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To clinically evaluate complex diseases at the point of care (POC), it is crucial to have multiplexed quantitative sensing of biomolecules. This need has led to the development of ultra-sensitive and cost-effective biosensors. Electronic biosensors based on extended gate field-effect transistor (EGFET) are promising candidates for multiplexed biosensing due to their excellent sensitivity, facile integration, and straightforward interfacing with the readout electronics. Although some high-performance biosensing applications of EGFET systems have been demonstrated [1,2], current EGFET-based biosensors still need to overcome practical issues to reach broad use in POC settings, such as readout at low current levels (~nA), limited multiplexing ability, and complex customized nanofabrication of FET transducers. We demonstrate a custom standalone multiplexed EGFET-based potentiometric biosensing platform relying on modular electronics constructed with off-the-shelf components and an innovative assay format employing bioconjugates of gold nanoparticles (AuNPs) and antibodies (Abs). Our platform comprises a disposable sensing chip containing an EG electrode array functionalized with bioreceptor molecules, a multiplexing module enabling reproducible scanning of up to 32 electrodes, and a readout module based on a commercial FET operating in constant charge mode to enable indirect monitoring of gate surface potential shifts caused by analyte binding. We observe a remarkable 5-fold amplification of the potentiometric response due to the labeling of target antibodies with AuNPs in comparison with the traditional non-labeled assay. We investigate the amplification mechanism by analyzing and modeling the impedimetric response of the system and propose that AuNPs act as localized regions of high surface charge mediating the diffusion barrier layer disruption. The AuNP-enhanced response brings the sensitivity of our platform to a level comparable with fully customized potentiometric

Electronic biosensors based on the extended-gate field-effect transistor (EGFET) concept show great promise for multiplexed biosensing in clinical screening and monitoring of complex diseases at the point of care. These biosensors offer high sensitivity, simplified integration, and easy interfacing with conventional readout electronics. However, EGFET biosensors face practical limitations that hinder their widespread use, such as the need for complex nanostructuring of extended gates (EGs) and FET transducers to achieve ultra-high sensitivity and operate at low current levels (in the ~nA range). We present a low-cost, standalone, and multiplexed EGFET immunosensor. Our system consists of a disposable sensing chip with an EG electrode array, a multiplexing module that allows reproducible switching between up to 32 EGs, and a readout module built around a commercial FET transducer using off-the-shelf electronic components. We detect the binding of IgG antibodies by indirectly monitoring the gate surface potential, operating the FET transducer in constant charge mode. To achieve high sensitivity levels (approximately 20 mV/dec) and a low detection limit (around 10 fM), comparable to state-of-the-art nanostructured EGFET biosensors, we employ an innovative assay approach. This involves labeling the analyte antibody through bioconjugation with gold nanoparticles (AuNPs), resulting in a detection limit approximately 10^4 times lower than with the gold-standard optical method for the same antibody. Remarkably, our approach leads to a 5-fold amplification of the potentiometric response compared to direct antibody detection without labeling. To understand the origin of this amplification, we analyze the impedimetric response and find that AuNPs exhibit nanoantennae-like behavior, disrupting charge uniformity within the diffusion barrier layer and producing signal amplification. These findings demonstrate the potential for creating a new cost-effective and highly sensitive po

Cell culture has been one of the most relevant techniques in biology, providing a platform to investigate fundamental research questions about biological processes [1-2]. However, the complexity of 3D tissue structure and the interactions between different cell types and signaling molecules in vivo make it evident that conventional 2D cell culture is not capable of properly recapitulating the physiological dynamics of the tissues in vivo [3]. The latter, in addition to the ethical concerns of animal and human testing, have driven the development of in vitro tissue models that can reassemble in vivo 3D tissue microenvironments known as microphysiological systems (MPSs) [2-3]. The integration of sensors in culture platforms enables in situ monitoring of MPSs providing high sensitivity, temporal, and spatial resolution [4]. In this work, we present the development of a 3D-printed in vitro culturing platform for in situ monitoring of microencapsulated spheroids (MCSs) [5]. Flexible interdigitated gold microelectrodes are integrated into the 3D-printed structures to locally monitor the changes in the environment of the microencapsulated spheroids [6]. The pH of the environment was monitored for different MCSs densities. This allowed us to study the relationship between acidification and MCSs density in controlled environmental settings. The development of novel sensing and culturing platforms provides the possibility to enhance the physiological understanding of in vivo systems through the study of MPSs. References [1] Segeritz, C. P., & Vallier, L. (2017). Cell culture: Growing cells as model systems in vitro. In Basic science methods for clinical researchers (pp. 151-172). Academic Press. [2] Wikswo, J. P. (2014). The relevance and potential roles of microphysiological systems in biology and medicine. Experimental biology and medicine, 239(9), 1061-1072. [3] Sohn, L. L., Schwille, P., Hierlemann, A., Tay, S., Samitier, J., Fu, J., & Loskill, P. (2020). How can microflu

Biomolecular and physical stimuli, such as stiffness and stress, of the extracellular environment, regulate collective cell dynamics and tissue patterning. The viscosity in the tumor microenvironment can increase due to the accumulation of macromolecules over time. Islands of rigid tumors are surrounded by soft cells that are more deformable than their healthy counterparts. Nonetheless, how the viscosities of the tumor microenvironment regulate collective cell spatial and temporal organization is not fully understood. Here, we used the human hepatoma (HepG2) cancer cells, the basic structural component of the liver, as an example to study the influence of viscosity (range from 0.8 cP to 15 cP) on cancer cell collective behavior in 3D microcapsules reactors. Alginate/Alginate-carboxymethylcellulose microcapsules (AL/AL-CMC MCs) with HepG2 cells were generated using a home-made high-throughput droplet-based microfluidic platform. Cell distribution, cell proliferation, spheroids growth, morphology change, and cytoskeleton difference were observed and quantified, showing a significant effect on viscosity change. Importantly, F-actin and keratin 8 intensity and distribution results can be a cue that viscosity increases enhancing the ability of cancer cells to squeeze through dense tissue. The results thus demonstrate that extracellular viscosity as an important physical cue regulates tumor development relevance to cancer biology.

Engineered T cells that express chimeric antigen receptors (CARs) show a big potential in cancer immunological research. They are synthetic receptors that can target antigens independently from the MHC complex. However, most tumor associated antigens are not only expressed on the tumor site but also on healthy tissues. To switch off CAR T cells in case of on-target/off-tumor effects occur, an adaptor CAR platform, called the universal CAR (UniCAR), was developed. In contrast to conventional CAR systems this platform consists of the engineered T cells and a linker molecule, called target module (TM), that is both binding to the target antigen and recognized by the UniCAR T cell. With this approach there is the possibility to switch the system on and off when needed. Fibroblast activation protein (FAP) is a protein of high interest in cancer research as it is upregulated in most of epithelial cancers but shows a comparably low expression in healthy tissues. It is shown to be expressed on cancer associated fibroblasts and therefore plays an important role in the tumor microenvironment where it has pro-tumorigenic activity both in the FAP positive and surrounding cells. Given this, the goal of this work was to develop new TMs against FAP. To achieve such aim, we cloned two TM formats, either based on a single-chain variable fragment (scFv) or an IgG4 based backbone. The difference in the size of the backbones translates into different half-lifes and kinetics. These different TM formats can be used to customize UniCAR therapy according to the given circumstances. We were able to purify and perform various experiments with the mentioned TMs. We could show binding to the target cells using flow cytometry. We could also show that the TMs could be used to induce specific lysis in vitro on monolayer cells and spheroids. We were also able to show UniCAR T cell infiltration into the spheroids and their consequent activation. Moreover, in mouse models the TMs could be used for P

Immunotherapy based on chimeric antigen receptor (CAR) T-cells has demonstrated remarkable therapeutic effects, particularly against some hematological cancers. A versatile adaptor CAR system called RevCAR, consisting of RevCAR T-cells and a bispecific target module (RevTM) has been developed to overcome severe side effects associated with conventional CAR T-cell therapy. As the activity of RevCAR T-cells can be steered based on the availability of RevTM, working as an on/off switch, the system can be immediately turned off if side effects occur. Furthermore, the RevCAR system is highly flexible, since the same RevCAR T-cell can be directed towards different tumor-associated antigens (TAA) simply by adding RevTMs with different specificities. However, the effectiveness of CAR T-cells against solid tumors remains limited particularly due to their immunosuppressive tumor microenvironment (TME). To overcome these hurdles, we have established a novel RevCAR system targeting immune checkpoint molecules such as PD-L1, which are frequently overexpressed by cancer cells to suppress immune responses. We have constructed novel RevTMs that can redirect RevCAR T-cells to kill tumor cells that express such immune checkpoint molecules. Furthermore, true AND gate tumor targeting was achieved by targeting a TAA in addition to PD-L1 in a combinatorial manner using our Dual-RevCAR system. In this way, targeting PD-L1 not only results in Dual-RevCAR T-cell activation but simultaneously blocks the immunosuppressive PD-L1/PD-1 axis. Altogether, we have turned an immunosuppressive marker into an immune-activating signal that might modulate the TME in a beneficial manner, showing promise for the development of an effective immunotherapy against solid tumors.

Bei diesem Datensatz handelt es sich um analytische Charakterisierungen (ESI-MS, HR-MS, MALDI-TOF-MS) und Radiomarkierungsuntersuchungen zum nonadentaten Bispidin-Chelator bzw. Bispidin-TATE Konjugat mit Lutetium-177, Indium-111 und/oder Actinium-225. 1H, 13 C NMR and crystallographic data stored by collaboration partner (Heidelberg University)

This publication includes datasets of electrical impedance spectroscopy measurements of the response of functionalized gold nanowires when interacting with antigens and antibodies related to SARS-CoV-2 in physiological conditions and in human plasma samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic with millions of infections and deaths worldwide and devastating impact on global economy. Up to now, vaccines and monoclonal antibody (mAb) therapies lack to provide a long-lasting protection against rapidly evolving new emerging SARS-CoV-2 variants. Thus, novel therapeutic options are pressingly needed especially for immunocompromised patients and/or patients with high risk for developing a severe coronavirus disease 2019 (COVID-19). In that regard, we developed a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2). This ACE2 decoy potently binds to the SARS-CoV-2 receptor binding domain (RBD), neutralizes SARS-CoV-2 as well as the Delta and Omicron variant and protects hamsters from a SARS-CoV-2 infection. To additionally use this ACE2 decoy for elimination of virus infected cells, we equipped it with an epitope tag. Thus, it can be applied as adapter molecule in the modular platform technologies UniMAB and UniCAR, which already demonstrated great success in the setting of malignant diseases. As adapter molecule the ACE2 decoy is able to efficiently recruit either universal chimeric antigen receptor (UniCAR) modified T cells or, in combination with an anti-peptide epitope-anti-CD3 bispecific Ab of the UniMAB system, unmodified T cells to efficiently kill SARS-CoV-2 RBD expressing human cells. Taken together, the ACE2 decoy represents a very promising immunotherapeutic drug for both SARS-CoV-2 variant neutralization and infected cell killing via the UniMAB and UniCAR system and might, therefore, clearly improve the treatment of COVID-19 patients.

Liver cancer had the fastest increasing mortality for decades. Compared to 2D models, that cannot simulate the microenvironment, and key functions of organs, 3D organoids models are now entering the field of in vitro analysis. Here, alginate-chitosan hybrid microcapsules are fabricated with a high reproducible cross junction based microfluidic droplet generation system. With high reproducibility and compartmentalization, 103 of microcapsules can be generated within a few minutes. The effects of fluid flow rates on its sizes and shell thicknesses were systematically studied, forming microcapsules with different capsule diameters (~ 300 to 700 µm) and thicknesses (~ 5 to 150 µm). The semi-permeability of these capsules has been well studied, combined a COMSOL model. These microcapsules, with a suitable diffusion rate for nutrients, are applied for human hepatoma cell line encapsulation. Metabolic of organoids in capsules are confirmed by tracking substrates (testosterone) and metabolites (androstenedione). Overall, highly repeatable alginate-chitosan hybrid microcapsules obtained by microfluidic droplet method are not only suitable for liver cancer cells culturing, but also promising for various cell organoids generation.

Liver cancer had the fastest increasing mortality for decades. Compared to 2D models, that cannot simulate the microenvironment, and key functions of organs, 3D organoids models are now entering the field of in vitro analysis. Here, alginate-chitosan hybrid microcapsules are fabricated with a high reproducible cross junction based microfluidic droplet generation system. With high reproducibility and compartmentalization, 103 of microcapsules can be generated within a few minutes. The effects of fluid flow rates on its sizes and shell thicknesses were systematically studied, forming microcapsules with different capsule diameters (~ 300 to 700 µm) and thicknesses (~ 5 to 150 µm). The semi-permeability of these capsules has been well studied, combined a COMSOL model. These microcapsules, with a suitable diffusion rate for nutrients, are applied for human hepatoma cell line encapsulation. Metabolic of organoids in capsules are confirmed by tracking substrates (testosterone) and metabolites (androstenedione). Overall, highly repeatable alginate-chitosan hybrid microcapsules obtained by microfluidic droplet method are not only suitable for liver cancer cells culturing, but also promising for various cell organoids generation.

Background: Functional interaction between cancer cells and the surrounding microenvironment is still not sufficiently understood, which motivates the tremendous interest in the development of numerous in vitro and in vivo tumor models. Study Aims: To study the influence of different permeabilities of microcapsules (MCs) on different cancer cell proliferations, and to design and engineer the formation of 3D tumor clusters in MCs. Materials and Methods: A fluidics-based low-cost methodology was used to reproducibly generate alginate (AL) and alginate-chitosan (AL-CS) MCs in a cross-junctions water-in-oil system. The diffusion through the shell of AL and AL-CS MCs was monitored using fluorescein sodium (376 Da), FITC-Dextran 70 (70 kDa), and FITC-Dextran 2000 (2000 kDa) as fluorescent probes representing small molecules, proteins, and macromolecules, respectively. HepG2 Red FLuc (human hepatoma cell line) and A375 (human melanoma cell line) cultured in high-glucose DMEM medium were used to study the proliferation differences in terms of dimensions and geometries in AL and AL-CS MCs. The metabolic activity of tumor clusters in MCs was confirmed by tracking the turnover of testosterone to androstenedione with lower case Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Results: HepG2 Red FLuc and A375 cells show different proliferation properties in AL and AL-CS MCs. A375 tumor clusters grow faster in more permeable AL MCs and slower in less permeable AL-CS MCs. In the case of HepG2 Red FLuc, a significant difference in proliferation rate was not observed between AL and AL-CS MCs at the early stage (1 week). Interestingly, it was observed that different loose and tight cell cluster morphologies can form, also including cell proliferation along radial directions in both MC types and both cell lines. Cytochrome P450 (CYP)-dependent metabolization of testosterone by both HepG2 Red FLuc and A375 tumor clusters in the AL and AL-CS MCs showed the same trends in good

Silicon nanowire sensors have demonstrated outstanding utility in biosensing, especially for small biomolecules at extremely low concentrations. However, the sensor is less commonly applied in whole-cell monitoring, such as CAR T-cell counting during cancer treatment. The patient’s T-cells are modified to express chimeric antigen receptors (CAR), targeting specific tumor cells in CAR T-cell treatment. Therefore, the CAR T-cell level in blood is an essential parameter when it comes to determining the immune system’s reactivity to fight cancer cells. Although nanosensors are typically beneficial for early cancer diagnosis and detection, we want to expand their application and explore their usage in cancer treatment monitoring and development. Our previous works showed promising results of using nanosensors to find the most effective immunotherapy. In this work, we study the response of silicon nanowire field-effect transistors (SiNW FET) to the binding of CAR T-cells and discuss the benefits and limitations of the sensors in cell monitoring. The SiNW FETs fabricated in a top-down manner showed superior sensitivity to IgG antibodies sensing in our previous study. A peptide with a high affinity to the designed CAR T-cells immobilized on SiNW FETs to detect the cell binding. We observed distinguished signals following the number of cells binding to the sensing area. The results pave the way for using nanosensors in monitoring cancer treatment, yet they suggest some room for improvement.

Objectives Bispidines (3,7-diazabicyclo[3.3.1]nonanes) are of great interest for the use in radiopharmaceutical applications. Combining the advantages of highly preorganised rigid macrocyclic ligands and the flexibility of open-chain ligands, they are able to form highly stable complexes at mild reaction conditions with a broad range of di- and trivalent metal ions.1,2 Here, we present a nonadentate bispidine ligand (Fig. 1) labelled with [177Lu]LuCl3, [225Ac]AcCl3 and [111In]InCl3 at mild conditions and report their stability and inertness in aqueous solution and human serum.3 The results in terms of radiolabelling conditions and serum stabilities are compared with the “gold standard” DOTA, which requires harsh radiolabelling conditions, and the octadentate bispidine ligand H2bispa.2,4 Methods The bispidine ligand was synthesised according to literature on a multigram scale, with an overall yield of 5% over 9 steps.2 Radiolabelling experiments of the nonadentate bispidine ligand with [177Lu]Lu3+ and [225Ac]Ac3+ were carried out at 40 °C using 150 mM NH4OAc buffer (pH 6). Radiolabelling with [111In]In3+ was performed in the same buffer at room temperature. For comparison, the DOTA and the octadentate bispidine ligand H2bispa2 were labelled with the trivalent radiometals as well. The radiochemical yields and purities were monitored via radio-TLC and radio-HPLC for different ligand concentrations after 5 min, 30 min and 60 min. Radiostabilities in human serum were studied by radio-TLC and radio-SEC after 1 h, 1 d, 3 d and 7 d. Results Radiolabelling experiments gave quantitative yields for the formation of both 177Lu- and 225Ac‑complexes at 40 °C after 5 minutes for ligand concentrations of 10-6 mol/L. With [111In]In3+ a quantitative conversion was obtained even at room temperature after 60 minutes with a ligand concentration of 5·10-6 mol/L. For [177Lu]Lu3+, a molar activity of >100 MBq/nmol, for [225Ac]Ac3+ 0.2 MBq/nmol and for [111In]In3+ >20 MBq/nmol was found at

The development of point-of-care (POC) testing platforms has increased during the COVID-19 pandemic due to their multiple benefits including low cost, rapid turnaround time, on-site testing, and minimal sample preparation [1-2]. Although POC tests are a good alternative to the gold standard technique (reverse-transcriptase-polymerase chain reaction, RT-PCR) for SARS-CoV-2 detection, there are challenges regarding their sensitivity and specificity that need to be addressed [3-4]. One strategy to improve the performance of POC is the integration of nanostructures as sensing elements [5]. In this work, we used interdigitated gold nanowires (Au NWs) in combination with electrical impedance spectroscopy (EIS) for the detection of the receptor-binding domain of the S1 protein of the SARS-CoV-2 virus and the respective antibodies that appear during and after infection. Our sensor system was composed of six sensing devices, each of these sensors containing six pairs of interdigitated gold nanowires of 120 nm in width. The surface of the Au NWs was functionalized with antigens or antibodies of SARS-CoV-2 so that the molecules of interest present in the sample can bind to them. The adhesion of molecules to the surface of the Au NWs modulates the physicochemical properties of the surface [6]. As a result, it was possible to correlate the changes in electrical impedance with the binding of specific analytes to the surface of the Au NWs using EIS. The developed sensing platform is an attractive system for screening during pandemics and can be adapted for the detection of relevant target-analyte pairs in different diseases. References [1] E. Valera et al., ?COVID-19 Point-of-Care Diagnostics: Present and Future,? ACS Nano, vol. 15, no. 5, pp. 7899?7906, 2021, doi: 10.1021/acsnano.1c02981. [2] E. Morales-Narváez and C. Dincer, ?The impact of biosensing in a pandemic outbreak: COVID-19,? Biosens. Bioelectron., vol. 163, p. 112274, 2020, doi: https://doi.org/10.1016/j.bios.2020.1122

Despite the fact that chimeric antigen receptor (CAR) engineered T cells have shown encouraging therapeutic effects in hematological setups, the targeting of solid tumor-related antigens still represents a challenge. One main issue is that the expression of this type of antigens is not restricted to cancer cells, but normal tissues express them as well to some degree. In order to avoid strong side-effects caused by on-target/off-tumor effects, more controllable and specific CAR T cell-derived technologies need to be developed. In this line of thought, we developed the switchable, flexible and programmable Reverse (Rev) CAR platform. This system is based on engineered T cells expressing RevCAR molecules, which have extracellular short peptide epitopes incapable of recognizing surface antigens. Thus, the RevCAR T cells are per se inert and will only recognize the target cell through the interaction with an antigen-specific target module, named RevTM. RevTMs are bispecific antibodies designed to bind simultaneously to the target antigen and the short epitopes on the RevCAR engineered T cells. This interaction triggers a specific activation and cytotoxic activity of the RevCAR T cells redirecting them to eradicate the cancer cells. Here, we adapted our RevCAR technology to target the carcinoembryonic antigen (CEA), which is a significant tumor marker for colorectal cancers and other carcinomas. Moreover, we developed two RevTMs with different structures: scFv- and IgG4-based. The first one is built by linking a scFv against CEA to a scFv that recognizes the RevCAR epitope through glycine and serine residues, resulting in a small sized molecule (<60 kDa) with two binding sites. The IgG4-based RevTM connects the same scFv-structures but in this case through the hinge and constant Fc regions (CH2 and CH3) of an IgG4 antibody. The formation of disulfide bridges on the hinge portion causes the formation of homodimers, resulting in a molecule with increased molecular weight (

Objectives: An upregulation of cannabinoid receptors type 2 (CB2) has been reported in association with inflammation processes, traumatic brain injury, neurodegeneration and cancer.[1] The activation of CB2 leads to an anti-inflammatory action. Therefore, the non-invasive assessment of the CB2 availability with PET could improve the decision-making for CB2-directed therapies. We developed a series of fluorinated naphthyridine-2-one-carboxamides as CB2 ligands, of which [18F]LU14[2] and [18F]LU13 were radiosynthesized and biologically evaluated. Methods: The two radioligands [18F]LU14[2] and [18F]LU13 were developed starting from appropriate precursor compounds.The fraction of radiometabolites was quantified in isolated plasma and brain samples at 30 min p.i. The CB2 binding affinities selectivities (expressed and determined from KD and Ki values of both radioligands were determined in vitro. PET studies were performed to evaluate the radioligand uptake into the brains of rats overexpressing the hCB2(D80N) in the right striatum[3]. Results: Favourable properties where achieved for [18F]LU14, which could be further improved for [18F]LU13 (AM, affinity, selectivity and metabolic stability). In rats bearing the local overexpression of the hCB2 a target-specific and reversible uptake for both radioligands was demonstrated. [18F]LU14 reached a TAC peak SUV of 3.3 ± 0.6 at 7.4 ± 2.8 min p.i., and the SUVr (target region–to–cerebellum) was stable between 6.6 and 7.0 after 30 min p.i. For [18F]LU13 a stable SUV of 3.6 ± 0.9 after 26 min p.i. was reached and a close to linearly increasing SUVr (target region–to–cerebellum) up to 8.8 ± 4.3 at 60 min p.i. was determined (slope = 0.15 SUV/min; R² =.0.8). Conclusion: [18F]LU14 and [18F]LU13 showed an excellent ability to image the CB2 receptors in vivo. Additionally, [18F]LU14 revealed a faster washout from the non-target regions in the brain, compared to [18F]LU13. References: [1] Stasiulewicz et al. IJMS, 2020, 21, 2778; [2] Te

Chimeric antigen receptor (CAR) T-cells are unquestionably considered one of the most promising approaches in cancer immunotherapy. Nonetheless, mild to severe toxicities are associated with this approach which include e.g. on-target/off-tumour toxicities and cytokine release syndrome. Aiming for increased clinical safety, a modular universal CAR (UniCAR) platform was developed by our group in which UniCAR T-cells are exclusively activated in the presence of a target module (TM) that specifically establishes the cross-link between target cells and UniCAR T-cells. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) present in the tumour stroma and also found to be overexpressed in tumour cells. This protein plays an important role in promoting tumour growth, metastasis, and immunosuppression and has therefore been studied as a target for cancer diagnosis and treatment. Given this and the demonstrated efficacy, flexibility and switchability of the UniCAR system, currently demonstrated in phase I clinical trials, we hereby aimed to develop TMs targeting FAP that can be used for both immunotherapeutic and theranostic approaches. For that, the single-chain variable fragment (scFv) of an anti-human FAP mAb was fused to the peptide epitope E5B9 that is recognized by the UniCAR T-cells, creating low molecular weight TMs that are rapidly eliminated allowing a specific and recurrent on/off switch of UniCAR T-cell activity via TM dosing. Additionally, extended half-life anti-FAP TMs based on the human IgG4 Fc-domain, including a mutated version, were created intending to strengthen anti-tumour responses and to ease the clinical TM administration at later stages of tumour therapy. All TMs were tested in vitro based on naturally and artificially overexpressing 2D and 3D models, and proven to specifically redirect UniCAR T-cells to FAP-expressing target cells. Positron emission tomography (PET) using 64Cu radiolabelled anti-FAP IgG4 TMs

As next generation for CAR T cells, adaptor CAR platforms have been developed, which are designed to improve safety, but at the same time maintain the high efficiency of the CAR T cell approach. In our lab, we developed the UniCAR system, which consists of universal (Uni)CAR T cells and tumor-specific target modules (TMs), which work as bridging molecules between the UniCAR T cells and the target cells. Until now, type 1 and type 2 UniCARs were developed. Both UniCAR types consist of an extracellular binding domain derived from a monoclonal antibody (mAb) directed to the nuclear La/SS-B protein. Type 1 UniCARs are derived from the anti-La mAb 5B9. Type 2 UniCARs from the anti-La mAb 7B6. As both anti-La mAbs are not able to precipitate native La protein, both anti-La mAbs are directed to a specific cryptic epitope which is not accessible on the cell surface. Thus, the UniCAR T cell is per se inert. To activate the UniCAR T cell for tumor cell killing a TM is needed as a second component. Typically, a TM is composed of a tumor-specific binding domain and the respective La epitope. Consequently, the affinity of the TM towards the target antigen but also towards the UniCAR T cell via the E5B9-tag plays an important role for functionality of the respective UniCAR system. In this study, we representatively aimed to elucidate if and how the affinity of the type 1 UniCAR domain to the E5B9 epitope impacts the functionality of the UniCAR system. To alter the interaction of UniCAR and TM, we designed different mutated E5B9 peptides (M1-M3) carrying one or two amino acid (aa) changes. In detail, aspartic acid and/or glutamic acid were mutated to glycine residues as they most probably are involved in epitope/paratope interactions. We subsequently fused these mutated peptides to an scFv domain, resulting in three different mutated TM versions. By conducting ELISA and flow-cytometry based binding studies, we showed that a single aa exchange (D3>G3) in M1 did not alter the affini

Chimeric antigen receptor (CAR) T-cells are undoubtedly a promising approach in cancer immunotherapy. Nevertheless, mild to severe toxicities are associated with this approach including on-target/off-tumor effects and cytokine release syndrome. Aiming for increased clinical safety, adaptor CAR technologies were developed which include the modular universal CAR (UniCAR) platform developed by our group. UniCAR T-cells are exclusively activated in the presence of a target module (TM), which establishes the cross-link between cancer cells and UniCAR T-cells. These TMs are highly versatile molecules that can be constructed not only by using antibody fragments but also e.g. peptides specifically targeting a receptor or molecule on the cell´s surface. Somatostatin receptor (SSTR) subtype 2 is highly expressed in a variety of malignancies and has therefore been studied as a marker and target for cancer diagnosis and treatment. Currently, SSTR2 agonists and antagonists, such as Tyr3-octreotate (TATE) and BASS or JR11, respectively, are particularly well established and clinically implemented mostly used for diagnostic nuclear medicine. Given this and the proven flexibility and efficacy of the UniCAR system, we hereby aimed to develop small peptide-derived TMs targeting SSTR2 that can be used for both immunotherapeutic and diagnostic approaches. For that, the abovementioned SSTR2 agonist and antagonists were chemically linked to the E5B9 peptide and equipped with the radiometal chelator NODAGA. These TMs were tested in vitro and in vivo, in which they have proven to specifically redirect UniCAR T-cells to human neuroendocrine and breast SSTR2-expressing cancer cells. Furthermore, the enrichment of these anti-SSTR2 peptide TMs at the tumor site was confirmed by positron emission tomography (PET) studies. We hereby designed novel small peptide-derived TMs that can be used for redirection of UniCAR T-cells to SSTR2-expressing cancer cells as well as for PET imaging, proving to b

In the midst of the COVID-19 pandemic, adaptive solutions are needed to allow us to make fast decisions and take effective sanitation measures, e.g., the fast screening of large groups (employees, passengers, pupils, etc.). Although being reliable, most of the existing SARS-CoV-2 detection methods, like polymerase chain reaction or paper-based immunosensors, lack the ability integrated into garments to be used on demand. Here, we report – at the proof-of-concept level – an organic field-effect transistor (OFET)-based biosensing device detecting of both SARS-CoV-2 antigens and anti-SARS-CoV-2 antibodies in less than 20 min. The biosensor was produced by functionalizing an intrinsically stretchable and semiconducting triblock copolymer (TBC) film either with the anti-S1 protein antibodies (S1 Abs) or receptor-binding domain (RBD) of the S1 protein, targeting CoV-2-specific RBDs and anti-S1 Abs, respectively. The obtained sensing platform is easy to realize due to the straightforward solution-based fabrication of the TBC film and the utilization of the reliable physical adsorption technique for the molecular immobilization. The device demonstrates a high sensitivity of about 19%/dec and a limit of detection (LOD) of 0.36 fg/mL for anti-SARS-Cov-2 antibodies and, at the same time, a sensitivity of 32%/dec and a LOD of 76.61 pg/mL for the virus antigen detection. The TBC used as active layer is soft, has a low modulus of 24 MPa, and can be stretched up to 90% with no crack formation of the film. With proper transfer to a stretchable-flexible substrate, the presented concept offers the possibility to realize stretchable biosensors, which might allow the fabrication of wearable platforms for on-the-fly detections of biomolecules to aid reducing – and eventually stopping – the spread of COVID-19 and future pandemics.

Liver cancer is the second most lethal malignancy worldwide. Recently, three-dimensional (3D) cancer organoids models have been constructed and applied to liver cancer research, to predict the therapy outcomes. However, there is still low success and reproducibility rate for generating patient-derived liver tumoroids due to limitations in current technologies. Herein, a high reproducible cross junction based microfluidic droplet generation system is applied for human hepatoma cell line encapsulation and organoids engineering. The fabrication of alginate and alginate-chitosan microcapsules is systematically studied, forming microcapsules with different shell thicknesses (~ 5 to 150 µm) and tunable permeability. In combination with a COMSOL model, the size selective permeability of different molecular complexes through the capsule membrane has been investigated, which is essential to ensure efficient mass transfer of small molecules, and prevent large substances from reaching the loaded cells. Finally, we demonstrate that the cells are prone to aggregate more tightly in capsules with a lower permeability, which causing more hypotonicity and lower viability. Because of the high reproducibility, compartmentalization, and easily permeability tuning, this system not only provides a great platform for liver patient-derived tumoroids forming, but also is promising for other cell organoids design and engineering.

CAR T-cell therapy achieved unparalleled clinical success rates for treatment of patients with hematological malignancies. However, progress and clinical translation towards solid tumors is slow and hampered by many factors e.g. increased complexity, high heterogeneity and an immunosuppressive tumor microenvironment. Thus, CAR T-cell therapy alone might not result in durable antitumor responses. Combinatorial approaches are promising strategies to improve CAR T-cell efficacy in solid tumor treatment. In this regard, we here aim to combine conventional cancer theranostics with CAR T-cell immunotherapy in one single approach. By using the well-established UniCAR system, a novel, multifunctional tool termed PSCA-IgG4 target module (TM) was developed for dual prostate cancer theranostics. It comprises a human PSCA-specific binding domain, the hinge and Fc-domain of human IgG4 molecules as well as the UniCAR epitope E5B9. As shown by in vitro assays with PSCA-positive and PSCA-negative prostate cancer cells, the novel TM redirected UniCAR T cells for efficient tumor cell lysis in a strictly antigen- and TM-dependent manner. After radiolabeling with copper-64 or actinium-225, the novel PSCA-IgG4 TM was successfully applied for diagnostic imaging and targeted radioimmunotherapy. The 64Cu-labeled PSCA-IgG4 TM showed maximal tumor accumulation with optimal tumor-to-background ratios after 1.5 days. Furthermore, targeted alpha-therapy with the 225Aclabeled TM significantly delayed the outgrowth of established tumors in mice. In summary, the here presented, novel PSCA-IgG4 TM is a promising candidate for dual theranostics of prostate cancer that may help to overcome present hurdles in solid tumor therapy. After radiolabeling it facilitates not only targeted alpha-therapy and diagnostic imaging of PSCA, but can be also repurposed as a TM for UniCAR T-cell immunotherapy.

Modifying of immune effector cells with chimeric antigen receptors (CARs) has revealed a promising therapeutic potential for targeting cancer, especially with CAR-modified T cells. However, the use of other immune cells like primary NK cells or NK cell lines appeared as another advantageous approach that can be combined with CAR technology. Unlike T cells, established NK cell lines can be used allogenically as an off-the-shelf product with reduced risk of toxicities. We have established previously a modular Universal CAR platform (UniCAR) which can be switched on/off and allows the flexible targeting of various tumor antigens. This system consists of two parts, the UniCAR-expressing immune effector cells and a target module (TM). The UniCAR-immune cells cannot recognize surface antigens but are only redirected with the TM which contains an antigen-binding moiety on one hand and an epitope recognized by the UniCAR molecules on the other hand. Here, we provide a proof of concept for using the UniCAR system in combination with the NK-92 cell line to target disialoganglioside GD2-expressing tumors. The UniCAR NK-92 cells induced increase in lysis of neuroblastoma and melanoma cell lines in the presence of scFv- or human IgG4-based TMs, associated with specific release of pro-inflammatory cytokines. Moreover, UniCAR NK-92 cells were shown to be functional in eradicating GD2-expressing tumors in experimental mice. In order to investigate the in vivo half-life of the scFv- and IgG4-based TMs, they were radiolabeled with 64Cu and detected using PET imaging. Dynamic PET scanning has shown that the IgG4 format increased the half-life of the TM to around 24 folds in comparison to the scFv-based TMs. In summary, UniCAR NK-92 provides an off-the-shelf universal platform that can be combined with various antibody formats, and can be easily expanded for therapeutic use.

Objectives: During the last 15 years several PSMA PET ligands for prostate cancer imaging have been developed resulting in the recent approvals of 68Ga-PSMA-11, 18F-PSMA-1007 and 18F-DCFPyL.[1,2] However, 99mTc remains a popular radiometal for SPECT imaging due to its longer half-life and availability from 99Mo/99mTc generators especially in outpatient imaging centers. Due to the widespread availability of SPECT cameras PSMA SPECT ligands have the potential to substantially extend the availability of PSMA-based imaging, but currently no PSMA SPECT ligand is approved. MIP-1404, PSMA-I&S and HYNIC-iPSMA are rare examples of PSMA SPECT tracers in clinical development. Accordingly, the aim of our study was to develop a PSMA ligand for 99mTc SPECT imaging of prostate cancer based on an N2S2 chelator, which enables reliable radiolabeling to form stable 99mTc complexes.[3] Methods: A number of compounds, which contain the PSMA binding motif Glu-urea-Lys, a varying linker and a bisaminothiol (BAT)-type N2S2 chelator, were synthesized. Selected compounds were radiolabeled with 99mTc, followed by assessment of in vitro stability of the formed complexes via radio-HPLC. The specific binding affinity towards PSMA and the internalization of the radioligands were examined in LNCaP cells, supplemented by autoradiographic studies. Tissue distribution and tumor accumulation were evaluated in LNCaP-tumor bearing mice via quantitative SPECT/CT imaging, benchmarked with [99mTc]Tc-PSMA-I&S[4] and [68Ga]Ga-PSMA-11. Results: Among the six 99mTc radioligands synthesized and examined, [99mTc]TcO-ABX474 showed most favorable properties. Radiolabeling of ABX474 (50 µg) was achieved starting from [99mTc]NaTcO4 (0.2-2 GBq) in saline (1.4 mL) using SnCl2 (1 µg in 0.01 M HCl) as reducing agent, in presence of calcium-D-heptagluconate (10 µg) and D-mannitol (1 mg) at pH 5-6. Incubation at r.t. for 20 min followed by heating at 80°C for 20 min afforded the product-containing solution with a radioche

Bispidines (3,7-diazabicyclo[3.3.1]nonane) and their derivatives act as bifunctional chelators (BFC), combining the advantages of multidentate macrocyclic and acyclic ligands (e.g., high kinetic inertness, rapid radiolabelling under mild conditions) [1]. This bicyclic chelator system shows a great diversity in terms of its denticity and type of functional groups, yielding a wide range of multidentate ligands that can bind a variety of different metal ions [1-3]. In addition, they allow a facile functionalisation of targeting molecules such as peptides, peptidomimetics, and bispecific antibodies which can also be used as target modules for adapter CAR T-cell cross-linkage [1, 4]. Herein, we present a nonadentate bispidine ligand labelled with [177Lu]Lu3+ and [225Ac]Ac3+ at mild conditions. The radiometal complexes have been obtained with high radiochemical yields (99%) and are stable in human serum [3]. This is unique so far, as many chelators are not able to bind both LuIII and AcIII under mild conditions (physiological pH, T<40°C) with fast complexation kinetics and high molar activities (>100 MBq/nmol for [177Lu]Lu3+ and ~0.2 MBq/nmol for [225Ac]Ac3+). For targeting, the chelator was functionalised with a peptidic somatostatin analogue (Tyr3 -octreotate), which addresses the somatostatin subtype receptor 2 in neuroendocrine tumours. Both 177Lu(III)- and 225Ac(III)-labelled conjugates were investigated towards their binding affinity and internalization in a murine pheochromocytoma (MPC) and human pancreatic carcinoid (BON1) tumour cell line and were compared with [177Lu]Lu(III)- and [ 225Ac]Ac(III)-DOTA-TATE. The presented 177Lu(III)- and 225Ac(III)-labelled bispidine-conjugates show favourable labelling kinetics and high radiostabilities in human serum. The radioconjugates exhibited dissociation constants in the lower nanomolar range (<10 nM) and high internalisation rates (>95 %) in both cell lines. In comparison to the corresponding DOTA-radioconjugates, milder

Background: Glioblastoma (GBM) is a very aggressive brain tumor, associated with poor prognosis and survival. So far, the efficiency of available therapies is limited. After proving their effectiveness in targeting hematological malignancies, chimeric antigen receptor (CAR) T cells might provide a promising therapeutic approach for GBM. Here, we present our switchable Reverse CAR technology (RevCAR). Unlike conventional CAR T cells, RevCAR T cells contain an epitope in their extracellular receptor domain, and can only be activated via bispecific target modules (RevTM) which recognize the RevCAR T cells on one side and tumor cells on the other side. Once these TMs are eliminated, RevCARs are switched off. In addition, we have developed dual-targeting RevCARs allowing the control of T cells according to the AND gate logic of Boolean algebra. Methods: RevTMs directed against GMB were expressed in eukaryotic cells and purified with affinity chromatography. The ability of these RevTMs to bind GBM cells and RevCAR T cells was analyzed using flow cytometry. Moreover, the capability of the mono-specific and the dual-targeting RevCAR T cells to kill GBM cells was analyzed using luminescence-based cytotoxicity assay in the absence or presence of a range of RevTM concentrations. Secreted pro-inflammatory cytokines were also evaluated by ELISA. In addition to the in vitro assays, a proof of concept co-injection experiment was performed in vivo. Results: In this study, we show that GBM-specific RevTMs can bind both the RevCAR T cells and the GBM cells. Importantly, the RevCAR T cells can be activated to secrete pro-inflammatory cytokines and to efficiently kill GBM cells via the RevTMs. Moreover, we were able to prove that dual-targeting RevCAR T cells can be activated only upon recognition of two different GBM targets, thereby allowing a highly specific and selective killing of GBM both in vitro and in vivo. Conclusion: In conclusion, the switchable RevCAR platform is a novel t

As next generation for CAR T cells, adaptor CAR platforms have been developed, which are designed to improve safety, but at the same time maintain the high efficiency of the CAR T cell approach. In our lab, we developed the UniCAR system, which consists of universal (Uni)CAR T cells and tumor-specific target modules (TMs), which work as bridging molecules between the UniCAR T cells and the target cells. Until now, type 1 and type 2 UniCARs were developed. Both UniCAR types consist of an extracellular binding domain derived from a monoclonal antibody (mAb) directed to the nuclear La/SS-B protein. Type 1 UniCARs are derived from the anti-La mAb 5B9. Type 2 UniCARs from the anti-La mAb 7B6. As both anti-La mAbs are not able to precipitate native La protein, both anti-La mAbs are directed to a specific cryptic epitope which is not accessible on the cell surface. Thus, the UniCAR T cell is per se inert. To activate the UniCAR T cell for tumor cell killing a TM is needed as a second component. Typically, a TM is composed of a tumor-specific binding domain and the respective La epitope. Consequently, the affinity of the TM towards the target antigen but also towards the UniCAR T cell via the E5B9-tag plays an important role for functionality of the respective UniCAR system. In this study, we representatively aimed to elucidate if and how the affinity of the type 1 UniCAR domain to the E5B9 epitope impacts the functionality of the UniCAR system. To alter the interaction of UniCAR and TM, we designed different mutated E5B9 peptides (M1-M3) carrying one or two amino acid (aa) changes. In detail, aspartic acid and/or glutamic acid were mutated to glycine residues as they most probably are involved in epitope/paratope interactions. We subsequently fused these mutated peptides to an scFv domain, resulting in three different mutated TM versions. By conducting ELISA and flow-cytometry based binding studies, we showed that a single aa exchange (D3>G3) in M1 did not alter the affini

Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic PET imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with 64Cu2+ the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with 225Ac3+ and applied for targeted alpha therapy. A single injection of the 225Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy.

Dendritic cells (DCs) play a key role in the orchestration of antitumor immunity. Activated DCs efficiently enhance antitumor effects mediated by natural killer cells and T lymphocytes. Conversely, tolerogenic DCs essentially contribute to an immunosuppressive tumor microenvironment. Thus, DCs can profoundly influence tumor progression and clinical outcome of tumor patients. To gain novel insights into the role of human DCs in pancreatic ductal adenocarcinoma (PDAC), we explored the frequency, spatial organization, and clinical significance of conventional DCs type 1 (cDC1s) and type 2 (cDC2s) and plasmacytoid DCs (pDCs) in primary PDAC tissues. A higher density of whole tumor area (WTA)- and tumor stroma (TS)-infiltrating cDC1s was significantly associated with better disease-free survival (DFS). In addition, an increased frequency of intraepithelial tumor-infiltrating cDC2s was linked to better DFS and overall survival (OS). Furthermore, an increased density of WTA- and TS-infiltrating pDCs tended to improve DFS. Moreover, a higher frequency of WTA- and TS-infiltrating cDC1s and pDCs emerged as an independent prognostic factor for better DFS and OS. These findings indicate that tumor-infiltrating DCs can significantly influence the clinical outcome of PDAC patients and may contribute to the design of novel treatment options that target PDAC-infiltrating DCs.

Radiation of tumor cells can lead to the selection and outgrowth of tumor escape variants. As radioresistant tumor cells are still sensitive to retargeting of T cells, it appears promising to combine radio- with immunotherapy keeping in mind that the radiation of tumors favors the local conditions for immunotherapy. However, radiation of solid tumors will not only hit the tumor cells but also the infiltrated immune cells. Therefore, we wanted to learn how radiation influences the functionality of T cells with respect to retargeting to tumor cells via a conventional bispecific T cell engager (BiTE) and our previously described modular BiTE format UNImAb. T cells were irradiated between 2 and 50 Gy. Low dose radiation of T cells up to about 20 Gy caused an increased release of the cytokines IL2, TNF and interferon-g and an improved capability to kill target cells. Although radiation with 50 Gy strongly reduced the function of the T cells, it did not completely abrogate the functionality of the T cells.

Molecular imaging offers the possibility to investigate biological and biochemical processes non-invasively and to obtain information on both anatomy and dysfunctions. Based on the data obtained, a fundamental understanding of various disease processes can be derived and treat-ment strategies can be planned. In this context, methods that combine several modalities in one probe are increasingly being used. Due to a comparable, very high sensitivity and provided complementary information, the combination of nuclear and optical probes has taken on a special significance. In this review article, dual-labelled systems for biomodal nuclear and optical imaging based on both modular ligands and nanomaterials are discussed. Particular attention is paid to radiometal-labelled molecules for single-photon emission computed tomography (SPECT) and positron emission tomography (PET), and metal complexes combined with fluorescent dyes for optical imaging. The clinical potential of such probes, especially for fluorescence-guided surgery is assessed.

Most patients with head and neck squamous cell carcinomas (HNSCC) are diagnosed at a locally advanced stage and show heterogeneous treatment responses. Low SLC3A2 (solute carrier family 3 member 2) mRNA and protein (CD98hc) expression levels are associated with higher locoregional control in HNSCC patients treated with primary radiochemotherapy or postoperative radiochemotherapy, suggesting that CD98hc could be a target for HNSCC radiosensitization. One of the targeted strategies for tumor radiosensitization is precision immunotherapy, e.g., the use of chimeric antigen receptor (CAR) T cells. This study aimed to define the potential clinical value of new treatment approaches combining conventional radiotherapy with CD98hc-targeted immunotherapy. To address this question, we analyzed the antitumor activity of the combination of fractionated irradiation and switchable universal CAR (UniCAR) system against radioresistant HNSCC cells in 3D culture. CD98hc-redirected UniCAR T cells showed the ability to destroy radioresistant HNSCC spheroids. Also, the infiltration rate of the UniCAR T cells was enhanced in the presence of the CD98hc target module. Furthermore, sequential treatment with fractionated irradiation followed by CD98hc-redirected UniCAR T treatment showed a synergistic effect. Taken together, our obtained data underline the improved antitumor effect of the combination of radiotherapy with CD98hc-targeted immunotherapy. Such a combination presents an attractive approach for the treatment of high-risk HNSCC patients.

The non-classical human leukocyte antigen (HLA)-G exerts immune-suppressive properties modulating both NK and T cell responses. While it is physiologically expressed at the maternal–fetal interface and in immune-privileged organs, HLA-G expression is found in tumors and in virus-infected cells. So far, there exists little information about the role of HLA-G and its interplay with immune cells in biopsies, surgical specimen or autopsy tissues of lung, kidney and/or heart muscle from SARS-CoV-2-infected patients compared to control tissues. Heterogeneous, but higher HLA-G protein expression levels were detected in lung alveolar epithelial cells of SARS-CoV-2-infected patients compared to lung epithelial cells from influenza-infected patients, but not in other organs or lung epithelia from non-viral-infected patients, which was not accompanied by high levels of SARS-CoV-2 nucleocapsid antigen and spike protein, but inversely correlated to the HLA-G-specific miRNA expression. High HLA-G expression levels not only in SARS-CoV-2-, but also in influenza-infected lung tissues were associated with a high frequency of tissue-infiltrating immune cells, but low numbers of CD8+ cells and an altered expression of hyperactivation and exhaustion markers in the lung epithelia combined with changes in the spatial distribution of macrophages and T cells. Thus, our data provide evidence for an involvement of HLA-G and HLA-G-specific miRNAs in immune escape and as suitable therapeutic targets for the treatment of SARS-CoV-2 infections.

We report a nonadentate bispidine (3,7-diazabicyclo[3.3.1]nonane) that unveils the potential to bind theranostically relevant radionuclides, including indium-111, lutetium-177 and actinium-225 at mild labeling conditions. This radiopharmaceutical candidate allows the simultaneous application of imaging and treatment (radionuclide theranostics) without changing the type of bioconjugate, i.e. it allows the strong binding to an imaging and a therapeutic radionuclide by the same chelator. Since sophisticated coordination chemistry is required to achieve high thermodynamic and kinetic stability, it is not surprising that only a few chelators have been reported that are able to strongly bind several radionuclides to a satisfactory extent. Bispidine-derived ligands have proven to be ideal for di- and trivalent metal ions with generally fast complexation kinetics and high in vitro and in vivo stabilities. The presented (radio)complexes are formed under mild conditions (pH 6, <40°C) and exhibit thermodynamic stability and inertness in human serum comparable to the corresponding DOTA complexes. The bispidine-based complexing agent was conjugated to a peptide, targeting somatostatin type 2 receptors (SSTR2), overexpressed on neuroendocrine tumors. The 177Lu- and 225Ac-labeled conjugates were investigated, considering their binding to two different SSTR2 positive cell lines, including the human pancreatic carcinoid tumor (BON-SSTR2+) and the murine pheochromocytoma cell line (MPC). The biodistribution and accumulation pattern in MPC tumor-bearing mice was also evaluated. The LuIII and AcIII complexes studied show how ligand structures can be optimized in general by extending the denticity and varying the donor set in order to allow for fast complex formation and medically relevant inertness.

Purpose 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) is utilized for staging and treatment planning of head and neck squamous cell carcinomas (HNSCC). Some older publications on the prognostic relevance showed inconclusive results, most probably due to small study sizes. This study evaluates the prognostic and potentially predictive value of FDG-PET in a large multi-center analysis. Methods Original analysis of individual FDG-PET and patient data from 16 international centers (8 institutional datasets, 8 public repositories) with 1104 patients. All patients received curative intent radiotherapy/chemoradiation (CRT) and pre-treatment FDG-PET imaging. Primary tumors were semi-automatically delineated for calculation of SUVmax, SUVmean, metabolic tumor volume (MTV) and total lesion glycolysis (TLG). Cox regression analyses were performed for event-free survival (EFS), overall survival (OS), loco-regional control (LRC) and freedom from distant metastases (FFDM). Results FDG-PET parameters were associated with patient outcome in the whole cohort regarding clinical endpoints (EFS, OS, LRC, FFDM), in uni- and multivariate Cox regression analyses. Several previously published cut-off values were successfully validated. Subgroup analyses identified tumor- and human papillomavirus (HPV) specific parameters. In HPV positive oropharynx cancer (OPC) SUVmax was well suited to identify patients with excellent LRC for organ preservation. Patients with SUVmax of 14 or less were unlikely to develop loco-regional recurrence after definitive CRT. In contrast FDG PET parameters deliver only limited prognostic information in laryngeal cancer. Conclusion FDG-PET parameters bear considerable prognostic value in HNSCC and potential predictive value in subgroups of patients, especially regarding treatment de-intensification and organ-preservation. The potential predictive value needs further validation in appropriate control groups. Further research on advanced imaging appro

The cannabinoid receptor type 2 (CB2R) is an attractive target for diagnosis and therapy of neurodegenerative diseases and cancer. Recently, we reported a novel naphthyrid-2-one based positron-emission tomography (PET) radioligand for imaging of the CB2R in the brain ([18F]5). In this study we aimed at the development of a novel 18F-labeled CB2R radioligand with improved binding properties and metabolic stability. Starting from the structure of 5, we developed a novel series of fluorinated derivatives by modifying the substituents at the naphthyrid-2-one subunit. Compound 28 (LU13) was identified with the highest binding affinity and selectivity versus CB1R (CB2RKi = 0.6 nM; CB1RKi/CB2RKi > 1000) and was selected for radiolabeling with 18F and biological characterization. The radiofluorination was performed starting from the corresponding bromo-precursor (31) bearing a fully deuterated N-alkyl chain to protect against defluorination. The in vitro evaluation of [18F]LU13 proved the high binding affinity of the radioligand towards rat (rCB2RKD = 0.2 nM) and human (hCB2RKD = 1.1 nM) CB2R. Metabolism studies in mice revealed a metabolic stability at 30 min p.i. with fractions of parent compound of >80% in the brain and 90% in the spleen with only trace of defluorination products detected in plasma. PET imaging in a rat model of vector-based/related overexpression in the striatum revealed a high signal to background ratio, demonstrating the ability of [18F]LU13 to reach and selectively label the hCB2R in the brain. Thus, [18F]LU13 is a novel and highly promising PET radioligand for the imaging of up regulated CB2R expression under pathological conditions in the brain.

Immunotherapy using CAR-T cells is a new paradigm technology for cancer treatment. To avoid severe side effects and tumor escape variants observed for conventional CAR-T cells approach, adaptor CAR technologies are under development, where intermediate target modules redirect immune cells against cancer cells. In this work, silicon nanowire field effect transistors are used to assist in the development of target modules for an optimized CAR-T cell operation. Focusing on a library of seven variants of E5B9 peptide that is used as CAR peptide epitope, we performed multiplexed binding tests in serum using nanosensor chips. Peptides have been immobilized onto the sensor to compare the signals of transistor upon titration with anti-E5B9 antibodies. Correlation analysis of binding affinities and sensitivities enabled a selection of best candidates for the interaction between CAR and target modules. Finally, cytotoxic functionality of CAR-T cells in combination with the selected target modules were successfully proven. Our results open the perspective for the nanobiosensorics to go beyond the early diagnostics in the field of clinical cancer research, and paves the way towards personalization and efficient monitoring of the immunotherapeutic treatment, where the quantitative analysis with the standard techniques is not an option.

The cannabinoid receptor type 2 (CB2R) is an attractive target for diagnosis and therapy of neurodegenerative diseases and cancer. Recently, we reported a novel naphthyrid-2-one based positron-emission tomography (PET) radioligand for imaging of the CB2R in the brain ([18F]5). In this study we aimed at the development of a novel 18F-labeled CB2R radioligand with improved binding properties and metabolic stability. Starting from the structure of 5, we developed a novel series of fluorinated derivatives by modifying the substituents at the naphthyrid-2-one subunit. Compound 28 (LU13) was identified with the highest binding affinity and selectivity versus CB1R (CB2RKi = 0.6 nM; CB1RKi/CB2RKi > 1000) and was selected for radiolabeling with 18F and biological characterization. The radiofluorination was performed starting from the corresponding bromo-precursor (31) bearing a fully deuterated N-alkyl chain to protect against defluorination. The in vitro evaluation of [18F]LU13 proved the high binding affinity of the radioligand towards rat (rCB2RKD = 0.2 nM) and human (hCB2RKD = 1.1 nM) CB2R. Metabolism studies in mice revealed a metabolic stability at 30 min p.i. with fractions of parent compound of >80% in the brain and 90% in the spleen with only trace of defluorination products detected in plasma. PET imaging in a rat model of vector-based/related overexpression in the striatum revealed a high signal to background ratio, demonstrating the ability of [18F]LU13 to reach and selectively label the hCB2R in the brain. Thus, [18F]LU13 is a novel and highly promising PET radioligand for the imaging of up regulated CB2R expression under pathological conditions in the brain.

Silicon nanowire sensors have demonstrated outstanding utility in biosensing, especially for small biomolecules at extremely low concentrations. However, the sensor is less commonly applied in whole-cell monitoring, such as CAR T-cell counting during cancer treatment. The patient’s T-cells are modified to express chimeric antigen receptors (CAR), targeting specific tumor cells in CAR T-cell treatment. Therefore, the CAR T-cell level in blood is an essential parameter when it comes to determining the immune system’s reactivity to fight cancer cells. Although nanosensors are typically beneficial for early cancer diagnosis and detection, we want to expand their application and explore their usage in cancer treatment monitoring and development. Our previous works showed promising results of using nanosensors to find the most effective immunotherapy. In this work, we study the response of silicon nanowire field-effect transistors (SiNW FET) to the binding of CAR T-cells and discuss the benefits and limitations of the sensors in cell monitoring. The SiNW FETs fabricated in a top-down manner showed superior sensitivity to IgG antibodies sensing in our previous study. A peptide with a high affinity to the designed CAR T-cells immobilized on SiNW FETs to detect the cell binding. We observed distinguished signals following the number of cells binding to the sensing area. The results pave the way for using nanosensors in monitoring cancer treatment, yet they suggest some room for improvement.

Chimeric antigen receptor (CAR)-expressing T-cells are without a doubt a breakthrough therapy for hematological malignancies. Despite their success, clinical experience has revealed several challenges, which include relapse after targeting single antigens such as CD19 in the case of B-cell acute lymphoblastic leukemia (B-ALL), and the occurrence of side effects that could be severe in some cases. Therefore, it became clear that improved safety approaches, and targeting multiple antigens, should be considered to further improve CAR T-cell therapy for B-ALL. In this paper, we address both issues by investigating the use of CD10 as a therapeutic target for B-ALL with our switchable UniCAR system. The UniCAR platform is a modular platform that depends on the presence of two elements to function. These include UniCAR T-cells and the target modules (TMs), which cross-link the T-cells to their respective targets on tumor cells. The TMs function as keys that control the switchability of UniCAR T-cells. Here, we demonstrate that UniCAR T-cells, armed with anti-CD10 TM, can efficiently kill B-ALL cell lines, as well as patient-derived B-ALL blasts, thereby highlighting the exciting possibility for using CD10 as an emerging therapeutic target for B-cell malignancies.

Liver cancer is the second most lethal malignancy worldwide. Recently, three-dimensional (3D) cancer organoids models have been constructed and applied to liver cancer research, to predict the therapy outcomes. However, there is still low success and reproducibility rate for generating patient-derived liver tumoroids due to limitations in current technologies. Herein, a high reproducible cross junction based microfluidic droplet generation system is applied for human hepatoma cell line encapsulation and organoids engineering. The fabrication of alginate and alginate-chitosan microcapsules is systematically studied, forming microcapsules with different shell thicknesses (~ 5 to 150 µm) and tunable permeability. In combination with a COMSOL model, the size selective permeability of different molecular complexes through the capsule membrane has been investigated, which is essential to ensure efficient mass transfer of small molecules, and prevent large substances from reaching the loaded cells. Finally, we demonstrate that the cells are prone to aggregate more tightly in capsules with a lower permeability, which causing more hypotonicity and lower viability. Because of the high reproducibility, compartmentalization, and easily permeability tuning, this system not only provides a great platform for liver patient-derived tumoroids forming, but also is promising for other cell organoids design and engineering.

CAR T-cell therapy achieved unparalleled clinical success rates for treatment of patients with hematological malignancies. However, progress and clinical translation towards solid tumors is slow and hampered by many factors e.g. increased complexity, high heterogeneity and an immunosuppressive tumor microenvironment. Thus, CAR T-cell therapy alone might not result in durable antitumor responses. Combinatorial approaches are promising strategies to improve CAR T-cell efficacy in solid tumor treatment. In this regard, we here aim to combine conventional cancer theranostics with CAR T-cell immunotherapy in one single approach. By using the well-established UniCAR system, a novel, multifunctional tool termed PSCA-IgG4 target module (TM) was developed for dual prostate cancer theranostics. It comprises a human PSCA-specific binding domain, the hinge and Fc-domain of human IgG4 molecules as well as the UniCAR epitope E5B9. As shown by in vitro assays with PSCA-positive and PSCA-negative prostate cancer cells, the novel TM redirected UniCAR T cells for efficient tumor cell lysis in a strictly antigen- and TM-dependent manner. After radiolabeling with copper-64 or actinium-225, the novel PSCA-IgG4 TM was successfully applied for diagnostic imaging and targeted radioimmunotherapy. The 64Cu-labeled PSCA-IgG4 TM showed maximal tumor accumulation with optimal tumor-to-background ratios after 1.5 days. Furthermore, targeted alpha-therapy with the 225Aclabeled TM significantly delayed the outgrowth of established tumors in mice. In summary, the here presented, novel PSCA-IgG4 TM is a promising candidate for dual theranostics of prostate cancer that may help to overcome present hurdles in solid tumor therapy. After radiolabeling it facilitates not only targeted alpha-therapy and diagnostic imaging of PSCA, but can be also repurposed as a TM for UniCAR T-cell immunotherapy.

Background: Functional interaction between cancer cells and the surrounding microenvironment is still not sufficiently understood, which motivates the tremendous interest in the development of numerous in vitro and in vivo tumor models. Study Aims: To study the influence of different permeabilities of microcapsules (MCs) on different cancer cell proliferations, and to design and engineer the formation of 3D tumor clusters in MCs. Materials and Methods: A fluidics-based low-cost methodology was used to reproducibly generate alginate (AL) and alginate-chitosan (AL-CS) MCs in a cross-junctions water-in-oil system. The diffusion through the shell of AL and AL-CS MCs was monitored using fluorescein sodium (376 Da), FITC-Dextran 70 (70 kDa), and FITC-Dextran 2000 (2000 kDa) as fluorescent probes representing small molecules, proteins, and macromolecules, respectively. HepG2 Red FLuc (human hepatoma cell line) and A375 (human melanoma cell line) cultured in high-glucose DMEM medium were used to study the proliferation differences in terms of dimensions and geometries in AL and AL-CS MCs. The metabolic activity of tumor clusters in MCs was confirmed by tracking the turnover of testosterone to androstenedione with lower case Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Results: HepG2 Red FLuc and A375 cells show different proliferation properties in AL and AL-CS MCs. A375 tumor clusters grow faster in more permeable AL MCs and slower in less permeable AL-CS MCs. In the case of HepG2 Red FLuc, a significant difference in proliferation rate was not observed between AL and AL-CS MCs at the early stage (1 week). Interestingly, it was observed that different loose and tight cell cluster morphologies can form, also including cell proliferation along radial directions in both MC types and both cell lines. Cytochrome P450 (CYP)-dependent metabolization of testosterone by both HepG2 Red FLuc and A375 tumor clusters in the AL and AL-CS MCs showed the same trends in good

Background: Glioblastoma (GBM) is a very aggressive brain tumor, associated with poor prognosis and survival. So far, the efficiency of available therapies is limited. After proving their effectiveness in targeting hematological malignancies, chimeric antigen receptor (CAR) T cells might provide a promising therapeutic approach for GBM. Here, we present our switchable Reverse CAR technology (RevCAR). Unlike conventional CAR T cells, RevCAR T cells contain an epitope in their extracellular receptor domain, and can only be activated via bispecific target modules (RevTM) which recognize the RevCAR T cells on one side and tumor cells on the other side. Once these TMs are eliminated, RevCARs are switched off. In addition, we have developed dual-targeting RevCARs allowing the control of T cells according to the AND gate logic of Boolean algebra. Methods: RevTMs directed against GMB were expressed in eukaryotic cells and purified with affinity chromatography. The ability of these RevTMs to bind GBM cells and RevCAR T cells was analyzed using flow cytometry. Moreover, the capability of the mono-specific and the dual-targeting RevCAR T cells to kill GBM cells was analyzed using luminescence-based cytotoxicity assay in the absence or presence of a range of RevTM concentrations. Secreted pro-inflammatory cytokines were also evaluated by ELISA. In addition to the in vitro assays, a proof of concept co-injection experiment was performed in vivo. Results: In this study, we show that GBM-specific RevTMs can bind both the RevCAR T cells and the GBM cells. Importantly, the RevCAR T cells can be activated to secrete pro-inflammatory cytokines and to efficiently kill GBM cells via the RevTMs. Moreover, we were able to prove that dual-targeting RevCAR T cells can be activated only upon recognition of two different GBM targets, thereby allowing a highly specific and selective killing of GBM both in vitro and in vivo. Conclusion: In conclusion, the switchable RevCAR platform is a novel t

Despite the fact that chimeric antigen receptor (CAR) engineered T cells have shown encouraging therapeutic effects in hematological setups, the targeting of solid tumor-related antigens still represents a challenge. One main issue is that the expression of this type of antigens is not restricted to cancer cells, but normal tissues express them as well to some degree. In order to avoid strong side-effects caused by on-target/off-tumor effects, more controllable and specific CAR T cell-derived technologies need to be developed. In this line of thought, we developed the switchable, flexible and programmable Reverse (Rev) CAR platform. This system is based on engineered T cells expressing RevCAR molecules, which have extracellular short peptide epitopes incapable of recognizing surface antigens. Thus, the RevCAR T cells are per se inert and will only recognize the target cell through the interaction with an antigen-specific target module, named RevTM. RevTMs are bispecific antibodies designed to bind simultaneously to the target antigen and the short epitopes on the RevCAR engineered T cells. This interaction triggers a specific activation and cytotoxic activity of the RevCAR T cells redirecting them to eradicate the cancer cells. Here, we adapted our RevCAR technology to target the carcinoembryonic antigen (CEA), which is a significant tumor marker for colorectal cancers and other carcinomas. Moreover, we developed two RevTMs with different structures: scFv- and IgG4-based. The first one is built by linking a scFv against CEA to a scFv that recognizes the RevCAR epitope through glycine and serine residues, resulting in a small sized molecule (<60 kDa) with two binding sites. The IgG4-based RevTM connects the same scFv-structures but in this case through the hinge and constant Fc regions (CH2 and CH3) of an IgG4 antibody. The formation of disulfide bridges on the hinge portion causes the formation of homodimers, resulting in a molecule with increased molecular weight (

Modifying of immune effector cells with chimeric antigen receptors (CARs) has revealed a promising therapeutic potential for targeting cancer, especially with CAR-modified T cells. However, the use of other immune cells like primary NK cells or NK cell lines appeared as another advantageous approach that can be combined with CAR technology. Unlike T cells, established NK cell lines can be used allogenically as an off-the-shelf product with reduced risk of toxicities. We have established previously a modular Universal CAR platform (UniCAR) which can be switched on/off and allows the flexible targeting of various tumor antigens. This system consists of two parts, the UniCAR-expressing immune effector cells and a target module (TM). The UniCAR-immune cells cannot recognize surface antigens but are only redirected with the TM which contains an antigen-binding moiety on one hand and an epitope recognized by the UniCAR molecules on the other hand. Here, we provide a proof of concept for using the UniCAR system in combination with the NK-92 cell line to target disialoganglioside GD2-expressing tumors. The UniCAR NK-92 cells induced increase in lysis of neuroblastoma and melanoma cell lines in the presence of scFv- or human IgG4-based TMs, associated with specific release of pro-inflammatory cytokines. Moreover, UniCAR NK-92 cells were shown to be functional in eradicating GD2-expressing tumors in experimental mice. In order to investigate the in vivo half-life of the scFv- and IgG4-based TMs, they were radiolabeled with 64Cu and detected using PET imaging. Dynamic PET scanning has shown that the IgG4 format increased the half-life of the TM to around 24 folds in comparison to the scFv-based TMs. In summary, UniCAR NK-92 provides an off-the-shelf universal platform that can be combined with various antibody formats, and can be easily expanded for therapeutic use.

Objectives: During the last 15 years several PSMA PET ligands for prostate cancer imaging have been developed resulting in the recent approvals of 68Ga-PSMA-11, 18F-PSMA-1007 and 18F-DCFPyL.[1,2] However, 99mTc remains a popular radiometal for SPECT imaging due to its longer half-life and availability from 99Mo/99mTc generators especially in outpatient imaging centers. Due to the widespread availability of SPECT cameras PSMA SPECT ligands have the potential to substantially extend the availability of PSMA-based imaging, but currently no PSMA SPECT ligand is approved. MIP-1404, PSMA-I&S and HYNIC-iPSMA are rare examples of PSMA SPECT tracers in clinical development. Accordingly, the aim of our study was to develop a PSMA ligand for 99mTc SPECT imaging of prostate cancer based on an N2S2 chelator, which enables reliable radiolabeling to form stable 99mTc complexes.[3] Methods: A number of compounds, which contain the PSMA binding motif Glu-urea-Lys, a varying linker and a bisaminothiol (BAT)-type N2S2 chelator, were synthesized. Selected compounds were radiolabeled with 99mTc, followed by assessment of in vitro stability of the formed complexes via radio-HPLC. The specific binding affinity towards PSMA and the internalization of the radioligands were examined in LNCaP cells, supplemented by autoradiographic studies. Tissue distribution and tumor accumulation were evaluated in LNCaP-tumor bearing mice via quantitative SPECT/CT imaging, benchmarked with [99mTc]Tc-PSMA-I&S[4] and [68Ga]Ga-PSMA-11. Results: Among the six 99mTc radioligands synthesized and examined, [99mTc]TcO-ABX474 showed most favorable properties. Radiolabeling of ABX474 (50 µg) was achieved starting from [99mTc]NaTcO4 (0.2-2 GBq) in saline (1.4 mL) using SnCl2 (1 µg in 0.01 M HCl) as reducing agent, in presence of calcium-D-heptagluconate (10 µg) and D-mannitol (1 mg) at pH 5-6. Incubation at r.t. for 20 min followed by heating at 80°C for 20 min afforded the product-containing solution with a radioche

Modification of autologous T cells with tumor-specific chimeric antigen receptors (CARs) has been shown to be an effective tool for treatment of hematologic malignancies. However, clinical translation towards solid tumors is still limited as demonstrated by the suboptimal performance of CAR T cells in first in-human studies. Combining advances in CAR-T cell therapy and cancer theranostics may provide a promising multimodal approach to increase therapeutic efficacy and achieve durable responses in cancer patients. Here we present the development and functional characterization of different peptide- or antibody constructs targeting tumor-associated antigens or different structures of the tumor microenvironment. Equipment of these molecules with the E5B9 peptide epitope, enabled their use in the well-established UniCAR system. Accordingly, the constructs were able to specifically activate UniCAR T cells for efficient killing of both hematological and solid tumors in vitro and in vivo. Upon conjugation of chelators like DOTAGA and NODAGA, the peptide- or antibody derivates were further successfully applied for cancer theranostics, using e.g. 68Ga, 64Cu as diagnostic radionuclides and e.g. 67Cu, 225Ac as therapeutic radionuclides. PET imaging in xenotransplanted mice has demonstrated high contrast tumor accumulation. Consistent with the stable tumor accumulation, 225Ac-labeled molecules demonstrated therapeutic efficacy in a xenograft mouse model. In summary, E5B9-tagged peptide- or antibody constructs open a new era of cancer immunotheranostics as they can be used multimodally for (i) UniCAR-T cell therapy, (ii) non-invasive diagnostic imaging and (iii) targeted radioimmunotherapy. Prospectively, they could thus bridge the gap between the fields of CAR-T cells and cancer theranostics.

Chimeric antigen receptor (CAR) T-cells show remarkable therapeutic effects especially in B-cell derived leukemias and lymphomas. However, clinical translation of such an innovative immunotherapeutic approach in highly heterogeneous hematological malignancies like acute myeloid leukemia (AML) or solid tumors is still challenging due to life-threatening side effects, immune escape and disease relapse. To overcome such major hurdles and to address the unmet need for further improvements in CAR therapy, we have established flexible, switchable and programmable adaptor CAR platform technologies named UniCAR and RevCAR. These modular strategies consist of T-cells engineered with adaptor CARs which are primarily inactive as they are incapable to recognize surface antigens. Universal adaptor CAR T-cells can be flexibly redirected to any tumor antigen and controlled by targeting modules (TMs) cross-linking adaptor CAR T- and tumor cells resulting in tumor cell lysis. As an advancement of UniCARs, RevCARs lack the extracellular antigen-binding moiety reducing the receptor size and facilitating the genetical modification of T-cells with several RevCARs possessing different specificity and functionality. Thus, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. So far, we have successfully shown preclinical applicability of the UniCAR and RevCAR approaches to target hematological malignancies as well as solid tumors in a flexible and specific manner using tumor cell lines and patient-derived materials. Remarkably, efficiency and switchability of UniCAR T-cells were even proven for the first time in patients in a clinical phase I study. Furthermore, by targeting of two different tumor antigens, combinatorial OR and AND gate logic targeting according to the rules of Boolean algebra was accomplished using the RevCAR platform. These achievements have a high potential for an improved and personalized tumor immunotherapy.