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.

T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation.

Since the first description of nuclear autoantigens in the late 1960s and early 1970s, researchers, including ourselves, have found it difficult to establish monoclonal antibodies (mabs) against nuclear antigens, including the La/SS-B (Sjögrens' syndrome associated antigen B) autoantigen. To date, only a few anti-La mabs have been derived by conventional hybridoma technology; however, those anti-La mabs were not bona fide autoantibodies as they recognize either human La specific, cryptic, or post-translationally modified epitopes which are not accessible on native mouse La protein. Herein, we present a series of novel murine anti-La mabs including truly autoreactive ones. These mabs were elicited from a human La transgenic animal through adoptive transfer of T cells from non-transgenic mice immunized with human La antigen. Detailed epitope and paratope analyses experimentally confirm the hypothesis that somatic hypermutations that occur during T cell dependent maturation can lead to autoreactivity to the nuclear La/SS-B autoantigen.

Two Be or Not Two Be: The Nuclear Autoantigen La/SS-B Is Able to Form Dimers and Oligomers in a Redox Dependent Manner.

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.

Integrated photodetectors for compact Fourier-transform waveguide spectrometers.

Extreme miniaturization of infrared spectrometers is critical for their integration into next-generation consumer electronics, wearables and ultrasmall satellites. In the infrared, there is a necessary compromise between high spectral bandwidth and high spectral resolution when miniaturizing dispersive elements, narrow band-pass filters and reconstructive spectrometers. Fourier-transform spectrometers are known for their large bandwidth and high spectral resolution in the infrared; however, they have not been fully miniaturized. Waveguide-based Fourier-transform spectrometers offer a low device footprint, but rely on an external imaging sensor such as bulky and expensive InGaAs cameras. Here we demonstrate a proof-of-concept miniaturized Fourier-transform waveguide spectrometer that incorporates a subwavelength and complementary-metal-oxide-semiconductor-compatible colloidal quantum dot photodetector as a light sensor. The resulting spectrometer exhibits a large spectral bandwidth and moderate spectral resolution of 50 cm-1 at a total active spectrometer volume below 100 µm × 100 µm × 100 µm. This ultracompact spectrometer design allows the integration of optical/analytical measurement instruments into consumer electronics and space devices.

Colloidal HgTe Quantum Dot/Graphene Phototransistor with a Spectral Sensitivity Beyond 3 µm.

Infrared light detection enables diverse technologies ranging from night vision to gas analysis. Emerging technologies such as low-cost cameras for self-driving cars require highly sensitive, low-cost photodetector cameras with spectral sensitivities up to wavelengths of 10 µm. For this purpose, colloidal quantum dot (QD) graphene phototransistors offer a viable alternative to traditional technologies owing to inexpensive synthesis and processing of QDs. However, the spectral range of QD/graphene phototransistors is thus far limited to 1.6 µm. Here, HgTe QD/graphene phototransistors with spectral sensitivity up to 3 µm are presented, with specific detectivities of 6 × 108 Jones at a wavelength of 2.5 µm and a temperature of 80 K. Even at kHz light modulation frequencies, specific detectivities exceed 108 Jones making them suitable for fast video imaging. The simple device architecture and QD film patterning in combination with a broad spectral sensitivity manifest an important step toward low-cost, multi-color infrared cameras.

Temperature-Dependent Charge Carrier Transfer in Colloidal Quantum Dot/Graphene Infrared Photodetectors.

Colloidal PbS quantum dot (QD)/graphene hybrid photodetectors are emerging QD technologies for affordable infrared light detectors. By interfacing the QDs with graphene, the photosignal of these detectors is amplified, leading to high responsivity values. While these detectors have been mainly operated at room temperature, low-temperature operation is required for extending their spectral sensitivity beyond a wavelength of 3 µm. Here, we unveil the temperature-dependent response of PbS QD/graphene phototransistors by performing steady-state and time-dependent measurements over a large temperature range of 80-300 K. We find that the temperature dependence of photoinduced charge carrier transfer from the QD layer to graphene is (i) not impeded by freeze-out of the (Schottky-like) potential barrier at low temperatures, (ii) tremendously sensitive to QD surface states (surface oxidation), and (iii) minimally affected by the ligand exposure time and QD layer thickness. Moreover, the specific detectivity of our detectors increases with cooling, with a maximum measured specific detectivity of at least 1010 Jones at a wavelength of 1280 nm and a temperature of 80 K, which is an order of magnitude larger compared to the corresponding room temperature value. The temperature- and gate voltage-dependent characterization presented here constitutes an important step in expanding our knowledge of charge transfer at interfaces of low-dimensional materials and toward the realization of next-generation optoelectronic devices.

Synaptic transistors with aluminum oxide dielectrics enabling full audio frequency range signal processing.

The rapid evolution of the neuromorphic computing stimulates the search for novel brain-inspired electronic devices. Synaptic transistors are three-terminal devices that can mimic the chemical synapses while consuming low power, whereby an insulating dielectric layer physically separates output and input signals from each other. Appropriate choice of the dielectric is crucial in achieving a wide range of operation frequencies in these devices. Here we report synaptic transistors with printed aluminum oxide dielectrics, improving the operation frequency of solution-processed synaptic transistors by almost two orders of magnitude to 50 kHz. Fabricated devices, yielding synaptic response for all audio frequencies (20 Hz to 20 kHz), are employed in an acoustic response system to show the potential for future research in neuro-acoustic signal processing with printed oxide electronics.

A theranostic PSMA ligand for PET imaging and retargeting of T cells expressing the universal chimeric antigen receptor UniCAR.

Chimeric antigen receptor (CAR) T cells have shown impressive therapeutic potential. Due to the lack of direct control mechanisms, therapy-related adverse reactions including cytokine release- and tumor lysis syndrome can even become life-threatening. In case of target antigen expression on non-malignant cells, CAR T cells can also attack healthy tissues. To overcome such side effects, we have established a modular CAR platform termed UniCAR: UniCAR T cells per se are inert as they recognize a peptide epitope (UniCAR epitope) that is not accessible on the surface of living cells. Bifunctional adapter molecules termed target modules (TM) can cross-link UniCAR T cells with target cells. In the absence of TMs, UniCAR T cells automatically turn off. Until now, all UniCAR TMs were constructed by fusion of the UniCAR epitope to an antibody domain. To open up the wide field of low-molecular-weight compounds for retargeting of UniCAR T cells to tumor cells, and to follow in parallel the progress of UniCAR T cell therapy by PET imaging we challenged the idea to convert a PET tracer into a UniCAR-TM. For proof of concept, we selected the clinically used PET tracer PSMA-11, which binds to the prostate-specific membrane antigen overexpressed in prostate carcinoma. Here we show that fusion of the UniCAR epitope to PSMA-11 results in a low-molecular-weight theranostic compound that can be used for both retargeting of UniCAR T cells to tumor cells, and for non-invasive PET imaging and thus represents a member of a novel class of theranostics.

Retargeting of UniCAR T cells with an in vivo synthesized target module directed against CD19 positive tumor cells.

Recent treatments of leukemias with T cells expressing chimeric antigen receptors (CARs) underline their impressive therapeutic potential but also their risk of severe side effects including cytokine release storms and tumor lysis syndrome. In case of cross-reactivities, CAR T cells may also attack healthy tissues. To overcome these limitations, we previously established a switchable CAR platform technology termed UniCAR. UniCARs are not directed against typical tumor-associated antigens (TAAs) but instead against a unique peptide epitope: Fusion of this peptide epitope to a recombinant antibody domain results in a target module (TM). TMs can cross-link UniCAR T cells with tumor cells and thereby lead to their destruction. So far, we constructed TMs with a short half-life. The fast turnover of such a TM allows to rapidly interrupt the treatment in case severe side effects occur. After elimination of most of the tumor cells, however, longer lasting TMs which have not to be applied via continous infusion would be more convenient for the patient. Here we describe and characterize a TM for retargeting UniCAR T cells to CD19 positive tumor cells. Moreover, we show that the TM can efficiently be produced in vivo from producer cells housed in a sponge-like biomimetic cryogel and, thereby, serving as an in vivo TM factory for an extended retargeting of UniCAR T cells to CD19 positive leukemic cells.

A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform.

Recent treatments of leukemias with chimeric antigen receptor (CAR) expressing T cells underline their impressive therapeutic potential. However, once adoptively transferred into patients, there is little scope left to shut them down after elimination of tumor cells or in case adverse side effects occur. This becomes of special relevance if they are directed against commonly expressed tumor associated antigens (TAAs) such as receptors of the ErbB family. To overcome this limitation, we recently established a modular CAR platform technology termed UniCAR. UniCARs are not directed against TAAs but instead against a unique peptide epitope on engineered recombinant targeting modules (TMs), which guide them to the target. In the absence of a TM UniCAR T cells are inactive. Thus an interruption of any UniCAR activity requires an elimination of unbound TM and the TM complexed with UniCAR T cells. Elimination of the latter one requires a disassembly of the UniCAR-TM complexes. Here, we describe a first nanobody (nb)-based TM directed against EGFR. The novel TM efficiently retargets UniCAR T cells to EGFR positive tumors and mediates highly efficient target-specific and target-dependent tumor cell lysis both in vitro and in vivo. After radiolabeling of the novel TM with 64Cu and 68Ga, we analyzed its biodistribution and clearance as well as the stability of the UniCAR-TM complexes. As expected unbound TM is rapidly eliminated while the elimination of the TM complexed with UniCAR T cells is delayed. Nonetheless, we show that UniCAR-TM complexes dissociate in vitro and in vivo in a concentration-dependent manner in line with the concept of a repeated stop and go retargeting of tumor cells via the UniCAR technology.

Retargeting of T lymphocytes to PSCA- or PSMA positive prostate cancer cells using the novel modular chimeric antigen receptor platform technology "UniCAR".

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