Fibroblast activation protein targeted radiotherapy induces an immunogenic tumor microenvironment and enhances the efficacy of PD-1 immune checkpoint inhibition.

PURPOSE: FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a PD-1 targeting antibody. METHODS: C57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with 177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of 177Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses. RESULTS: 177Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors, 177Lu-FAP-2287 increased CD8+ T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8+ T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86. CONCLUSION: In a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8+ T cells. These findings provide a rationale for clinical studies of combined 177Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.

Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy.

PURPOSE: Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies. METHODS: FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule-based FAP-targeting agent. RESULTS: Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of 68Ga-FAP-2286, 111In-FAP-2286, and 177Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. 177Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46. CONCLUSION: In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of 68Ga-FAP-2286 for imaging and 177Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.

A prospective multicenter validation study for a novel angiography-derived physiological assessment software: Rationale and design of the radiographic imaging validation and evaluation for Angio-iFR (ReVEAL iFR) study.

Angiography-derived physiological assessment of coronary lesions has emerged as an alternative to wire-based assessment aiming at less-invasiveness and shorter procedural time as well as cost effectiveness in physiology-guided decision making. However, current available image-derived physiology software have limitations including the requirement of multiple projections and are time consuming. METHODS/DESIGN: The ReVEAL iFR (Radiographic imaging Validation and EvALuation for Angio-iFR) trial is a multicenter, multicontinental, validation study which aims to validate the diagnostic accuracy of the Angio-iFR medical software device (Philips, San Diego, US) in patients undergoing angiography for Chronic Coronary Syndrome (CCS). The Angio-iFR will enable operators to predict both the iFR and FFR value within a few seconds from a single projection of cine angiography by using a lumped parameter fluid dynamics model. Approximately 440 patients with at least one de-novo 40% to 90% stenosis by visual angiographic assessment will be enrolled in the study. The primary endpoint is the sensitivity and specificity of the iFR and FFR for a given lesion compared to the corresponding invasive measures. The enrollment started in August 2019, and was completed in March 2021. SUMMARY: The Angio-iFR system has the potential of simplifying physiological evaluation of coronary stenosis compared with available systems, providing estimates of both FFR and iFR. The ReVEAL iFR study will investigate the predictive performance of the novel Angio-iFR software in CCS patients. Ultimately, based on its unique characteristics, the Angio-iFR system may contribute to improve adoption of functional coronary assessment and the workflow in the catheter laboratory.

In vitro-to-in vivo extrapolation (IVIVE) by PBTK modeling for animal-free risk assessment approaches of potential endocrine-disrupting compounds.

While in vitro testing is used to identify hazards of chemicals, nominal in vitro assay concentrations may misrepresent potential in vivo effects and do not provide dose-response data which can be used for a risk assessment. We used reverse dosimetry to compare in vitro effect concentrations-to-in vivo doses causing toxic effects related to endocrine disruption. Ten compounds (acetaminophen, bisphenol A, caffeine, 17α-ethinylestradiol, fenarimol, flutamide, genistein, ketoconazole, methyltestosterone, and trenbolone) have been tested in the yeast estrogen screening (YES) or yeast androgen-screening (YAS) assays for estrogen and androgen receptor binding, as well as the H295R assay (OECD test guideline no. 456) for potential interaction with steroidogenesis. With the assumption of comparable concentration-response ratios of these effects in the applied in vitro systems and the in vivo environment, the lowest observed effect concentrations from these assays were extrapolated to oral doses (LOELs) by reverse dosimetry. For extrapolation, an eight-compartment Physiologically Based Toxicokinetic (PBTK) rat model based on in vitro and in silico input data was used. The predicted LOEL was then compared to the LOEL actually observed in corresponding in vivo studies (YES/YAS assay versus uterotrophic or Hershberger assay and steroidogenesis assay versus pubertal assay or generation studies). This evaluation resulted in 6 out of 10 compounds for which the predicted LOELs were in the same order of magnitude as the actual in vivo LOELs. For four compounds, the predicted LOELs differed by more than tenfold from the actual in vivo LOELs. In conclusion, these data demonstrate the applicability of reverse dosimetry using a simple PBTK model to serve in vitro-in silico-based risk assessment, but also identified cases and test substance were the applied methods are insufficient.

Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.

Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T(594)) for high-affinity interaction. We confirm that T(594) is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T(594)-specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface.

Directed supramolecular surface assembly of SNAP-tag fusion proteins.

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized SNAP-fusion proteins on cyclodextrin-coated surfaces yielded stable monolayers. The binding of the fusion proteins is specific and occurs with an affinity in the order of 10(6) M(-1) as determined by surface plasmon resonance. Reversible micropatterns of the fusion proteins on micropatterned cyclodextrin surfaces were visualized by using fluorescence microscopy. Furthermore, the guest-functionalized proteins could be assembled out of solution specifically onto the surface of cyclodextrin vesicles. The SNAP-tag labeling of proteins thus allows for assembly of modified proteins through a host-guest interaction on different surfaces. This provides a new strategy in fabricating protein patterns on surfaces and takes advantage of the high labeling efficiency of the SNAP-tag with designed supramolecular elements.

Finding the Needle in the Haystack: High-Resolution Techniques for Characterization of Mixed Protein Particles Containing Shed Silicone Rubber Particles Generated During Pumping.

During the manufacturing process of biopharmaceuticals, peristaltic pumps are employed at different stages for transferring and dosing of the final product. Commonly used silicone tubings are known for particle shedding from the inner tubing surface due to friction in the pump head. These nanometer sized silicone rubber particles could interfere with proteins. Until now, only mixed protein particles containing micrometer-sized contaminations such as silicone oil have been characterized, detected, and quantified. To overcome the detection limits in particle sizes of contaminants, this study aimed for the definite identification of protein particles containing nanometer sized silicone particles in qualitative and quantitative manner. The mixed particles consisted of silicone rubber particles either coated with a protein monolayer or embedded into protein aggregates. Confocal Raman microscopy allows label free chemical identification of components and 3D particle imaging. Labeling the tubing enables high-resolution imaging via confocal laser scanning microscopy and counting of mixed particles via Imaging Flow Cytometry. Overall, these methods allow the detection and identification of particles of unknown origin and composition and could be a forensic tool for solving problems with contaminations during processing of biopharmaceuticals.

Rapid Alloy Development of Extremely High-Alloyed Metals Using Powder Blends in Laser Powder Bed Fusion.

The design of new alloys by and for metal additive manufacturing (AM) is an emerging field of research. Currently, pre-alloyed powders are used in metal AM, which are expensive and inflexible in terms of varying chemical composition. The present study describes the adaption of rapid alloy development in laser powder bed fusion (LPBF) by using elemental powder blends. This enables an agile and resource-efficient approach to designing and screening new alloys through fast generation of alloys with varying chemical compositions. This method was evaluated on the new and chemically complex materials group of multi-principal element alloys (MPEAs), also known as high-entropy alloys (HEAs). MPEAs constitute ideal candidates for the introduced methodology due to the large space for possible alloys. First, process parameters for LPBF with powder blends containing at least five different elemental powders were developed. Secondly, the influence of processing parameters and the resulting energy density input on the homogeneity of the manufactured parts were investigated. Microstructural characterization was carried out by optical microscopy, electron backscatter diffraction (EBSD), and energy-dispersive X-ray spectroscopy (EDS), while mechanical properties were evaluated using tensile testing. Finally, the applicability of powder blends in LPBF was demonstrated through the manufacture of geometrically complex lattice structures with energy absorption functionality.

Modeling the Effect of Primary and Secondary Twinning on Texture Evolution during Severe Plastic Deformation of a Twinning-Induced Plasticity Steel.

Modeling the effect of deformation twinning and the ensuing twin-twin- and slip-twin-induced hardening is a long-standing problem in computational mechanical metallurgy of materials that deform by both slip and twinning. In this work, we address this effect using the twin volume transfer method, which obviates the need of any cumbersome criterion for twin variant selection. Additionally, this method is capable of capturing, at the same time, secondary or double twinning, which is particularly important for modeling in large strain regimes. We validate our modeling methodology by simulating the behavior of an Fe-23Mn-1.5Al-0.3C twinning-induced plasticity (TWIP) steel under large strain conditions, experimentally achieved in this work through equal-channel angular pressing (ECAP) for up to two passes in a 90° die following route BC at 300 °C. Each possible twin variant, whether nucleating inside the parent grain or inside a potential primary twin variant was predefined in the initial list of orientations as possible grain of the polycrystal with zero initial volume fraction. A novelty of our approach is to take into account the loss of coherency of the twins with their parent matrix under large strains, obstructing progressively their further growth. This effect has been captured by attenuating growth rates of twins as a function of their rotation away from their perfect twin orientation, dubbed here as "disorientation" with respect to the mother grain's lattice. The simulated textures and the hardening under tensile strain showed very good agreement with experimental characterization and mechanical testing results. Furthermore, upper-bound Taylor deformation was found to be operational for the TWIP steel deformation when all the above ingredients of twinning are captured, indicating that self-consistent schemes can be bypassed.

Exploiting Process-Related Advantages of Selective Laser Melting for the Production of High-Manganese Steel.

Metal additive manufacturing has strongly gained scientific and industrial importance during the last decades due to the geometrical flexibility and increased reliability of parts, as well as reduced equipment costs. Within the field of metal additive manufacturing methods, selective laser melting (SLM) is an eligible technique for the production of fully dense bulk material with complex geometry. In the current study, we addressed the application of SLM for processing a high-manganese TRansformation-/TWinning-Induced Plasticity (TRIP/TWIP) steel. The solidification behavior was analyzed by careful characterization of the as-built microstructure and element distribution using optical and scanning electron microscopy (SEM). In addition, the deformation behavior was studied using uniaxial tensile testing and SEM. Comparison with conventionally produced TRIP/TWIP steel revealed that elemental segregation, which is normally very pronounced in high-manganese steels and requires energy-intensive post processing, is reduced due to the high cooling rates during SLM. Also, the very fast cooling promoted ε- and α'-martensite formation prior to deformation. The superior strength and pronounced anisotropy of the SLM-produced material was correlated with the microstructure based on the process-specific characteristics.

Subtype-specific modulation of estrogen receptor-coactivator interaction by phosphorylation.

The estrogen receptor (ER) is the number one target for the treatment of endocrine responsive breast cancer and remains a highly attractive target for new drug development. Despite considerable efforts to understand the role of ER post-translational modifications (PTMs), the complexity of these modifications and their impact, at the molecular level, are poorly understood. Using a chemical biology approach, fundamentally rooted in an efficient protein semisynthesis of tyrosine phosphorylated ER constructs, the complex role of the ER tyrosine phosphorylation is addressed here for the first time on a molecular level. The semisynthetic approach allows for the site-specific introduction of PTMs as well as biophysical probes. A combination of biophysical techniques, including NMR, with molecular dynamics studies reveals the role of the phosphorylation of the clinically relevant tyrosine 537 (Y537) in ERα and the analogous tyrosine (Y488) in ERß. Phosphorylation has important effects on the dynamics of the ER Helix 12, which is centrally involved in receptor activity regulation, and on its interplay with ligand and cofactor binding, but with differential regulatory effects of the analogous PTMs on the two ER subtypes. Combined, the results bring forward a novel molecular model of a phosphorylation-induced subtype specific ER modulatory mechanism, alternative to the widely accepted ligand-induced activation mechanism.

Model based 3D CS-catheter tracking from 2D X-ray projections: binary versus attenuation models.

Tracking the location of medical devices in interventional X-ray data solves different problems. For example the motion information of the devices is used to determine cardiac or respiratory motion during X-ray guided procedures or device features are used as landmarks to register images. In this publication an approach using a 3D deformable catheter model is presented and used to track a coronary sinus (CS) catheter in 3D plus time through a complete rotational angiography sequence. The benefits of using voxel based models with attenuation information for 2D/3D registration are investigated in comparison to binary catheter models. The 2D/3D registration of the model allows to extract a 3D catheter shape from every individual 2D projection. The tracking accuracy is evaluated on simulated and clinical rotational angiography data of the contrast enhanced left atrium. The quantitative evaluation of the experiments delivers an average registration accuracy for all catheter electrodes of 0.23 mm in 2D and 0.95 mm in 3D when using an attenuation model of the catheter. The overall tracking accuracy is lower when using binary catheter models.