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.

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.

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.

Cyclooxygenase 2 (COX-2) is a key enzyme of the tumorigenesis-inflammation interface and can be induced by hypoxia. A pseudohypoxic transcriptional signature characterizes pheochromocytomas and paragangliomas (PPGLs) of the cluster I, mainly represented by tumors with mutations in von Hippel-Lindau (VHL), endothelial PAS domain-containing protein 1 (EPAS1), or succinate dehydrogenase (SDH) subunit genes. The aim of this study was to investigate a possible association between underlying tumor driver mutations and COX-2 in PPGLs. COX-2 gene expression and immunoreactivity were examined in clinical specimens with documented mutations, as well as in spheroids and allografts derived from mouse pheochromocytoma (MPC) cells. COX-2 in vivo imaging was performed in allograft mice. We observed significantly higher COX-2 expression in cluster I, especially in VHL-mutant PPGLs, however, no specific association between COX-2 mRNA levels and a hypoxia-related transcriptional signature was found. COX-2 immunoreactivity was present in about 60% of clinical specimens as well as in MPC spheroids and allografts. A selective COX-2 tracer specifically accumulated in MPC allografts. This study demonstrates that, although pseudohypoxia is not the major determinant for high COX-2 levels in PPGLs, COX-2 is a relevant molecular target. This potentially allows for employing selective COX-2 inhibitors as targeted chemotherapeutic agents and radiosensitizers. Moreover, available models are suitable for preclinical testing of these treatments.

COX-2 can be considered as a clinically relevant molecular target for adjuvant, in particular radiosensitizing treatments. In this regard, using selective COX-2 inhibitors, e.g., in combination with radiotherapy or endoradiotherapy, represents an interesting treatment option. Based on our own findings that nitric oxide (NO)-releasing and celecoxib-derived COX-2 inhibitors (COXIBs) showed promising radiosensitizing effects in vitro, we herein present the development of a series of eight novel NO-COXIBs differing in the peripheral substitution pattern and their chemical and in vitro characterization. COX-1 and COX-2 inhibition potency was found to be comparable to the lead NO-COXIBs, and NO-releasing properties were demonstrated to be mainly influenced by the substituent in 4-position of the pyrazole (Cl vs. H). Introduction of the N-propionamide at the sulfamoyl residue as a potential prodrug strategy lowered lipophilicity markedly and abolished COX inhibition while NO-releasing properties were not markedly influenced. NO-COXIBs were tested in vitro for a combination with single-dose external X-ray irradiation as well as [177Lu]LuCl3 treatment in HIF2α-positive mouse pheochromocytoma (MPC-HIF2a) tumor spheroids. When applied directly before X-ray irradiation or 177Lu treatment, NO-COXIBs showed radioprotective effects, as did celecoxib, which was used as a control. Radiosensitizing effects were observed when applied shortly after X-ray irradiation. Overall, the NO-COXIBs were found to be more radioprotective compared with celecoxib, which does not warrant further preclinical studies with the NO-COXIBs for the treatment of pheochromocytoma. However, evaluation as radioprotective agents for healthy tissues could be considered for the NO-COXIBs developed here, especially when used directly before irradiation.

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.

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.

Hereditary spastic paraplegias (HSPs) cause characteristic gait impairment leading to an increased risk of stumbling or even falling. Biomechanically, gait deficits are characterized by reduced ranges of motion in lower body joints, limiting foot clearance and ankle range of motion. To date, there is no standardized approach to continuously and objectively track the degree of dysfunction in foot elevation since established clinical rating scales require an experienced investigator and are considered to be rather subjective. Therefore, digital disease-specific biomarkers for foot elevation are needed.

Pheochromocytomas/paragangliomas (PPGLs) with pathogenic mutations in the succinate dehydrogenase subunit B (SDHB) are associated with a high metastatic risk. Somatostatin receptor 2 (SSTR2)-dependent imaging is the most sensitive imaging modality for SDHB-related PPGLs, suggesting that SSTR2 expression is a significant cell surface therapeutic biomarker of such tumors.

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.

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.

Radiotherapy is one of the most frequently applied treatments in oncology. Tissue-absorbed ionizing radiation damages not only targeted cells but the surrounding cells too. The consequent long-term induced oxidative stress, irreversible tissue damage, or second malignancies draw attention to the urgent need of a follow-up medical method by which personalized treatment could be attained and the actually dose-limiting organ could be monitored in the clinical practice. We worked out a special hemisphere irradiation technique for mice which mimics the radiation exposure during radiotherapy. We followed up the changes of possible brain imaging biomarkers of side effects, such as cerebral blood flow, vascular endothelial function, and cellular metabolic processes for 60 days. BALB/c mice were divided into two groups (n=6 per group) based on the irradiation doses (5 and 20 Gy). After the irradiation procedure arterial spin labeling (ASL), diffusion-weighted imaging (DWI) in magnetic resonance modality and [18F]fluoro-deoxy-D-glucose positron emission tomography (FDG-PET) scans of the brain were obtained at several time points (3, 7, 30, and 60 days after the irradiation). Significant physiological changes were registered in the brain of animals following the irradiation by both applied doses. Elevated standard uptake values were detected all over the brain by FDG-PET studies 2 months after the irradiation. The apparent diffusion coefficients from DWI scans significantly decreased one month after the irradiation procedure, while ASL studies did not show any significant perfusion changes in the brain. Altogether, our sensitive multimodal imaging protocol seems to be an appropriate method for follow-up of the health status after radiation therapy. The presented approach makes possible parallel screening of healthy tissues and the effectiveness of tumor therapy without any additional radiation exposure.

Recently, we established the controllable modular UniCAR platform technology to advance the efficacy and safety of CAR T cell therapy. The UniCAR system is composed of (i) target modules (TMs) and (ii) UniCAR armed T cells. TMs are bispecific molecules that are able to bind to the tumor cell surface and simultaneously to UniCAR T cells. For interaction with UniCAR T cells, TMs contain a peptide epitope sequence which is recognised by UniCAR T cells. So far, a series of TMs against a variety of tumor targets including against the prostate stem cell antigen (PSCA) were constructed and functionally characterised. In order to facilitate their purification all these TMs are expressed as recombinant proteins equipped with an oligo-His-tag. The aim of the here presented manuscript was to learn whether or not the oligo-His-tag of the TM influences the UniCAR system. For this purpose, we constructed TMs against PSCA equipped with or lacking an oligo-His-tag. Both TMs were compared side by side including for functionality and biodistribution. According to our data, an oligo-His-tag of a UniCAR TM has only little if any effect on its binding affinity, in vitro and in vivo killing capability and in vivo biodistribution.

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).

New treatment options especially of solid tumors including for metastasized prostate cancer (PCa) are urgently needed. Recent treatments of leukemias with chimeric antigen receptors (CARs) underline their impressive therapeutic potential. However CARs currently applied in the clinics cannot be repeatedly turned on and off potentially leading to severe life threatening side effects. To overcome these problems, we recently described a modular CAR technology termed UniCAR: UniCAR T cells are inert but can be turned on by application of one or multiple target modules (TMs). Here we present preclinical data summarizing the retargeting of UniCAR T cells to PCa cells using TMs directed to prostate stem cell- (PSCA) or/and prostate specific membrane antigen (PSMA). In the presence of the respective TM(s), we see a highly efficient target-specific and target-dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and PCa cell lysis both in vitro and experimental mice.

Accumulating evidence suggests an unequivocal role of lysyl oxidases as key players of tumor progression and metastasis, which renders this enzyme family highly attractive for targeted non-invasive functional imaging of tumors. Considering their function in matrix remodeling, malignant melanoma appears as particularly interesting neoplasia in this respect. For the development of radiotracers that enable PET imaging of the melanoma-associated lysyl oxidase activity, substrates derived from the type I collagen α1 N-telopeptide were labeled with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) as prosthetic reagent. With regards to potential crosslinking to tumor-associated collagen in vivo, their interaction with triple-helical type I collagen was studied by SPR. A mouse model of human melanoma was established on the basis of the A375 cell line, for which the expression of the oncologically relevant lysyl oxidase isoforms LOX and LOXL2 was demonstrated in Western blot and immunohistochemical experiments. The radiopharmacological profiles of the peptidic radiotracers were evaluated in normal rats and A375 melanoma-bearing mice by ex vivo metabolite analysis, whole-body biodistribution studies and dynamic PET imaging. Out of three 18F-labeled telopeptide analogs, the one with the most favorable substrate properties has shown favorable tumor uptake and tumor-to-muscle ratio. Lysyl oxidase-mediated tumor uptake was proven by pharmacological inhibition using β-aminopropionitrile and by employing negative-control analogs of impeded or abolished targeting capability. The latter were obtained by substituting the lysine residue by ornithine and norleucine, respectively. Comparing the tumor uptake of the lysine-containing peptide with that of the non-functional analogs indicate the feasibility of lysyl oxidase imaging in melanoma using substrate-based radiotracers.

Physiological and pathophysiological functions of the phospholipase A2 receptor 1 (PLA2R1) are still not completely understood. To elucidate PLA2R1's function in prostate carcinoma, the receptor was ectopically overexpressed in LNCaP with silenced PLA2R1, and diminished in PC-3 cells with constitutively increased PLA2R1 expression relative to normal prostate epithelial cells. LNCaP cells were transfected to overexpress PLA2R1 (LNCaP-PLA2R1) and compared to control vector transfected cells (LNCaP-Ctrl). Alternatively, a CRISPR/Cas9-knockdown of PLA2R1 was achieved in PC-3 cells (PC-3 KD) and compared to the corresponding control-transfected cells (PC-3 Ctrl). The impact of PLA2R1 expression on proliferative and metastatic parameters was analysed in vitro. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP and PC-3 cells. Cell viability/proliferation and motility were significantly increased in LNCaP-PLA2R1 and PC-3 Ctrl compared to LNCaP-Ctrl and PC-3 KD cells, respectively. However, levels of apoptosis, clonogenicity and cell invasion were reduced in LNCaP-PLA2R1 and PC-3 Ctrl cells. Gene expression analysis revealed an up-regulation of fibronectin 1 (FN1), TWIST homolog 1 (TWIST1), and cyclin-dependent kinase 6 (CDK6) in LNCaP-PLA2R1. In LNCaP xenografts, PLA2R1-dependent regulation of clonogenicity appeared to outweigh the receptor's pro-oncogenic properties, resulting in decreased tumour growth, supporting the tumour-suppressive role of PLA2R1. Alternatively, PC-3 Ctrl xenografts exhibited faster tumour growth compared to PC-3 KD cells, suggesting a pro-oncogenic effect of endogenous PLA2R1 expression. The differential growth-regulatory effects of PLA2R1 may be mediated by FN1, TWIST1, and CDK6 expression, although further investigation is required.