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The extracellular environment regulates the structures and functions of cells, from the molecular to the tissue level. However, the underlying mechanisms influencing the organization and adaptation of cancer in three-dimensional (3D) environments are not yet fully understood. In this study, the influence of the viscosity of the environment is investigated on the mechanical adaptability of human hepatoma cell (HepG2) spheroids in vitro, using 3D microcapsule reactors formed with droplet-based microfluidics. To mimic the environment with different mechanical properties, HepG2 cells are encapsulated in alginate core–shell reservoirs (i.e., microcapsules) with different core viscosities tuned by incorporating carboxymethylcellulose. The significant changes in cell and spheroid distribution, proliferation, and cytoskeleton are observed and quantified. Importantly, changes in the expression and distribution of F-actin and keratin 8 indicate the relation between spheroid stiffness and viscosity of the surrounding medium. The increase of F-actin levels in the viscous medium can indicate an enhanced ability of tumor cells to traverse dense tissue. These results demonstrate the ability of cancer cells to dynamically adapt to the changes in extracellular viscosity, which is an important physical cue regulating tumor development, and thus of relevance in cancer biology.

summary over: (a) raw data for the metabolic assays (b) raw data for the analysis of the permeability of the capsules (c) unpublished images: typical cross section analysis of the capsules and organoids

Discussed are two picolinate appended bispidine ligands (3,7-diazabicyclo[3.3.1]nonane derivatives) in comparison with an earlier described bis-pyridine derivative, which are all known to strongly bind CuII. The radiopharmacological characterization of the two isomeric bispidine complexes includes quantitative labeling with 64CuII at ambient conditions with high radiochemical purities and yields (molar activities > 200 MBq/nmol). Challenge experiments in presence of EDTA, cyclam, human serum and SOD demonstrate high stability and inertness of the 64Cu-bispidine complexes. Biodistribution studies performed in Wistar rats indicate a rapid renal elimination for both 64Cu-labeled chelates. The bispidine ligand with the picolinate group in N7 position was selected for further biological experiments, and its backbone was therefore substituted with a benzyl-NCS group at C9. Two tumor target modules (TMs), targeting prostate stem cell antigen (PSCA), overexpressed in prostate cancer, and the fibroblast activation protein (FAP) in fibrosarcoma, were selected for thiourea coupling with the NCS-functionalized ligand and lysine residues of TMs. Small animal PET experiments on tumor-bearing mice showed very good and specific accumulation of the 64Cu-labeled TMs in tumors.

Immunotherapies are already successfully used for cancer treatment. In our lab two modular platform technologies were developed which demonstrated high success against various types of malignant cells: the universal chimeric antigen receptor (UniCAR) system, and the bispecific antibody (bsAb) based UniMAB system. Both approaches have the aim to recruit T cells to kill the malignant cells carrying a tumor associated antigen (TAA) on their surface. In the first case, UniCAR modified T cells are linked via a TAA specific target module (TM) to cancer cells. In the case of the UniMAB system, natural T cells are recruited to cancer cells via a TAA specific TM and a bsAb based effector module (EM) which binds to CD3 of T cells and to an epitope tag of the TM. In both cases, an immune complex is formed leading to T cell activation and tumor cell elimination. Most importantly, the T cell activation is dependent on the cross-linkage of T cells and tumor cells via the TM or TM/EM complex. Thus, T cell activation is steerable which increases the safety of these approaches. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to millions of deaths worldwide, we aimed to apply the UniCAR and UniMAB technology against SARS-CoV-2 and virus infected cells as novel immunotherapeutic treatment approach. For this purpose, we designed two TMs binding to the receptor binding domain (RBD) of SARS-CoV-2. The first TM is based on a single chain fragment variable (scFv) with specificity for RBD, whereas the second one contains the extracellular domain of the human ACE2 receptor which is the entry receptor of SARS-CoV-2. Both TMs were able to efficiently recruit either UniCAR T cells or, in combination with the EM of the UniMAB system, natural T cells to efficiently kill human cells having the RBD of SARS-CoV-2 on their surface. Remarkably, the ACE2-Mb-TM is additionally able to block RBD/ACE2 interaction and potently neutralizes pseudo- as well as live S

Immunotherapies are already successfully used for cancer treatment. In our lab two modular platform technologies were developed which demonstrated high success against various types of malignant cells: the universal chimeric antigen receptor (UniCAR) system, and the bispecific antibody (bsAb) based UniMAB system. Both approaches have the aim to recruit T cells to kill the malignant cells carrying a tumor associated antigen (TAA) on their surface. In the first case, UniCAR modified T cells are linked via a TAA specific target module (TM) to cancer cells. In the case of the UniMAB system, natural T cells are recruited to cancer cells via a TAA specific TM and a bsAb based effector module (EM) which binds to CD3 of T cells and to an epitope tag of the TM. In both cases, an immune complex is formed leading to T cell activation and tumor cell elimination. Most importantly, the T cell activation is dependent on the cross-linkage of T cells and tumor cells via the TM or TM/EM complex. Thus, T cell activation is steerable which increases the safety of these approaches. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to millions of deaths worldwide, we aimed to apply the UniCAR and UniMAB technology against SARS-CoV-2 and virus infected cells as novel immunotherapeutic treatment approach. For this purpose, we designed two TMs binding to the receptor binding domain (RBD) of SARS-CoV-2. The first TM is based on a single chain fragment variable (scFv) with specificity for RBD, whereas the second one contains the extracellular domain of the human ACE2 receptor which is the entry receptor of SARS-CoV-2. Both TMs were able to efficiently recruit either UniCAR T cells or, in combination with the EM of the UniMAB system, natural T cells to efficiently kill human cells having the RBD of SARS-CoV-2 on their surface. Remarkably, the ACE2-Mb-TM is additionally able to block RBD/ACE2 interaction and potently neutralizes pseudo- as well as live S

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

Oncology research has been an exciting area for the application of electrical biosensors over the last few decades. Extremely high sensitivity of field-effect transistor-based (FET) biosensors directed the interest of scientists mostly to early cancer detection applications. However, the potential of electrical biosensors should not be limited to cancer diagnosis. With immunotherapy on the way to become the fourth pillar of cancer treatment, there will be an increasing demand for immunoassay testing before, during, and after the conduction of therapeutic treatment. Our previous results have demonstrated the usefulness of FET-based biosensors in the research and development of a safer engineered chimeric antigen receptor (CAR) T-cells-based therapy. In this work, we continue to explore the practicality of electrical biosensors in the potential therapeutic setting of immunotherapy. Here, we present a complete stand-alone FET-based biosensing system relying on the extended gate (EG) concept. Our platform has an independent readout which is portable, compatible with multiplexed measurement, and self-sufficient for point-of-care sensing purposes. The extended gate sensing chip is disposable and can be tailored for the specific sensing application. Compared to the traditional ion-sensitive FET setup, EG FET provides improved flexibility, cost-effectiveness, and adaptiveness to individual needs making it a promising candidate for clinical application. During a universal CAR T-cells (UniCAR) therapy, “switch” molecules are injected into the patient’s bloodstream to activate the T-cell processes for attacking tumors. Therefore, it is crucial to monitor the level of CAR T-cells and switch molecules for better control and decision-making related to personalized treatment. Here, we demonstrate the application of our EG FET platform in testing the therapeutic components of this treatment and discuss the related outlook for FET-based biosensors in immunotherapy. Our work endeavor

Chimeric antigen receptors (CARs) are used for redirection of T cells against tumor cells and have demonstrated remarkable therapeutic effects against some hematological cancers. The modular system termed RevCAR can overcome the dangerous side effects associated with conventional CAR T cell therapy, such as cytokine release syndrome. It is composed of RevCAR T cells and a bispecific molecule target module, termed RevTM. This system is highly safe since RevCAR T cell activity can be controlled by RevTM and immediately switched off if side effects are detected. It is also versatile, since the same RevCAR T cell can be directed to different tumor-associated antigens (TAA) simply by adding RevTMs with different specificities. However, to improve the effectiveness in the treatment of solid tumors, the immunosuppressive tumor microenvironment (TME) still needs to be addressed. For this purpose, we have developed novel RevTMs targeting immune checkpoint molecules such as PD-L1, which are often upregulated by cancer cells to escape the immune cells. We have demonstrated that our novel RevTMs can redirect RevCAR T cells to specifically kill cell lines expressing immune checkpoint molecules. In addition to this strategy, the combinatorial targeting of a TAA and an immune checkpoint is very promising strategy to overcome the immunosuppressive microenvironment of solid tumors and thus to improve the treatment outcome.

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

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

To improve postoperative monitoring of patients undergoing pancreatic surgery, we propose a bedside, droplet-based millifluidic device that facilitates near real-time accurate and rapid determination of drain amylase activity. Drain liquid is co-encapsulated with a starch substrate in multiple nanoliter-sized reactors to track the fluorescence intensity released upon reaction with amylase. Comparing the amylase levels of 32 patient samples, 31 out of 32 millifluidic system results (96.9%) matched the clinical data within the 95% confidence interval. Importantly, the new millifluidic strategy significantly reduces the amylase assay time from several hours to approximately 3-4 minutes and offers a ca. 10 times lower limit of detection of 0.007 µmol/sL compared to that of current clinical techniques. Finally, we for the first time demonstrate the possibility of dynamic monitoring of amylase levels in the patient samples. Thus, the proposed droplet-based instant assay system could augment state-of-the-art clinical methods and significantly improve diagnosis of postoperative complications in patients after pancreatic surgery via near-real-time bedside monitoring.

The La-protein is a multifunctional abundantly expressed protein in the nucleus of all human cells, where it for example functions as a chaperone for newly synthesized RNA-polymerase III transcripts. Recently, it has been shown that La is overexpressed in malignant cells and can be translocated to the cell surface under certain conditions, e. g. radio- or chemotherapy-induced necrosis, where it becomes a promising, inducible target for so-called “Death Targeting” approaches. The aim of this study was to investigate whether the La-protein could also be used as an inducible target for CAR T cell therapy. Therefore, three different CAR T cells directed against the La-protein (La-CARs) were developed and tested regarding their potential for “Death Targeting” of tumor cells. For construction of the La-CARs, we selected three different anti-La mAbs (5B9, 7B6 and 312B), which recognize distinct epitopes within the La-protein in a redox-dependent manner. This allowed us to determine, if the redox-state influences the ability of the La-CARs to target cell surface-bound La-protein. We first examined if La-CARs could eliminate tumor cells that were artificially labeled with wildtype La-protein or its oxidation-resistant triple-cysteine-mutant (TCM). By performing luciferase-based killing assays, we showed, that all La-CARs were specifically activated for lysis of La- or TCM-decorated tumor cells, which also resulted in significant secretion of pro-inflammatory cytokines. Redox-dependent differences of the La-CARs could be observed, whereby the non-redox-dependent 5B9-CAR was activated by La-wt- and TCM-labeled cells, the 7B6-CAR preferably by La-wt-labeled and the 312B-CAR preferably by TCM-labeled tumor cells. We could further prove that La-release from tumor cells could be induced by treatment with the cytotoxic drug cisplatin and La-CARs could be redirected against remaining tumor-cells after cisplatin treatment. Thereby, the 5B9-CAR, which recognizes La-protein independent

Biosensors based on the extended gate field-effect transistor (EG FET) differ from the traditional FET- based biosensors in terms of creating a spatial separation between the EG sensing element and the FET transducer. Thus, EG can be employed as a cost-effective and disposable unit, while the FET transducer is retained as a reusable component. These features make EG particularly attractive for multiplexed biosensing since an EG electrode array can be easily integrated within a single chip, and individual electrodes can be separately modified for the recognition of diverse analyte types. Although the use of EG FET-based platforms has been successfully demonstrated in advanced biosensing applications,¹˒² they still suffer from practical disadvantages including limited multiplexing, complex nanofabrication of FET transducers, and reliance on bulky specialized high-performance measurement instruments for readout of low current levels (~nA range), all substantially reducing their suitability for many applications in point-of-care (POC) diagnostics and monitoring. We present a custom-designed multiplexed standalone EG FET-based potentiometric biosensing platform relying on a commercial FET transducer, portable modular electronics, and innovative assay format for potentiometric response enhancement based on the conjugation of analyte antibodies (Abs) to gold nanoparticles (AuNPs). To achieve simplified signal readout, we employ an in-house fabricated common reference electrode for all sensing points and operate the FET in constant charge mode. Potential shifts at the EG surface during sensing are indirectly detected as voltage changes between the reference electrode and the FET source terminal. We demonstrate the sensing of antibody-antibody interactions on the immunoglobulin G system and realize the 5-fold amplification of the potentiometric response compared to the traditional label-free assay by introducing AuNP-based labeling of the target analyte. Using the described

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

Novel immunotherapeutic approaches such as chimeric antigen receptor (CAR)-expressing immune cells are showing promising results against cancer. Among these are the CAR Natural Killer (NK) cells that can be produced from either established cell lines such as NK-92 or allogenic NK cells, which can attack cancer cells. However, some challenges are encountered, when targeting solid tumors, in general and in particular glioblastoma, including the immunosuppressive tumor microenvironment, the antigen escape, the on-target/off-tumor toxicity and neurotoxicities. In addition to the inability to control the activity of conventional CAR NK cells once they are transferred into the patient in case of toxicities occur. Therefore, we aimed to develop a safer and more tumor-specific CAR NK cell platform called the Reverse CAR (RevCAR) NK-92 system. This system consists of two components, the NK-92 cells expressing RevCARs having an extracellular short peptide epitope incapable of recognizing surface antigens by itself, unless redirected by the other component of the system, which is a bispecific molecule called Rev Target Module (RevTM). The RevTM contains two scFvs, one is targeting the respective E5B9 or E7B6 epitope of the RevCAR NK-92 cells and the other one binds to the tumor associated antigen (TAA) of interest. Only in the presence of the respective RevTM, RevCAR NK cells can be directed towards tumor cells, resulting in tumor lysis. By dosing of the short-living RevTMs, we enabled the control of the RevCAR NK cell activity. Furthermore, the modular character of the RevCAR system provides the flexibility for targeting of different TAAs of interest simply by exchanging RevTMs with different specificities. Out of these TAAs, the fibroblast growth factor-inducible 14 (Fn14) surface receptor and its ligand (tumor necrosis factor-related weak inducer of apoptosis (TWEAK)) have been shown to be upregulated in many solid tumors, like glioblastoma. The activation of TWEAK/Fn14 pat

Genetically engineered T cells expressing chimeric antigen receptors (CARs) have shown promising results particularly when targeting tumor associated antigens (TAAs) related to hematological malignancies. However, TAAs are usually expressed to some extend also on healthy tissues leading to on-target/off-tumor effects. To overcome this important safety issue along with improving targeting specificity and efficient killing of tumor escape variants, we adapted our Reverse CAR (RevCAR) system to follow an AND-gate Boolean logic. For that, Dual-RevCAR T cells were designed and armed with (I) a signaling (SIG) RevCAR, that includes the intracellular domain (ICD) of CD3 zeta; and (II) a costimulatory (COS) RevCAR, which contains a domain derived from CD28. Because the extracellular domains of both RevCARs are derived from the La/SS-B nuclear protein, Dual-RevCAR T cells will remain inactive until they encounter matching target modules (RevTMs). The bispecific antibody (bsAb)-like structure of the RevTMs allows their binding to RevCAR molecules and to specific antigens. However, only the simultaneous binding of RevTMs to SIG and COS RevCARs will promote the full activation of the Dual-RevCAR T cells. The epithelial cell adhesion molecule (EpCAM) and the carcinoembryonic antigen (CEA) have become appealing markers due to their overexpression in various solid tumor entities such as colorectal cancer, therefore representing promising target antigens for cancer immunotherapies following such a Dual Targeting CAR approach. Having this in mind, the aim of this work was to assess the potential therapeutic application of the Dual-RevCAR system to target EpCAM and CEA following an AND-gating approach.

T-cells genetically modified to express chimeric antigen receptors (CARs) are playing a more and more important role in targeted cancer immunotherapy. However, these living drugs can also cause lifethreatening side effects. To overcome such limitations and improve the safety of CAR T-cell therapy, adaptor CAR platforms such as the universal CAR (UniCAR) have been developed. This platform consists of the UniCAR T-cell and a target module (TM) cross-linkage effector and tumor cells. Here, we have established a novel UniCAR system targeting Fibroblast Activation Protein (FAP) that is highly expressed in the tumor microenvironment of epithelial cancers and a marker for cancer-associated fibroblasts. For that, we constructed two novel FAP-directed TMs possessing different sizes and pharmacokinetic properties, in which one is based on a single-chain variable fragment (scFv), and the other is based on an IgG4 backbone. We have shown that both TMs were able to bind to FAP-expressing cells and redirect UniCAR T-cells in vitro to monolayer and spheroid target cells inducing effective killing. Furthermore, we could show infiltration and activation of T-cells in the spheroid setting. Using in vivo models, the TMs were proven to be suitable to be used for PET imaging showing FAP-specific accumulation at the tumor site. Moreover, the immunotherapeutic effect of UniCAR T-cells in combination with FAP TMs was demonstrated using mouse models. In conclusion, in this work, we could show that the two novel anti- FAP TMs prove to hold great theranostic potential for diagnostic imaging and immunotherapy.

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.

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.

TheraSTAR – Project Overview

This presentation was given in Lisbon for the first OncoProTools Summer School.

Chimeric antigen receptor (CAR)-engineered immune effector cells constitute a promising approach for adoptive cancer immunotherapy. Nevertheless, on-target/off-tumor toxicity and immune escape due to antigen loss represent considerable challenges. These may be overcome by adaptor CARs that are selectively triggered by bispecific molecules that crosslink the CAR with a tumor-associated surface antigen. Here, we generated NK cells carrying a first- or second-generation universal CAR (UniCAR) and redirected them to tumor cells with so-called target modules (TMs) which harbor an ErbB2 (HER2)-specific antibody domain for target cell binding and the E5B9 peptide recognized by the UniCAR. To investigate differential effects of the protein design on activity, we developed homodimeric TMs with one, two or three E5B9 peptides per monomer, and binding domains either directly linked or separated by an IgG4 Fc domain. The adaptor molecules were expressed as secreted proteins in Expi293F cells, purified from culture supernatants and their bispecific binding to UniCAR and ErbB2 was confirmed by flow cytometry. In cell killing experiments, all tested TMs redirected NK cell cytotoxicity selectively to ErbB2-positive tumor cells. Nevertheless, we found considerable differences in the extent of specific cell killing depending on TM design and CAR composition, with adaptor proteins carrying two or three E5B9 epitopes being more effective when combined with NK cells expressing the first-generation UniCAR, while the second-generation UniCAR was more active in the presence of TMs with one E5B9 sequence. These results may have important implications for the further development of optimized UniCAR and target module combinations for cancer immunotherapy.

UV/Vis of natural Bi(III)- and Pb(II)-L2 and Bi(III)- and Pb(II)-L3; radio-SEC of 133La-L2; radiolabeling kinetics

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced deleterious effects to healthy tissues. This applies in particular to the kidneys as primary organs of elimination, which requires dosimetric estimates to justify internal radionuclide therapy. In this context, the targeting of enzymes of the renal brush border membrane by cleavable linkers between target molecule and radiolabel that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of ⁶⁴Cu-labeled cleavable linkers and quantified their substrate potentials toward neprilysin (NEP), a highly abundant peptidase at the renal brush border membrane. This allowed for the derivation of structure-activity relationships and selected cleavable linkers were attached to the somatostatin receptor subtype 2 ligand [Tyr³]octreotate. Subsequent radiopharmacological characterization revealed that a substrate-based targeting of NEP in the kidneys with small molecules or peptides entails a certain degree of premature cleavage in the blood circulation by soluble and endothelium-derived NEP. However, for a tissue-specific targeting of NEP in the kidneys, the additional targeting of albumin in the blood by albumin-binding moieties is highlighted.

Octa- and specifically nonadentate ligands with a bispidine scaffold (3,7-diazabicyclo[3.3.1]nonane) are known to be efficiently coordinated to a range of metal ions of interest in radiopharmaceutical chemistry and lead to exceedingly stable and inert complexes. The nonadentate bispidine L2 (with a tridentate bipyridine acetate appended to N3 and a picolinate at N7) has been shown before to be an ideal chelator for 111In3+, 177Lu3+ and 225Ac3+, nuclides of interest for diagnosis and therapy, and a proof-of-principle study with an SSTR2-specific octreotate has shown potential for theranostic applications. We now have extended these studies in two directions. Firstly, we present the ligand derivative L3, where the bipyridine acetate is substituted with terpyridine, a softer donor for metal ions with a preference for more covalency. L3 did not fulfill the hopes because complexation is much less efficient: while for Bi3+ and Pb2+ the ligand is an excellent chelator with similar properties to L2, Lu3+ and La3+ show very slow and inefficient complexation with L3 in contrast to L2, and 225Ac3+ is not fully coordinated, even at an elevated temperature (92% radiochemical yield (RCY) at 80 °C, 60 min, [L3] = 10-4 M). These observations have led to a hypothesis for the complexation pathway that is in line with all experimental data and supported by a preliminary DFT analysis, which is of importance for the design of further optimized bispidine chelators. Secondly, the coordination chemistry of L2 has been extended to Bi3+, La3+ and Pb2+, including solid state and solution structural work, complex stabilities, radiolabeling and radiostability studies. All complexes of this ligand (La3+, Ac3+, Lu3+, Bi3+, In3+, Pb2+), including nuclides for targeted alpha therapy (TAT), single photon emission computed tomography (SPECT), and positron emission tomography (PET) are formed efficiently at physiological conditions, i.e., suitable for the labeling of delicate biological vectors such a

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced deleterious effects to healthy tissues. This applies in particular to the kidneys as primary organs of elimination, which requires dosimetric estimates to justify internal radionuclide therapy. In this context, the targeting of enzymes of the renal brush border membrane by cleavable linkers between target molecule and radiolabel that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of ⁶⁴Cu-labeled cleavable linkers and quantified their substrate potentials toward neprilysin (NEP), a highly abundant peptidase at the renal brush border membrane. This allowed for the derivation of structure-activity relationships and selected cleavable linkers were attached to the somatostatin receptor subtype 2 ligand [Tyr³]octreotate. Subsequent radiopharmacological characterization revealed that a substrate-based targeting of NEP in the kidneys with small molecules or peptides entails a certain degree of premature cleavage in the blood circulation by soluble and endothelium-derived NEP. However, for a tissue-specific targeting of NEP in the kidneys, the additional targeting of albumin in the blood by albumin-binding moieties is highlighted.

The COVID-19 pandemic caused by SARS-CoV-2 led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options, which can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2 infected animal models, but also for (ii) clearance of virus infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Despite the success of chimeric antigen receptor (CAR) T-cells especially for treating hematological malignancies, critical drawbacks, such as “on-target, off-tumor” toxicities, need to be addressed to improve safety in translating to clinical application. This is especially true, when targeting tumor-associated antigens (TAAs) that are not exclusively expressed by solid tumors but also on healthy tissues. To improve the safety profile, we developed switchable adaptor CAR systems including the RevCAR system. RevCAR T-cells are activated by cross-linking of bifunctional adaptor molecules termed target modules (RevTM). In a further development, we established a Dual-RevCAR system for an AND-gated combinatorial targeting by splitting the stimulatory and co-stimulatory signals of the RevCAR T-cells on two individual CARs. Examples of common markers for colorectal cancer (CRC) are the carcinoembryonic antigen (CEA) and the epithelial cell adhesion molecule (EpCAM), while these antigens are also expressed by healthy cells. Here we describe four novel structurally different RevTMs for targeting of CEA and EpCAM. All anti-CEA and anti-EpCAM RevTMs were validated and the simultaneous targeting of CEA+ and EpCAM+ cancer cells redirected specific in vitro and in vivo killing by Dual-RevCAR T-cells. In summary, we describe the development of CEA and EpCAM specific adaptor RevTMs for monospecific and AND-gated targeting of CRC cells via the RevCAR platform as an improved approach to increase tumor specificity and safety of CAR T-cell therapies.

Nanogels open up access to a wide range of applications and offer among others hopeful approaches for use in the field of biomedicine. This review provides a brief overview of current developments of nanogels in general, particularly in the fields of drug delivery, therapeutic applications, tissue engineering and sensor systems. Specifically, cyclodextrin (CD)-based nanogels are important because they have exceptional complexation properties and are highly biocompatible. Nanogels as a whole and CD-based nanogels in particular can be customized in a wide range of sizes and equipped with a desired surface charge as well as containing additional molecules inside and outside, such as dyes, solubility-mediating groups or even biological vector molecules for pharmaceutical targeting. Currently, biological investigations are mainly carried out in vitro, but more and more in vivo applications are gaining importance. Modern molecular imaging methods are increasingly being used for the latter. Due to an extremely high sensitivity and the possibility of obtaining quantitative data on pharmacokinetic and pharmacodynamic properties, nuclear methods such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) using radiolabeled compounds are particularly suitable here. The use of radiolabeled nanogels for imaging, but also for therapy, is being discussed.

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform “UniCAR” is currently under early clinical investigation. Recently, first proof-of-concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to a high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off-switch of UniCAR T-cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T-cells for the therapy of AML.

Background: Chimeric antigen receptor (CAR) T-cells are a promising approach in cancer immunotherapy, particularly for treating hematologic malignancies. Yet, their effectiveness is limited when tackling solid tumors, where immune cell infiltration and immunosuppressive tumor microenvironments (TME) are major hurdles. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) and various tumor cells, playing an important role in tumor growth and immunosuppression. Aiming to modulate the TME with increased clinical safety and effectiveness, we developed novel small and size-extended immunotheranostic UniCAR target modules (TMs) targeting FAP. Methods: The specific binding and functionality of the anti-FAP-scFv TM and the size extended anti-FAP-IgG4 TM were assessed using 2D and 3D in vitro models as well as in vivo. Their specific tumor accumulation and diagnostic potential was evaluated using PET studies after functionalization with a chelator and suitable radionuclide. Results: The anti-FAP-scFv and -IgG4 TMs effectively and specifically redirected UniCAR T-cells using 2D, 3D, and in vivo models. Moreover, a remarkably high and specific accumulation of radiolabeled FAP-targeting TMs at the tumor site of xenograft mouse models was observed. Conclusions: These findings demonstrate that the novel anti-FAP TMs are promising immunotheranostic tools to foster cancer imaging and treatment, paving the way for a more convenient, individualized, and safer treatment of cancer patients.

The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib is an emerging cancer therapeutic that just recently gained Food and Drug Administration approval for treatment of estrogen receptor (ER)-positive, human epidermal growth factor receptor (Her)2-negative breast cancer in combination with the ER degrader fulvestrant. However, CDK4/6 inhibitors are not cancer-specific and may affect also other proliferating cells. Given the importance of T cells in antitumor defense, we studied the influence of palbociclib/fulvestrant on human CD3+ T cells and novel emerging T cell-based cancer immunotherapies. Palbociclib considerably inhibited the proliferation of activated T cells by mediating G0/G1 cell cycle arrest. However, after stopping the drug supply this suppression was fully reversible. In light of combination approaches, we further investigated the effect of palbociclib/fulvestrant on T cell-based immunotherapies by using a CD3-PSCA bispecific antibody or universal chimeric antigen receptor (UniCAR) T cells. Thereby, we observed that palbociclib clearly impaired T cell expansion. This effect resulted in a lower total concentration of interferon-g and tumor necrosis factor, while palbociclib did not inhibit the average cytokine release per cell. In addition, the cytotoxic potential of the redirected T cells was unaffected by palbociclib and fulvestrant. Overall, these novel findings may have implications for the design of treatment modalities combining CDK4/6 inhibition and T cell-based cancer immunotherapeutic strategies.

Detection of antigens and antibodies (Abs) is of great importance in determining the infection and immunity status of the population, as they are key parameters guiding the handling of pandemics. Current point-of-care (POC) devices are a convenient option for rapid screening; however, their sensitivity requires further improvement. We present an interdigitated gold nanowire-based impedance nanobiosensor to detect COVID-19-associated antigens (receptor-binding domain of S1 protein of the SARS-CoV-2 virus) and respective Abs appearing during and after infection. The electrochemical impedance spectroscopy technique was used to assess the changes in measured impedance resulting from the binding of respective analytes to the surface of the chip. After 20 min of incubation, the sensor devices demonstrate a high sensitivity of about 57 pS·sn per concentration decade and a limit of detection (LOD) of 0.99 pg/mL for anti-SARS-CoV-2 Abs and a sensitivity of around 21 pS·sn per concentration decade and an LOD of 0.14 pg/mL for the virus antigen detection. Finally, the analysis of clinical plasma samples demonstrates the applicability of the developed platform to assist clinicians and authorities in determining the infection or immunity status of the patients.

Uncrewed aerial vehicles (UAVs), also known as drones, are ubiquitous and their use cases extend today from governmental applications to civil applications such as the agricultural, medical, and transport sectors, etc. In accordance with the requirements in terms of demand, it is possible to carry out various missions involving several types of UAVs as well as various onboard sensors. According to the complexity of the mission, some configurations are required both in terms of hardware and software. This task becomes even more complex when the system is composed of autonomous UAVs that collaborate with each other without the assistance of an operator. Several factors must be considered, such as the complexity of the mission, the types of UAVs, the communication architecture, the routing protocol, the coordination of tasks, and many other factors related to the environment. Unfortunately, although there are many research works that address the use cases of multi-UAV systems, there is a gap in the literature regarding the difficulties involved with the implementation of these systems from scratch. This review article seeks to examine and understand the communication issues related to the implementation from scratch of autonomous multi-UAV systems for collaborative decisions. The manuscript will also provide a formal definition of the ecosystem of a multi-UAV system, as well as a comparative study of UAV types and related works that highlight the use cases of multi-UAV systems. In addition to the mathematical modeling of the collaborative target detection problem in distributed environments, this article establishes a comparative study of communication architectures and routing protocols in a UAV network. After reading this review paper, readers will benefit from the multicriteria decisionmaking roadmaps to choose the right architectures and routing protocols adapted for specific missions. The open challenges and future directions described in this manuscript can be us

We present a portable multiplexed biosensor platform based on the extended gate field-effect transistor and demonstrate its amplified response thanks to gold nanoparticle-based bioconjugates introduced as a part of the immunoassay. The platform comprises a disposable chip hosting an array of 32 extended gate electrodes, a readout module based on a single transistor operating in constant charge mode, and a multiplexer to scan sensing electrodes one-by-one. Although employing only off-the-shelf electronic components, our platform achieves sensitivities comparable to fully customized nanofabricated potentiometric sensors. In particular, it reaches a detection limit of 0.2 fM for the pure molecular assay when sensing horseradish peroxidase-linked secondary antibody (∼0.4 nM reached by standard microplate methods). Furthermore, with the gold nanoparticle bioconjugation format, we demonstrate ca. 5-fold amplification of the potentiometric response compared to a pure molecular assay, at the detection limit of 13.3 fM. Finally, we elaborate on the mechanism of this amplification and propose that nanoparticle-mediated disruption of the diffusion barrier layer is the main contributor to the potentiometric signal enhancement. These results show the great potential of our portable, sensitive, and cost-efficient biosensor for multidimensional diagnostics in the clinical and laboratory settings, including e.g., serological tests or pathogen screening.

Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hor-mone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor pro-gression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide 177Lu. The re-sulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images re-vealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.

Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo.

We demonstrate the fluidics-based low-cost methodology to reproducibly generate the alginate and alginate-chitosan microcapsules and apply it to grow human hepatoma (HepG2) spheroids of different dimensions and geometries. Focusing specifically on the composition and thickness of the hydrogel shell, permeability of the microcapsules was selectively tuned. The diffusion of the selected benchmark molecules through the shell has been systematically investigated using both, experiments and simulations, which is essential to ensure efficient mass transfer and/or filtering of the biochemical species. Depending on available space, phenotypically different 3D cell assemblies have been observed inside the capsules, varying in the tightness of cell aggregations and their shapes. Metabolic activity of spheroids in microcapsules was confirmed by tracking the turnover of testosterone to androstenedione with chromatography studies in a metabolic assay. Because of the facile tuning of the shell thickness and permeability, we believe that our system is suitable for studying the formation of cancer spheroids and their functional interaction with the surrounding microenvironment.

Bacteria primarily live in structured environments, such as colonies and biofilms, attached to surfaces or growing within soft tissues. They are engaged in local competitive and cooperative interactions impacting our health and well-being, for example, by affecting population-level drug resistance. Our knowledge of bacterial competition and cooperation within soft matrices is in-complete, partly because we lack high-throughput tools to quantitatively study their interactions. Here, we introduce a method to generate a large amount of agarose microbeads that mimic the natural culture conditions experienced by bacteria to co-encapsulate two strains of fluores-cence-labeled Escherichia coli. Focusing specifically on low bacterial inoculum (1–100 cells/capsule), we demonstrate a study on the formation of colonies of both strains within these 3D scaffolds and follow their growth kinetics and interaction using fluorescence microscopy in highly replicated experiments. We confirmed that the average final colony size is inversely proportional to the inoculum size in this semi-solid environment as a result of limited available resources. Further-more, the colony shape and fluorescence intensity per colony are distinctly different in mono-culture and co-culture. The experimental observations in mono- and co-culture are compared with predictions from a simple growth model. We suggest that our high throughput and small foot-print microbead system is an excellent platform for future investigation of competitive and co-operative interactions in bacterial communities under diverse conditions, including antibiotics stress.

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.

T-cells genetically modified to express chimeric antigen receptors (CARs) are playing a more and more important role in targeted cancer immunotherapy. However, these living drugs can also cause lifethreatening side effects. To overcome such limitations and improve the safety of CAR T-cell therapy, adaptor CAR platforms such as the universal CAR (UniCAR) have been developed. This platform consists of the UniCAR T-cell and a target module (TM) cross-linkage effector and tumor cells. Here, we have established a novel UniCAR system targeting Fibroblast Activation Protein (FAP) that is highly expressed in the tumor microenvironment of epithelial cancers and a marker for cancer-associated fibroblasts. For that, we constructed two novel FAP-directed TMs possessing different sizes and pharmacokinetic properties, in which one is based on a single-chain variable fragment (scFv), and the other is based on an IgG4 backbone. We have shown that both TMs were able to bind to FAP-expressing cells and redirect UniCAR T-cells in vitro to monolayer and spheroid target cells inducing effective killing. Furthermore, we could show infiltration and activation of T-cells in the spheroid setting. Using in vivo models, the TMs were proven to be suitable to be used for PET imaging showing FAP-specific accumulation at the tumor site. Moreover, the immunotherapeutic effect of UniCAR T-cells in combination with FAP TMs was demonstrated using mouse models. In conclusion, in this work, we could show that the two novel anti- FAP TMs prove to hold great theranostic potential for diagnostic imaging and immunotherapy.

To improve postoperative monitoring of patients undergoing pancreatic surgery, we propose a bedside, droplet-based millifluidic device that facilitates near real-time accurate and rapid determination of drain amylase activity. Drain liquid is co-encapsulated with a starch substrate in multiple nanoliter-sized reactors to track the fluorescence intensity released upon reaction with amylase. Comparing the amylase levels of 32 patient samples, 31 out of 32 millifluidic system results (96.9%) matched the clinical data within the 95% confidence interval. Importantly, the new millifluidic strategy significantly reduces the amylase assay time from several hours to approximately 3-4 minutes and offers a ca. 10 times lower limit of detection of 0.007 µmol/sL compared to that of current clinical techniques. Finally, we for the first time demonstrate the possibility of dynamic monitoring of amylase levels in the patient samples. Thus, the proposed droplet-based instant assay system could augment state-of-the-art clinical methods and significantly improve diagnosis of postoperative complications in patients after pancreatic surgery via near-real-time bedside monitoring.

Novel immunotherapeutic approaches such as chimeric antigen receptor (CAR)-expressing immune cells are showing promising results against cancer. Among these are the CAR Natural Killer (NK) cells that can be produced from either established cell lines such as NK-92 or allogenic NK cells, which can attack cancer cells. However, some challenges are encountered, when targeting solid tumors, in general and in particular glioblastoma, including the immunosuppressive tumor microenvironment, the antigen escape, the on-target/off-tumor toxicity and neurotoxicities. In addition to the inability to control the activity of conventional CAR NK cells once they are transferred into the patient in case of toxicities occur. Therefore, we aimed to develop a safer and more tumor-specific CAR NK cell platform called the Reverse CAR (RevCAR) NK-92 system. This system consists of two components, the NK-92 cells expressing RevCARs having an extracellular short peptide epitope incapable of recognizing surface antigens by itself, unless redirected by the other component of the system, which is a bispecific molecule called Rev Target Module (RevTM). The RevTM contains two scFvs, one is targeting the respective E5B9 or E7B6 epitope of the RevCAR NK-92 cells and the other one binds to the tumor associated antigen (TAA) of interest. Only in the presence of the respective RevTM, RevCAR NK cells can be directed towards tumor cells, resulting in tumor lysis. By dosing of the short-living RevTMs, we enabled the control of the RevCAR NK cell activity. Furthermore, the modular character of the RevCAR system provides the flexibility for targeting of different TAAs of interest simply by exchanging RevTMs with different specificities. Out of these TAAs, the fibroblast growth factor-inducible 14 (Fn14) surface receptor and its ligand (tumor necrosis factor-related weak inducer of apoptosis (TWEAK)) have been shown to be upregulated in many solid tumors, like glioblastoma. The activation of TWEAK/Fn14 pat

Oncology research has been an exciting area for the application of electrical biosensors over the last few decades. Extremely high sensitivity of field-effect transistor-based (FET) biosensors directed the interest of scientists mostly to early cancer detection applications. However, the potential of electrical biosensors should not be limited to cancer diagnosis. With immunotherapy on the way to become the fourth pillar of cancer treatment, there will be an increasing demand for immunoassay testing before, during, and after the conduction of therapeutic treatment. Our previous results have demonstrated the usefulness of FET-based biosensors in the research and development of a safer engineered chimeric antigen receptor (CAR) T-cells-based therapy. In this work, we continue to explore the practicality of electrical biosensors in the potential therapeutic setting of immunotherapy. Here, we present a complete stand-alone FET-based biosensing system relying on the extended gate (EG) concept. Our platform has an independent readout which is portable, compatible with multiplexed measurement, and self-sufficient for point-of-care sensing purposes. The extended gate sensing chip is disposable and can be tailored for the specific sensing application. Compared to the traditional ion-sensitive FET setup, EG FET provides improved flexibility, cost-effectiveness, and adaptiveness to individual needs making it a promising candidate for clinical application. During a universal CAR T-cells (UniCAR) therapy, “switch” molecules are injected into the patient’s bloodstream to activate the T-cell processes for attacking tumors. Therefore, it is crucial to monitor the level of CAR T-cells and switch molecules for better control and decision-making related to personalized treatment. Here, we demonstrate the application of our EG FET platform in testing the therapeutic components of this treatment and discuss the related outlook for FET-based biosensors in immunotherapy. Our work endeavor

Genetically engineered T cells expressing chimeric antigen receptors (CARs) have shown promising results particularly when targeting tumor associated antigens (TAAs) related to hematological malignancies. However, TAAs are usually expressed to some extend also on healthy tissues leading to on-target/off-tumor effects. To overcome this important safety issue along with improving targeting specificity and efficient killing of tumor escape variants, we adapted our Reverse CAR (RevCAR) system to follow an AND-gate Boolean logic. For that, Dual-RevCAR T cells were designed and armed with (I) a signaling (SIG) RevCAR, that includes the intracellular domain (ICD) of CD3 zeta; and (II) a costimulatory (COS) RevCAR, which contains a domain derived from CD28. Because the extracellular domains of both RevCARs are derived from the La/SS-B nuclear protein, Dual-RevCAR T cells will remain inactive until they encounter matching target modules (RevTMs). The bispecific antibody (bsAb)-like structure of the RevTMs allows their binding to RevCAR molecules and to specific antigens. However, only the simultaneous binding of RevTMs to SIG and COS RevCARs will promote the full activation of the Dual-RevCAR T cells. The epithelial cell adhesion molecule (EpCAM) and the carcinoembryonic antigen (CEA) have become appealing markers due to their overexpression in various solid tumor entities such as colorectal cancer, therefore representing promising target antigens for cancer immunotherapies following such a Dual Targeting CAR approach. Having this in mind, the aim of this work was to assess the potential therapeutic application of the Dual-RevCAR system to target EpCAM and CEA following an AND-gating approach.

High-fidelity (computer-) experiments are very expensive to perform, hence extracting as much information as possible from the collected data is vital. We present methods to estimate experiment outcomes based on ingested past data, to steer further sample acquisition both by suggesting paths for maximized uncertainty reduction and highlighting sensitivities on inputs, with use-cases from laser-light propagation and laser-plasma interactions to electron-bunch kinetics.

Droplet-based microfluidics that emerged a decade ago has offered a new route to boost the efficiency of biochemical methods in terms of detection time and sensitivity [Kaminski et al., Chem. Soc. Rev. 46, 2017]. Droplet-based fluidic technology saves materials consumption and reduces experimental wastes, allowing for real-time and individual tracking of each reactor, and has found numerous applications in biology and biotechnology [Baraban et al., Lab. Chip, 11, 2011]. In our lab, we developed a droplet-based microfluidic system to detect the bioreaction (e.g., bacterial growth, antibiotic effect, enzyme reaction dynamic, and so on) in high throughput and sensitivity by detecting fluorescence signals. The system achieves monitoring the bioreaction in the hundred to thousand droplets with the size of 200 nL with multi-channel automatically. Depending on the different purposes, this system can be adjusted to monitor the same batch of analytes over time in the long term (days), or continuously real-time detect analytes level change, as well as to switch between various optical settings. For example, we used the millifluidic droplets reactor system to study the bacterial coexistence by monitoring two Escherichia coli strains' growth simultaneously in real time [Zhao et al., Lab. Chip, 21, 2021]. Our system offers an environment with high statistical output, unaffected bacteria growth, and long-time measurements in a well-mixed liquid inhabit. By reading fluorescence in two parallel detector channels, we obtained and analyzed the monoculture and co-culture of these two strains E. coli BFP and E. coli YFP and explained the interaction and relationship between them. In another work, we investigated the antibiotic effect on bacterial coexistence. Our automatic nanoliter droplet analyzer is used to study the interactions of the sensitive and resistant strains of E. coli in the presence of antibiotics and to define the criteria leading to the emergence of cross-protection ph

Chimeric antigen receptors (CARs) are used for redirection of T cells against tumor cells and have demonstrated remarkable therapeutic effects against some hematological cancers. The modular system termed RevCAR can overcome the dangerous side effects associated with conventional CAR T cell therapy, such as cytokine release syndrome. It is composed of RevCAR T cells and a bispecific molecule target module, termed RevTM. This system is highly safe since RevCAR T cell activity can be controlled by RevTM and immediately switched off if side effects are detected. It is also versatile, since the same RevCAR T cell can be directed to different tumor-associated antigens (TAA) simply by adding RevTMs with different specificities. However, to improve the effectiveness in the treatment of solid tumors, the immunosuppressive tumor microenvironment (TME) still needs to be addressed. For this purpose, we have developed novel RevTMs targeting immune checkpoint molecules such as PD-L1, which are often upregulated by cancer cells to escape the immune cells. We have demonstrated that our novel RevTMs can redirect RevCAR T cells to specifically kill cell lines expressing immune checkpoint molecules. In addition to this strategy, the combinatorial targeting of a TAA and an immune checkpoint is very promising strategy to overcome the immunosuppressive microenvironment of solid tumors and thus to improve the treatment outcome.

Biosensors based on the extended gate field-effect transistor (EG FET) differ from the traditional FET- based biosensors in terms of creating a spatial separation between the EG sensing element and the FET transducer. Thus, EG can be employed as a cost-effective and disposable unit, while the FET transducer is retained as a reusable component. These features make EG particularly attractive for multiplexed biosensing since an EG electrode array can be easily integrated within a single chip, and individual electrodes can be separately modified for the recognition of diverse analyte types. Although the use of EG FET-based platforms has been successfully demonstrated in advanced biosensing applications,¹˒² they still suffer from practical disadvantages including limited multiplexing, complex nanofabrication of FET transducers, and reliance on bulky specialized high-performance measurement instruments for readout of low current levels (~nA range), all substantially reducing their suitability for many applications in point-of-care (POC) diagnostics and monitoring. We present a custom-designed multiplexed standalone EG FET-based potentiometric biosensing platform relying on a commercial FET transducer, portable modular electronics, and innovative assay format for potentiometric response enhancement based on the conjugation of analyte antibodies (Abs) to gold nanoparticles (AuNPs). To achieve simplified signal readout, we employ an in-house fabricated common reference electrode for all sensing points and operate the FET in constant charge mode. Potential shifts at the EG surface during sensing are indirectly detected as voltage changes between the reference electrode and the FET source terminal. We demonstrate the sensing of antibody-antibody interactions on the immunoglobulin G system and realize the 5-fold amplification of the potentiometric response compared to the traditional label-free assay by introducing AuNP-based labeling of the target analyte. Using the described

The La-protein is a multifunctional abundantly expressed protein in the nucleus of all human cells, where it for example functions as a chaperone for newly synthesized RNA-polymerase III transcripts. Recently, it has been shown that La is overexpressed in malignant cells and can be translocated to the cell surface under certain conditions, e. g. radio- or chemotherapy-induced necrosis, where it becomes a promising, inducible target for so-called “Death Targeting” approaches. The aim of this study was to investigate whether the La-protein could also be used as an inducible target for CAR T cell therapy. Therefore, three different CAR T cells directed against the La-protein (La-CARs) were developed and tested regarding their potential for “Death Targeting” of tumor cells. For construction of the La-CARs, we selected three different anti-La mAbs (5B9, 7B6 and 312B), which recognize distinct epitopes within the La-protein in a redox-dependent manner. This allowed us to determine, if the redox-state influences the ability of the La-CARs to target cell surface-bound La-protein. We first examined if La-CARs could eliminate tumor cells that were artificially labeled with wildtype La-protein or its oxidation-resistant triple-cysteine-mutant (TCM). By performing luciferase-based killing assays, we showed, that all La-CARs were specifically activated for lysis of La- or TCM-decorated tumor cells, which also resulted in significant secretion of pro-inflammatory cytokines. Redox-dependent differences of the La-CARs could be observed, whereby the non-redox-dependent 5B9-CAR was activated by La-wt- and TCM-labeled cells, the 7B6-CAR preferably by La-wt-labeled and the 312B-CAR preferably by TCM-labeled tumor cells. We could further prove that La-release from tumor cells could be induced by treatment with the cytotoxic drug cisplatin and La-CARs could be redirected against remaining tumor-cells after cisplatin treatment. Thereby, the 5B9-CAR, which recognizes La-protein independent