tregs can enhance antitumor immunity, it also triggers undesirable autoimmune responses. Therefore, there is a need for therapeutic agents that selectively target Tregs within the TME without affecting systemic Tregs. In this study, as shown also by others, the chemokine (C-C motif) receptor 8 (CCR8) was found to be predominantly expressed on Tregs within the TME of both humans and mice, representing a unique target for selective depletion of tumor-residing Tregs. Based on this, we developed BAY 3375968, a novel anti-human CCR8 antibody, along with respective surrogate anti-mouse CCR8 antibodies, and demonstrated their in vitro mode-of-action through induction of potent antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities. In vivo, anti-mouse CCR8 antibodies effectively depleted Tregs within the TME primarily via ADCP, leading to increased CD8+ T cell infiltration and subsequent tumor growth inhibition across various cancer models. This monotherapeutic efficacy was significantly enhanced in combination with ICIs. Collectively, these findings suggest that CCR8 targeting represents a promising strategy for Treg depletion in cancer therapies. BAY 3375968 is currently under investigation in a Phase I clinical trial (NCT05537740).

The repertoire and structure of adhesion GPCR transcript variants assembled from publicly available deep-sequenced human samples.

Alternative splicing and multiple transcription start and termination sites can produce a diverse repertoire of mRNA transcript variants from a given gene. While the full picture of the human transcriptome is still incomplete, publicly available RNA datasets have enabled the assembly of transcripts. Using publicly available deep sequencing data from 927 human samples across 48 tissues, we quantified known and new transcript variants, provide an interactive, browser-based application Splice-O-Mat and demonstrate its relevance using adhesion G protein-coupled receptors (aGPCRs) as an example. On average, 24 different transcript variants were detected for each of the 33 human aGPCR genes, and several dominant transcript variants were not yet annotated. Variable transcription starts and complex exon-intron structures encode a flexible protein domain architecture of the N- and C termini and the seven-transmembrane helix domain (7TMD). Notably, we discovered the first GPCR (ADGRG7/GPR128) with eight transmembrane helices. Both the N- and C terminus of this aGPCR were intracellularly oriented, anchoring the N terminus in the plasma membrane. Moreover, the assessment of tissue-specific transcript variants, also for other gene classes, in our application may change the evaluation of disease-causing mutations, as their position in different transcript variants may explain tissue-specific phenotypes.

The dark sides of the GPCR tree - research progress on understudied GPCRs.

A large portion of the human GPCRome is still in the dark and understudied, consisting even of entire subfamilies of GPCRs such as odorant receptors, class A and C orphans, adhesion GPCRs, Frizzleds and taste receptors. However, it is undeniable that these GPCRs bring an untapped therapeutic potential that should be explored further. Open questions on these GPCRs span diverse topics such as deorphanisation, the development of tool compounds and tools for studying these GPCRs, as well as understanding basic signalling mechanisms. This review gives an overview of the current state of knowledge for each of the diverse subfamilies of understudied receptors regarding their physiological relevance, molecular mechanisms, endogenous ligands and pharmacological tools. Furthermore, it identifies some of the largest knowledge gaps that should be addressed in the foreseeable future and lists some general strategies that might be helpful in this process.

The N Terminus of Adhesion G Protein-Coupled Receptor GPR126/ADGRG6 as Allosteric Force Integrator.

The adhesion G protein-coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates the binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown. In this study, we provide this information by combining classic second-messenger detection with single-cell atomic force microscopy. We established a monoclonal antibody targeting the N terminus to stimulate GPR126 and compared it to the activation through its known ECM ligands, collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be regarded as an allosteric module that can fine-tune receptor activation in a context-specific manner.

A Novel NAMPT Inhibitor-Based Antibody-Drug Conjugate Payload Class for Cancer Therapy.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody-drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure-activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate─to our knowledge for the first time─the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.

Antibody-drug conjugates harboring a kinesin spindle protein inhibitor with immunostimulatory properties.

Antibody-drug conjugates (ADCs) are used to target cancer cells by means of antibodies directed to tumor-associated antigens, causing the incorporation of a cytotoxic payload into target cells. Here, we characterized the mode of action of ADC costing of a TWEAKR-specific monoclonal antibody conjugated to a small molecule kinesin spindle protein (KSP) inhibitor (KSPi). These TWEAKR-KSPi-ADCs showed strong efficacy in a TWEAKR expressing CT26 colon cancer model in mice. TWEAKR-KSPi-ADCs controlled the growth of CT26 colon cancers in immunodeficient as well as in immunocompetent mice. However, when treated with suboptimal doses, TWEAKR-KSPi-ADCs were still active in immunocompetent but not in immunodeficient mice, indicating that TWEAKR-KSPi-ADCs act - in addition to the cytotoxic mode of action - through an immunological mechanism. Indeed, in vitro experiments performed with a cell-permeable small molecule KSPi closely related to the active payload released from the TWEAKR-KSPi-ADCs revealed that KSPi was capable of stimulating several hallmarks of immunogenic cell death (ICD) on three different human cancer cell lines: cellular release of adenosine triphosphate (ATP) and high mobility group B1 protein (HMGB1), exposure of calreticulin on the cell surface as well as a transcriptional type-I interferon response. Further, in vivo experiments confirmed that treatment with TWEAKR-KSPi-ADCs activated immune responses via enhancing the infiltration of CD4+ and CD8+ T lymphocytes in tumors and the local production of interferon-γ, interleukin-2, and tumor necrosis factor-α. In conclusion, the antineoplastic effects of TWEAKR-KSPi-ADCs can partly be attributed to its ICD-stimulatory properties.

One step ahead: mapping the Italian and German cybersecurity laws against the proposal for a NIS2 directive.

With the COVID-19 pandemic accelerating digital transformation of the Single Market, the European Commission also speeded up the review of the first piece of European Union (EU)-wide cybersecurity legislation, the NIS Directive. Originally foreseen for May 2021, the Commission presented the review as early as December 2020 together with a Proposal for a NIS2 Directive. Almost in parallel, some Member States strengthened (or adopted) national laws beyond the scope of the NIS Directive to respond adequately to the fast-paced digital threat landscape. Against this backdrop, the article investigates the national interventions in the field of cybersecurity recently adopted by Italy and Germany. In order to identify similarities and divergences of the Italian and German national frameworks with the European Commission's Proposal for a NIS2 Directive, the analysis will focus on selected aspects extrapolated from the Commission Proposal, namely: i) the enlarged scope; ii) detailed cybersecurity risk-management measures; iii) more stringent supervisory measures; and, iv) stricter enforcement requirements, including harmonised sanctions across the EU. The article concludes that the national cybersecurity legal frameworks under scrutiny already match the core of the proposed changes envisaged by the NIS2 Proposal.

A Model for the Signal Initiation Complex Between Arrestin-3 and the Src Family Kinase Fgr.

Arrestins regulate a wide range of signaling events, most notably when bound to active G protein-coupled receptors (GPCRs). Among the known effectors recruited by GPCR-bound arrestins are Src family kinases, which regulate cellular growth and proliferation. Here, we focus on arrestin-3 interactions with Fgr kinase, a member of the Src family. Previous reports demonstrated that Fgr exhibits high constitutive activity, but can be further activated by both arrestin-dependent and arrestin-independent pathways. We report that arrestin-3 modulates Fgr activity with a hallmark bell-shaped concentration-dependence, consistent with a role as a signaling scaffold. We further demonstrate using NMR spectroscopy that a polyproline motif within arrestin-3 interacts directly with the SH3 domain of Fgr. To provide a framework for this interaction, we determined the crystal structure of the Fgr SH3 domain at 1.9 Å resolution and developed a model for the GPCR-arrestin-3-Fgr complex that is supported by mutagenesis. This model suggests that Fgr interacts with arrestin-3 at multiple sites and is consistent with the locations of disease-associated Fgr mutations. Collectively, these studies provide a structural framework for arrestin-dependent activation of Fgr.

Immunostimulatory effects of targeted thorium-227 conjugates as single agent and in combination with anti-PD-L1 therapy.

BACKGROUND: Targeted thorium-227 conjugates (TTCs) are an emerging class of targeted alpha therapies (TATs). Their unique mode of action (MoA) is the induction of difficult-to-repair clustered DNA double-strand breaks. However, thus far, their effects on the immune system are largely unknown. Here, we investigated the immunostimulatory effects of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC) in vitro and in vivo in monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice. METHODS: The murine cell line MC38 was transfected with the human gene encoding for MSLN (hMSLN) to enable binding of the non-cross-reactive MSLN-TTC. The immunostimulatory effects of MSLN-TTC were studied in vitro on human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studied in vivo as monotherapy or in combination with anti-PD-L1 in MC38-hMSLN tumor-bearing immunocompetent C57BL/6 mice. Experiments were supported by RNA sequencing, flow cytometry, immunohistochemistry, mesoscale, and TaqMan PCR analyses to study the underlying immunostimulatory effects. In vivo depletion of CD8+ T cells and studies with Rag2/Il2Rg double knockout C57BL/6 mice were conducted to investigate the importance of immune cells to the efficacy of MSLN-TTC. RESULTS: MSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6, CCL20, CXCL10, and stimulator of interferon genes (STING)-related genes) in vitro as determined by RNASeq analysis. The results, including phospho-STING activation, were confirmed on the protein level. Danger-associated molecular pattern molecules were upregulated in parallel, leading to dendritic cell (DC) activation in vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, p<0.05) as a single agent in human MSLN-expressing MC38 tumor-bearing immunocompetent mice. Combining MSLN-TTC with anti-PD-L1 further enhanced the efficacy (T:C 0.08, p<0.001) as evidenced by the increased number of tumor-free surviving animals. MSLN-TTC monotherapy caused migration of CD103+ cDC1 DCs and infiltration of CD8+ T cells into tumors, which was enhanced on combination with anti-PD-L1. Intriguingly, CD8+ T-cell depletion decreased antitumor efficacy. CONCLUSIONS: These in vitro and in vivo data on MSLN-TTC demonstrate that the MoA of TTCs involves activation of the immune system. The findings are of relevance for other targeted radiotherapies and may guide clinical combination strategies.

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR-SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

An Eight Amino Acid Segment Controls Oligomerization and Preferred Conformation of the two Non-visual Arrestins.

G protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP6). IP6 interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP6 on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP6, arrestin-2 forms "infinite" chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP6; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP6, the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP6 effect. Because IP6 is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins.

Tailored Linker Chemistries for the Efficient and Selective Activation of ADCs with KSPi Payloads.

Several antibody-drug conjugates (ADCs) have failed to achieve a sufficiently large therapeutic window in patients due to toxicity induced by unspecific payload release in the circulation or ADC uptake into healthy organs. Herein, we describe the successful engineering of ADCs consisting of novel linkers, which are efficiently and selectively cleaved by the tumor-associated protease legumain. ADCs generated via this approach demonstrate high potency and a preferential activation in tumors compared to healthy tissue, thus providing an additional level of safety. A remarkable tolerance of legumain for different linker peptides, including those with just a single asparagine residue, together with a modifier of the physicochemical metabolite profile, proves the broad applicability of this approach for a tailored design of ADCs.

Crystal structure of the SH3 domain of human Lyn non-receptor tyrosine kinase.

Lyn kinase (Lck/Yes related novel protein tyrosine kinase) belongs to the family of Src-related non-receptor tyrosine kinases. Consistent with physiological roles in cell growth and proliferation, aberrant function of Lyn is associated with various forms of cancer, including leukemia, breast cancer and melanoma. Here, we determine a 1.3 Å resolution crystal structure of the polyproline-binding SH3 regulatory domain of human Lyn kinase, which adopts a five-stranded ß-barrel fold. Mapping of cancer-associated point mutations onto this structure reveals that these amino acid substitutions are distributed throughout the SH3 domain and may affect Lyn kinase function distinctly.

Antibody-Prodrug Conjugates with KSP Inhibitors and Legumain-Mediated Metabolite Formation.

Many antibody-drug conjugates (ADCs) have failed to achieve a sufficient therapeutic window in clinical studies either due to target-mediated or off-target toxicities. To achieve an additional safety level, a new class of antibody-prodrug conjugates (APDCs) directed against different targets in solid tumors is here described. The tumor-associated lysosomal endopeptidase legumain with a unique cleavage sequence was utilized for APDC metabolism. Legumain-activatable APDCs were as potent as their cathepsin B-activatable analogues. The peptide sequence susceptible to legumain cleavage was optimized for further discrimination of the formation of active metabolites within tumor cells versus healthy tissues, leveraging different tissue-specific legumain activities. Optimized APDCs with slow legumain-mediated conversion reduced preclinically the levels of active metabolite in healthy organs while retaining high activity against different TWEAKR- and B7H3-expressing tumors.

Arrestin-3 scaffolding of the JNK3 cascade suggests a mechanism for signal amplification.

Scaffold proteins tether and orient components of a signaling cascade to facilitate signaling. Although much is known about how scaffolds colocalize signaling proteins, it is unclear whether scaffolds promote signal amplification. Here, we used arrestin-3, a scaffold of the ASK1-MKK4/7-JNK3 cascade, as a model to understand signal amplification by a scaffold protein. We found that arrestin-3 exhibited >15-fold higher affinity for inactive JNK3 than for active JNK3, and this change involved a shift in the binding site following JNK3 activation. We used systems biochemistry modeling and Bayesian inference to evaluate how the activation of upstream kinases contributed to JNK3 phosphorylation. Our combined experimental and computational approach suggested that the catalytic phosphorylation rate of JNK3 at Thr-221 by MKK7 is two orders of magnitude faster than the corresponding phosphorylation of Tyr-223 by MKK4 with or without arrestin-3. Finally, we showed that the release of activated JNK3 was critical for signal amplification. Collectively, our data suggest a "conveyor belt" mechanism for signal amplification by scaffold proteins. This mechanism informs on a long-standing mystery for how few upstream kinase molecules activate numerous downstream kinases to amplify signaling.

The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains.

Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.

Antibody-Drug Conjugates with Pyrrole-Based KSP Inhibitors as the Payload Class.

The number of cytotoxic payload classes successfully employed in antibody-drug conjugates (ADCs) is still rather limited. The identification of ADC payloads with a novel mode of action will increase therapeutic options and potentially increase the therapeutic window. Herein, we describe the utilization of kinesin spindle protein inhibitors (KSPi) as a novel payload class providing highly potent ADCs against different targets, for instance HER-2 or TWEAKR/Fn14. Aspects of technical optimization include the development of different linker attachment sites, the stabilization of ADC linkage to avoid payload deconjugation and finally, the tailor-made design of active metabolites with a long lasting intracellular exposure in the tumor matching the mode of action of KSP inhibition. These KSPi-ADCs are highly potent and selective in vitro and demonstrate in vivo efficacy in a broad panel of tumor models including complete regressions in a patient-derived urothelial cancer model.

Structural basis of arrestin-3 activation and signaling.

A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP6) is a non-receptor activator of arrestin-3 and report the structure of IP6-activated arrestin-3 at 2.4-Å resolution. IP6-activated arrestin-3 exhibits an inter-domain twist and a displaced C-tail, hallmarks of active arrestin. IP6 binds to the arrestin phosphate sensor, and is stabilized by trimerization. Analysis of the trimerization surface, which is also the receptor-binding surface, suggests a feature called the finger loop as a key region of the activation sensor. We show that finger loop helicity and flexibility may underlie coupling to hundreds of diverse receptors and also promote arrestin-3 activation by IP6. Importantly, we show that effector-binding sites on arrestins have distinct conformations in the basal and activated states, acting as switch regions. These switch regions may work with the inter-domain twist to initiate and direct arrestin-mediated signaling.

Antitumor effects of regorafenib and sorafenib in preclinical models of hepatocellular carcinoma.

The purpose of this study was to investigate the antitumor activity of regorafenib and sorafenib in preclinical models of HCC and to assess their mechanism of action by associated changes in protein expression in a HCC-PDX mouse model. Both drugs were administered orally once daily at 10 mg/kg (regorafenib) or 30 mg/kg (sorafenib), which recapitulate the human exposure at the maximally tolerated dose in mice. In a H129 hepatoma model, survival times differed significantly between regorafenib versus vehicle (p=0.0269; median survival times 36 vs 27 days), but not between sorafenib versus vehicle (p=0.1961; 33 vs 28 days). Effects on tumor growth were assessed in 10 patient-derived HCC xenograft (HCC-PDX) models. Significant tumor growth inhibition was observed in 8/10 models with regorafenib and 7/10 with sorafenib; in four models, superior response was observed with regorafenib versus sorafenib which was deemed not to be due to lower sorafenib exposure. Bead-based multiplex western blot analysis was performed with total protein lysates from drug- and vehicle-treated HCC-PDX xenografts. Protein expression was substantially different in regorafenib- and sorafenib-treated samples compared with vehicle. The pattern of upregulated proteins was similar with both drugs and indicates an activated RAF/MEK/ERK pathway, but more proteins were downregulated with sorafenib versus regorafenib. Overall, both regorafenib and sorafenib were effective in mouse models of HCC, although several cases showed better regorafenib activity which may explain the observed efficacy of regorafenib in sorafenib-refractory patients.

Comparison of [(11)C]Choline ([(11)C]CHO) and [(18)F]Bombesin (BAY 86-4367) as Imaging Probes for Prostate Cancer in a PC-3 Prostate Cancer Xenograft Model.

PURPOSE: Carbon-11- and fluorine-18-labeled choline derivatives are commonly used in prostate cancer imaging in the clinical setting for staging and re-staging of prostate cancer. Due to a limited detection rate of established positron emission tomography (PET) tracers, there is a clinical need for innovative tumor-specific PET compounds addressing new imaging targets. The aim of this study was to compare the properties of [(18)F]Bombesin (BAY 86-4367) as an innovative biomarker for prostate cancer imaging targeting the gastrin-releasing peptide receptor and [(11)C]Choline ([(11)C]CHO) in a human prostate tumor mouse xenograft model by small animal PET/X-ray computed tomography (CT). PROCEDURES: We carried out a dual-tracer small animal PET/CT study comparing [(18)F]Bombesin and [(11)C]CHO. The androgen-independent human prostate tumor cell line PC-3 was implanted subcutaneously in the flanks of nu/nu NMRI mice (n = 10) (PET/CT measurements of two [(11)C]Choline mice could not be analyzed due to technical reasons). [(18)F]Bombesin and [(11)C]CHO PET/CT imaging was performed about 3-4 weeks after the implantation of PC-3 cells on two separate days. After the intravenous tail vein injection of 14 MBq [(18)F]Bombesin and 37 MBq [(11)C]CHO, respectively, a dynamic study over 60 min was acquired in list mode using an Inveon animal PET/CT scanner (Siemens Medical Solutions). The sequence of [(18)F]Bombesin and [(11)C]CHO was randomized. Image analysis was performed using summed images as well as dynamic data. To calculate static and dynamic tumor-to-muscle (T/M), tumor-to-blood (T/B), liver-to-blood (L/B), and kidney-to-blood (K/B) ratios, 4 × 4 × 4 mm(3) volumes of interest (VOIs) of tumor, muscle (thigh), liver, kidney, and blood derived from transversal slices were used. RESULTS: The mean T/M ratio of [(18)F]Bombesin and [(11)C]CHO was 6.54 ± 2.49 and 1.35 ± 0.30, respectively. The mean T/B ratio was 1.83 ± 0.79 for [(18)F]Bombesin and 0.55 ± 0.10 for [(11)C]CHO. The T/M ratio as well as the T/B ratio for [(18)F]Bombesin were significantly higher compared to those for [(11)C]CHO (p < 0.001, respectively). Kidney and liver uptake was statistically significantly lower for [(18)F]Bombesin (K/B 3.41 ± 0.81, L/B 1.99 ± 0.38) compared to [(11)C]CHO [K/B 7.91 ± 1.85 (p < 0.001), L/B 6.27 ± 1.99 (p < 0.001)]. The magnitudes of the time course of T/M and T/B ratios (T/M and T/Bdyn ratios) were statistically significantly different (showing a higher uptake of [(18)F]Bombesin compared to [(11)C]CHO); additionally, also the change of the T/M and T/B ratios over time was significantly different between both tracers in the dynamic analysis (p < 0.001, respectively). Furthermore, there was a statistically significantly different change of the K/B and L/B ratios over time between the two tracers in the dynamic analysis (p = 0.026 and p < 0.001, respectively). CONCLUSIONS: [(18)F]Bombesin (BAY 86-4367) visually and semi-quantitatively outperforms [(11)C]CHO in the PC-3 prostate cancer xenograft model. [(18)F]Bombesin tumor uptake was significantly higher compared to [(11)C]CHO. [(18)F]Bombesin showed better imaging properties compared to the clinically utilized [(11)C]CHO due to a higher tumor uptake as well as a lower liver and kidney uptake.