On the basis of a pyrazine core structure, three new adenosine A2B receptor ligands (7a-c) were synthesized containing a 2-fluoropyridine moiety suitable for 18F-labeling. Compound 7a was docked into a homology model of the A2B receptor based on X-ray structures of the related A2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A1 receptor, 7a was radiolabeled as the most suitable candidate (Ki(A2B) = 4.24 nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [18F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [18F]7a in mice revealed the formation of a blood-brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.

(-)-[(18)F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α4β2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer's disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood.

Hydrogels based on gelatin have evolved as promising multifunctional biomaterials. Gelatin is crosslinked with lysine diisocyanate ethyl ester (LDI) and the molar ratio of gelatin and LDI in the starting material mixture determines elastic properties of the resulting hydrogel. In order to investigate the clinical potential of these biopolymers, hydrogels with different ratios of gelatin and diisocyanate (3-fold (G10_LNCO3) and 8-fold (G10_LNCO8) molar excess of isocyanate groups) were subcutaneously implanted in mice (uni- or bilateral implantation). Degradation and biomaterial-tissue-interaction were investigated in vivo (MRI, optical imaging, PET) and ex vivo (autoradiography, histology, serum analysis). Multimodal imaging revealed that the number of covalent net points correlates well with degradation time, which allows for targeted modification of hydrogels based on properties of the tissue to be replaced. Importantly, the degradation time was also dependent on the number of implants per animal. Despite local mechanisms of tissue remodeling no adverse tissue responses could be observed neither locally nor systemically. Finally, this preclinical investigation in immunocompetent mice clearly demonstrated a complete restoration of the original healthy tissue.

The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.

The [(18)F]fluorocyclobutyl group has the potential to be a metabolically stable prosthetic group for PET tracers. The synthesis of the radiolabeling precursor cis-cyclobutane-1,3-diyl bis(toluene-4-sulfonate) 8 was obtained from epibromohydrin in 7 steps (2% overall yield). The radiolabeling of this precursor 8 and its conjugation to L-tyrosine as a model system was successfully achieved to give the new non-natural amino acid 3-[(18)F]fluorocyclobutyl-L-tyrosine (L-3-[(18)F]FCBT) [(18)F]17 in 8% decay-corrected yield from the non-carrier-added [(18)F]fluoride. L-3-[(18)F]FCBT was investigated in vitro in different cancer cell lines to determine the uptake and stability. The tracer [(18)F]17 showed a time dependent uptake into different tumor cell lines (A549, NCI-H460, DU145) with the best uptake of 5.8% injected dose per 5×10(5) cells after 30min in human lung carcinoma cells A549. The stability of L-3-[(18)F]FCBT in human and rat plasma and the stability of the non-radioactive L-3-FCBT in rat hepatocytes were both found to be excellent. These results show that the non-natural amino acid L-3-[(18)F]FCBT is a promising metabolically stable radiotracer for positron emission tomography.

Assessment of dual time point (DTP) positron emission tomography was carried out with the aim of a quantitative determination of Km, the metabolic uptake rate of [18F]fluorodeoxyglucose as a measure of glucose consumption.

As the expression of a tumor associated antigen (TAA) is commonly not restricted to tumor cells, adoptively transferred T cells modified to express a conventional chimeric antigen receptor (CAR) might not only destroy the tumor cells but also attack target-positive healthy tissues. Furthermore, CAR T cells in patients with large tumor bulks will unpredictably proliferate and put the patients at high risk of adverse side effects including cytokine storms and tumor lysis syndrome. To overcome these problems, we previously established a modular CAR technology termed UniCAR: UniCAR T cells can repeatedly be turned on and off via dosing of a target module (TM). TMs are bispecific molecules which cross-link UniCAR T cells with target cells. After elimination of the respective TM, UniCAR T cells automatically turn off. Here we describe novel TMs against the disialoganglioside GD2 which is overexpressed in neuroectodermal but also many other tumors. In the presence of GD2-specific TMs, we see a highly efficient target-specific and -dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and tumor cell lysis both in vitro and experimental mice. According to PET-imaging, anti-GD2 TM enrich at the tumor site and are rapidly eliminated thus fulfilling all prerequisites of a UniCAR TM.

Combining stem cells with biomaterial scaffolds provides a promising strategy for the development of drug delivery systems. Here we propose an innovative immunotherapeutic organoid by housing human mesenchymal stromal cells (MSCs), gene-modified for the secretion of an anti-CD33-anti-CD3 bispecific antibody (bsAb), in a small biocompatible star-shaped poly(ethylene glycol)-heparin cryogel scaffold as a transplantable and low invasive therapeutic machinery for the treatment of acute myeloid leukemia (AML). The macroporous biohybrid cryogel platform displays effectiveness in supporting proliferation and survival of bsAb-releasing-MSCs overtime in vitro and in vivo, avoiding cell loss and ensuring a constant release of sustained and detectable levels of bsAb capable of triggering T-cell-mediated anti-tumor responses and a rapid regression of CD33+ AML blasts. This therapeutic device results as a promising and safe alternative to the continuous administration of short-lived immunoagents and paves the way for effective bsAb-based therapeutic strategies for future tumor treatments.

The adenosine A2B receptor has been proposed as a novel therapeutic target in cancer, as its expression is drastically elevated in several tumors and cancer cells. Noninvasive molecular imaging via positron emission tomography (PET) would allow the in vivo quantification of this receptor in pathological processes and most likely enable the identification and clinical monitoring of respective cancer therapies. On the basis of a bicyclic pyridopyrimidine-2,4-dione core structure, the new adenosine A2B receptor ligand 9 was synthesized, containing a 2-fluoropyridine moiety suitable for labeling with the short-lived PET radionuclide fluorine-18. Compound 9 showed a high binding affinity for the human A2B receptor (Ki(A2B) = 2.51 nM), along with high selectivities versus the A1, A2A, and A3 receptor subtypes. Therefore, it was radiofluorinated via nucleophilic aromatic substitution of the corresponding nitro precursor using [18F]F-/K2.2.2./K2CO3 in DMSO at 120 °C. Metabolic studies of [18F]9 in mice revealed about 60% of radiotracer intact in plasma at 30 minutes p.i. A preliminary PET study in healthy mice showed an overall biodistribution of [18F]9, corresponding to the known ubiquitous but low expression of the A2B receptor. Consequently, [18F]9 represents a novel PET radiotracer with high affinity and selectivity toward the adenosine A2B receptor and a suitable in vivo profile. Subsequent studies are envisaged to investigate the applicability of [18F]9 to detect alterations in the receptor density in certain cancer-related disease models.

Since epithelial growth factor receptor (EGFR) overexpression is linked to a variety of malignancies, it is an attractive target for immune therapy including chimeric antigen receptor (CAR)-engineered T cells. Unfortunately, CAR T cell therapy harbors the risk of severe, even life-threatening side effects. Adaptor CAR T cell platforms such as the previously described UniCAR system might be able to overcome these problems. In contrast to conventional CARs, UniCAR T cells are per se inert. Their redirection towards target cells occurs only in the presence of a tumor-specific target molecule (TM). TMs are bifunctional molecules being able to recognize a tumor-associated antigen and to cross-link the CAR T cell via a peptide epitope recognized by the UniCAR domain.

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Adoptive transfer of chimeric antigen receptor (CAR)-equipped T cells have demonstrated astonishing clinical efficacy in hematological malignancies recently culminating in the approval of two CAR T cell products. Despite this tremendous success, CAR T cell approaches have still achieved only moderate efficacy against solid tumors. As a major obstacle, engineered conventional T cells (Tconvs) face an anti-inflammatory, hostile tumor microenvironment often infiltrated by highly suppressive regulatory T cells (Tregs). Thus, potent CAR T cell treatment of solid tumors requires efficient activation of Tconvs via their engrafted CAR to overcome Treg-mediated immunosuppression. In that regard, selecting an optimal intracellular signaling domain might represent a crucial step to achieve best clinical efficiency. To shed light on this issue and to investigate responsiveness to Treg inhibition, we engrafted Tconvs with switchable universal CARs (UniCARs) harboring intracellularly the CD3ζ domain alone or in combination with costimulatory CD28 or 4-1BB. Our studies reveal that UniCAR ζ-, and UniCAR BB/ζ-engineered Tconvs are strongly impaired by activated Tregs, whereas UniCARs providing CD28 costimulation overcome Treg-mediated suppression both in vitro and in vivo. Compared to UniCAR ζ- and UniCAR BB/ζ-modified cells, UniCAR 28/ζ-armed Tconvs secrete significantly higher amounts of Th1-related cytokines and, furthermore, levels of these cytokines are elevated even upon exposure to Tregs. Thus, in contrast to 4-1BB costimulation, CD28 signaling in UniCAR-transduced Tconvs seems to foster a pro-inflammatory milieu, which contributes to enhanced resistance to Treg suppression. Overall, our results may have significant implications for CAR T cell-based immunotherapies of solid tumors strongly invaded by Tregs.

Tyrosine is an attractive target for chemo- and site-selective protein modification. The particular chemical nature of tyrosine residues allows bioconjugation chemistry with reactive aryl diazonium salts via electrophilic aromatic substitution to produce diazo compounds. In this work, we describe the preparation of 64Cu- and 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-diazonium salts as building blocks for azo coupling chemistry with tyrosine and tyrosine-containing peptides and proteins under mild conditions. 2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH2-Bn-NOTA) was used to form the corresponding 64Cu- and 68Ga-labeled complexes, followed by diazotization with NaNO2 in the presence of HCl. 64Cu- and 68Ga-labeled NOTA complexes were prepared in high radiochemical yields >80% starting from 20 μg of p-NH2-Bn-NOTA. Conversion of p-NH2-Bn-NOTA complexes into diazonium salts followed by azo coupling with l-tyrosine afforded 64Cu- and 68Ga-labeled tyrosine in radiochemical yields of 80 and 56%, respectively. Azo coupling with tyrosine-containing hexapeptide neurotensin NT(8-13) afforded 64Cu- and 68Ga-labeled NT(8-13) in radiochemical yields of 45 and 11%, respectively. Azo coupling of 64Cu-labeled NOTA-diazonium salt with human serum albumin (HSA) gave 64Cu-labeled HSA in radiochemical yields of 20%. The described azo coupling chemistry represents an innovative and versatile bioconjugation strategy for selective targeting of tyrosine residues in peptides and proteins.

We present the first in-human brain PET imaging data of the new α4β2 nicotinic acetylcholine receptor (nAChR)-targeting radioligand (+)-[18F]Flubatine. Aims were to develop a kinetic modeling-based approach to quantify (+)-[18F]Flubatine and compare the data of healthy controls (HCs) and patients with Alzheimer's disease (AD); to investigate the partial volume effect (PVE) on regional (+)-[18F]Flubatine binding; and whether (+)-[18F]Flubatine binding and cognitive test data respective β-amyloid radiotracer accumulation were correlated.

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs.

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.

Pheochromocytomas and extra-adrenal paragangliomas (PHEO/PGLs) are rare catecholamine-producing chromaffin cell tumors. For metastatic disease, no effective therapy is available. Overexpression of somatostatin type 2 receptors (SSTR2) in PHEO/PGLs promotes interest in applying therapies using somatostatin analogs linked to radionuclides and/or cytotoxic compounds, such as [(177)Lu]Lu-DOTA-(Tyr(3))octreotate (DOTATATE) and AN-238. Systematic evaluation of such therapies for the treatment of PHEO/PGLs requires sophisticated animal models. In this study, the mouse pheochromocytoma (MPC)-mCherry allograft model showed high tumor densities of murine SSTR2 (mSSTR2) and high tumor uptake of [(64)Cu]Cu-DOTATATE. Using tumor sections, we assessed mSSTR2-specific binding of DOTATATE, AN-238, and somatostatin-14. Therapeutic studies showed substantial reduction of tumor growth and tumor-related renal monoamine excretion in tumor-bearing mice after treatment with [(177)Lu]Lu-DOTATATE compared to AN-238 and doxorubicin. Analyses did not show agonist-dependent receptor downregulation after single mSSTR2-targeting therapies. This study demonstrates that the MPC-mCherry model is a uniquely powerful tool for the preclinical evaluation of SSTR2-targeting theranostic applications in vivo. Our findings highlight the therapeutic potential of somatostatin analogs, especially of [(177)Lu]Lu-DOTATATE, for the treatment of metastatic PHEO/PGLs. Repeated treatment cycles, fractionated combinations of SSTR2-targeting radionuclide and cytotoxic therapies, and other adjuvant compounds addressing additional mechanisms may further enhance therapeutic outcome.

Changes in the expression of α₇ nicotinic acetylcholine receptors (α₇ nAChRs) in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET). We report on a series of new ligands for α₇ nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR) of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT₃ receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a) for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10) has high potential to provide clinically relevant information about the expression and availability of α₇ nAChR in the brain.

Arterial spin labeling magnetic resonance imaging (ASL-MRI) has been recognised as a valuable method for non-invasive assessment of cerebral blood flow but validation studies regarding quantification accuracy by comparison against an accepted gold standard are scarce, especially in small animals. We have conducted the present study with the aim of comparing ASL flow-sensitive alternating inversion recovery (FAIR)-derived unidirectional water uptake (K1) and 68Ga/64Cu microsphere (MS)-derived blood flow (f) in the rat brain.