Extended-Aperture Shape Measurements Using Spatially Partially Coherent Illumination (ExASPICE).

We have recently demonstrated that the 3D shape of micro-parts can be measured using LED illumination based on speckle contrast evaluation in the recently developed SPICE profilometry (shape measurements based on imaging with spatially partially coherent illumination). The main advantage of SPICE is its improved robustness and measurement speed compared to confocal or white light interferometry. The limited spatial coherence of the LED illumination is used for depth discrimination. An electrically tunable lens in a 4f-configuration is used for fast depth scanning without mechanically moving parts. The approach is efficient, takes less than a second to capture required images, is eye-safe and offers a depth of focus of a few millimeters. However, SPICE's main limitation is its assumption of a small illumination aperture. Such a small illumination aperture affects the axial scan resolution, which dominates the measurement uncertainty. In this paper, we propose a novel method to overcome the aperture angle limitation of SPICE by illuminating the object from different directions with several independent LED sources. This approach reduces the full width at half maximum of the contrast envelope to one-eighth, resulting in a twofold improvement in measurement accuracy. As a proof of concept, shape measurements of various metal objects are presented.

RevCAR-expressing immune effector cells for targeting of Fn14-positive glioblastoma.

In recent studies, we have established the unique adapter chimeric antigen receptor (CAR) platform RevCAR which uses, as an extracellular CAR domain, a peptide epitope instead of an antibody domain. RevCAR adapters (termed RevCAR target modules, RevTMs) are bispecific antibodies that enable the reversible ON/OFF switch of the RevCAR system, improving the safety compared to conventional CARs. Here, we describe for the first time its use for retargeting of both T and NK-92 cells. In addition, we describe the development and preclinical validation of a novel RevTM for targeting of the fibroblast growth factor-inducible 14 (Fn14) surface receptor which is overexpressed on Glioblastoma (GBM) cells, and therefore serves as a promising target for the treatment of GBM. The novel RevTM efficiently redirects RevCAR modified T and NK-92 cells and leads to the killing of GBM cells both in vitro and in vivo. Tumor cell killing is associated with increased IL-2, TNF-α and/or IFN-γ secretion. Hence, these findings give an insight into the complementary potential of both RevCAR T and NK-92 systems as a safe and specific immunotherapeutic approach against GBM.

Radiolabeling of Platelets with 99mTc-HYNIC-Duramycin for In Vivo Imaging Studies.

Following the in vivo biodistribution of platelets can contribute to a better understanding of their physiological and pathological roles, and nuclear imaging methods, such as single photon emission tomography (SPECT), provide an excellent method for that. SPECT imaging needs stable labeling of the platelets with a radioisotope. In this study, we report a new method to label platelets with 99mTc, the most frequently used isotope for SPECT in clinical applications. The proposed radiolabeling procedure uses a membrane-binding peptide, duramycin. Our results show that duramycin does not cause significant platelet activation, and radiolabeling can be carried out with a procedure utilizing a simple labeling step followed by a size-exclusion chromatography-based purification step. The in vivo application of the radiolabeled human platelets in mice yielded quantitative biodistribution images of the spleen and liver and no accumulation in the lungs. The performed small-animal SPECT/CT in vivo imaging investigations revealed good in vivo stability of the labeling, which paves the way for further applications of 99mTc-labeled-Duramycin in platelet imaging.

Molecular imaging of bacterial outer membrane vesicles based on bacterial surface display.

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

Flash-profilometry: fullfield lensless acquisition of spectral holograms for coherence scanning profilometry.

Flash-profilometry is a novel measurement approach based on the fullfield lensless acquisition of spectral holograms. It is based on spectral sampling of the mutual coherence function and the subsequent calculation of its propagation along the optical axis several times the depth-of-field. Numerical propagation of the entire coherence function, rather than solely the complex amplitude, allows to digitally reproduce a complete scanning white-light interferometric (WLI) measurement. Hence, the corresponding 3D surface profiling system presented here achieves precision in the low nanometer range along an axial measurement range of several hundred micrometers. Due to the lensless setup, it is compact, immune against dispersion effects and lightweight. Additionally, because of the spectral sampling approach, it is faster than conventional coherence scanning WLI and robust against mechanical distortions, such as vibrations and rigid body movements. Flash-profilometry is therefore suitable for a wide range of applications, such as surface metrology, optical inspection, and material science and appears to be particularly suitable for a direct integration into production environments.

A novel ACE2 decoy for both neutralization of SARS-CoV-2 variants and killing of infected cells.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that 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.

Preclinical Characterization of the 177Lu-Labeled Prostate Stem Cell Antigen (PSCA)-Specific Monoclonal Antibody 7F5.

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 hormone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor progression. 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 resulting 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 revealed 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.

Functional pixels: a pathway towards true holographic displays using today's display technology.

Today's 3D dynamic holographic display techniques suffer from severe limitations due to an available number of pixels that is several orders of magnitude lower than required by conventional approaches. We introduce a solution to this problem by introducing the concept of functional pixels. This concept is based on pixels that individually spatially modulate the amplitude and phase of incident light with a polynomial function, rather than just a constant phase or amplitude. We show that even in the simple case of a linear modulation of the phase, the pixel count can be drastically reduced up to 3 orders of magnitude while preserving most of the image details. This scheme can be easily implemented with already existing technology, such as micro mirror arrays that provide tip, tilt and piston movement. Even though the individual pixels need to be technologically more advanced, the comparably small number of such pixels required to form a display may pave the way towards true holographic dynamic 3D displays.

Extending vision ray calibration by determination of focus distances.

The application of cameras as sensors in optical metrology techniques for three-dimensional topography measurement, such as fringe projection profilometry and deflectometry, presumes knowledge regarding the metric relationship between image space and object space. This relation is established by camera calibration and a variety of techniques are available. Vision ray calibration achieves highly precise camera calibration by employing a display as calibration target, enabling the use of active patterns in the form of series of phase-shifted sinusoidal fringes. Besides the required spatial coding of the display surface, this procedure yields additional full-field contrast information. Exploiting the relation between full-field contrast and defocus, we present an extension of vision ray calibration providing the additional information of the focus distances of the calibrated camera. In our experiments we achieve a reproducibility of the focus distances in the order of mm. Using a modified Laplacian based focus determination method, we confirm our focus distance results within a few mm.

Chocolate inspection by means of phase-contrast imaging using multiple-plane terahertz phase retrieval.

Terahertz (THz) radiation has shown enormous potential for non-destructive inspection in many contexts. Here, we present a method for imaging defects in chocolate bars that can be extended to many other materials. Our method requires only a continuous wave (CW) monochromatic source and detector at relatively low frequencies (280 GHz) corresponding to a relatively long wavelength of 1.1 mm. These components are used to construct a common-path configuration enabling the capturing of several images of THz radiation diffracted by the test object at different axial depths. The captured diffraction-rich images are used to constrain the associated phase retrieval problem enabling full access to the wave field, i.e., real amplitude and phase distributions. This allows full-field diffraction-limited phase-contrast imaging. Thus, we experimentally demonstrate the possibility of identifying contaminant particles with dimensions comparable to the wavelength.

Combining Radiation- with Immunotherapy in Prostate Cancer: Influence of Radiation on T Cells.

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 IL-2, TNF and interferon-γ 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.

Targeting CD10 on B-Cell Leukemia Using the Universal CAR T-Cell Platform (UniCAR).

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.

Development and Functional Characterization of a Versatile Radio-/Immunotheranostic Tool for Prostate Cancer Management.

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 a target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic positron emission tomography (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.

Nanosensors in clinical development of CAR-T cell immunotherapy.

Immunotherapy using CAR-T cells is a new technological paradigm 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. In this work, silicon nanowire field-effect transistors are used to develop target modules for an optimized CAR-T cell operation. Focusing on a library of seven variants of E5B9 peptide that is used as CAR targeting epitope, we performed multiplexed binding tests using nanosensor chips. These peptides had been immobilized onto the sensor to compare the transistor signals upon titration with anti-La 5B9 antibodies. The correlation of binding affinities and sensor sensitivities enabled a selection of candidates for the interaction between CAR and target modules. An extremely low detection limit was observed for the sensor, down to femtomolar concentration, outperforming the current assay of the same purpose. Finally, the CAR T-cells redirection capability of selected peptides in target modules was proven successful in an in-vitro cytotoxicity assay. Our results open the perspective for the nanosensors to go beyond the early diagnostics in clinical cancer research towards developing and monitoring immunotherapeutic treatment, where the quantitative analysis with the standard techniques is limited.

In Vitro Determination of Inhibitory Effects of Humic Substances Complexing Zn and Se on SARS-CoV-2 Virus Replication.

(1) Background: Humic substances are well-known human nutritional supplement materials and they play an important performance-enhancing role as animal feed additives. For decades, ingredients of humic substances have been proven to carry potent antiviral effects against different viruses. (2) Methods: Here, the antiviral activity of a humic substance containing ascorbic acid, Se- and Zn2+ ions intended as a nutritional supplement material was investigated against SARS-CoV-2 virus B1.1.7 Variant of Concern ("Alpha Variant") in a VeroE6 cell line. (3) Results: This combination has a significant in vitro antiviral effect at a very low concentration range of its intended active ingredients. (4) Conclusions: Even picomolar concentration ranges of humic substances, Vitamin C and Zn/Se ions in the given composition, were enough to achieve 50% viral replication inhibition in the applied SARS-CoV-2 virus inhibition test.

Terahertz referenceless wavefront sensing by means of computational shear-interferometry.

In this contribution, we demonstrate the first referenceless measurement of a THz wavefront by means of shear-interferometry. The technique makes use of a transmissive Ronchi phase grating to generate the shear. We fabricated the grating by mechanical machining of high-density polyethylene. At the camera plane, the +1 and -1 diffraction orders are coherently superimposed, generating an interferogram. We can adjust the shear by selecting the period of the grating and the focal length of the imaging system. We can also alter the direction of the shear by rotating the grating. A gradient-based iterative algorithm is used to reconstruct the wavefront from a set of shear interferograms. The results presented in this study demonstrate the first step towards wavefield sensing in the terahertz band without using a reference wave.

Γ-profilometry: a new paradigm for precise optical metrology.

We show that the shape of a surface can be unambiguously determined from investigating the coherence function of a wave-field reflected by the surface and without the requirement of a reference wave. Spatio-temporal sampling facilitates the identification of temporal shifts of the coherence function that correspond to finite height differences of the surface. Evaluating these finite differences allows for the reconstruction of the surface using a numerical integration procedure. Spatial sampling of the coherence function is provided by a shear interferometer whereas temporal sampling is achieved by means of a Soleil-Babinet compensator. This low coherence profiling method allows to determine the shape of an object with sub-micrometer resolution and over a large unambiguity range, although it does not require any isolation against mechanical vibration. The approach therefore opens up a new avenue for precise, rugged optical metrology suitable for industrial in-line applications.

Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy.

Clinical translation of novel immunotherapeutic strategies such as chimeric antigen receptor (CAR) T-cells in acute myeloid leukemia (AML) is still at an early stage. Major challenges include immune escape and disease relapse demanding for further improvements in CAR design. To overcome such hurdles, we have invented the switchable, flexible and programmable adaptor Reverse (Rev) CAR platform. This consists of T-cells engineered with RevCARs that are primarily inactive as they express an extracellular short peptide epitope incapable of recognizing surface antigens. RevCAR T-cells can be redirected to tumor antigens and controlled by bispecific antibodies cross-linking RevCAR T- and tumor cells resulting in tumor lysis. Remarkably, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. We herein show for the first time the applicability of the RevCAR platform to target myeloid malignancies like AML. Applying in vitro and in vivo models, we have proven that AML cell lines as well as patient-derived AML blasts were efficiently killed by redirected RevCAR T-cells targeting CD33 and CD123 in a flexible manner. Furthermore, by targeting both antigens, a Boolean AND gate logic targeting could be achieved using the RevCAR platform. These accomplishments pave the way towards an improved and personalized immunotherapy for AML patients.

And Yet It Moves: Oxidation of the Nuclear Autoantigen La/SS-B Is the Driving Force for Nucleo-Cytoplasmic Shuttling.

Decades ago, we and many other groups showed a nucleo-cytoplasmic translocation of La protein in cultured cells. This shuttling of La protein was seen after UV irradiation, virus infections, hydrogen peroxide exposure and the Fenton reaction based on iron or copper ions. All of these conditions are somehow related to oxidative stress. Unfortunately, these harsh conditions could also cause an artificial release of La protein. Even until today, the shuttling and the cytoplasmic function of La/SS-B is controversially discussed. Moreover, the driving mechanism for the shuttling of La protein remains unclear. Recently, we showed that La protein undergoes redox-dependent conformational changes. Moreover, we developed anti-La monoclonal antibodies (anti-La mAbs), which are specific for either the reduced form of La protein or the oxidized form. Using these tools, here we show that redox-dependent conformational changes are the driving force for the shuttling of La protein. Moreover, we show that translocation of La protein to the cytoplasm can be triggered in a ligand/receptor-dependent manner under physiological conditions. We show that ligands of toll-like receptors lead to a redox-dependent shuttling of La protein. The shuttling of La protein depends on the redox status of the respective cell type. Endothelial cells are usually resistant to the shuttling of La protein, while dendritic cells are highly sensitive. However, the deprivation of intracellular reducing agents in endothelial cells makes endothelial cells sensitive to a redox-dependent shuttling of La protein.