Myocardial Perfusion SPECT and ATTR imaging 2021 in Germany: Results of the 9th Survey.

AIM: This paper presents the results of the 9th survey of myocardial perfusion SPECT (MPS) from the reporting year 2021. METHODS: 218 questionnaires (131 practices (PR), 58 hospitals (HO), 29 university hospitals (UH)) were evaluated. Results of the last survey 2018 are set in squared brackets. RESULTS: MPS data from a total of 133,057 [145,930] patients (-8.8%) with 131,868 [143,707] stress and 106,546 [121,899] rest MPS were analysed. A comparison with official data revealed that 54% all MPS were recorded. From 2018 to 2021, official data showed a every year an increase in MPS numbers. On average, 610 [502] MPS patients (+22%) were examined in each department. 74% [69%] of the responders reported an increase or no changes in their MPS patient numbers. Ambulatory care cardiologists represented as always, the mayor referral group (68% [69%]). For the first time, pharmacological stress was more frequently applied than ergometry (42% [51]). Regadenoson was mostly used. The use of the different protocols remained nearly unchanged. Two-day protocols were predominantly applied (49% [48%]). A shift from multi-headed cameras (58% [72%]) to SPECT-CT systems (24% [17%]) was found. Attenuation correction was performed in 33% [26%] of all MPS. 88% [86%] of all stress, 88% [87%] of all rest and 87% [83%] of all stress and rest MPS were acquired as gated SPECT. 72% [67%] of all departments performed scoring by default. The number of departments without scoring decreased to 13% [16%]. CONCLUSIONS: The MPS Study 2021 shows that the long-term positive development of MPS imaging in Germany is continuing. The COVID-19 pandemia did not change this trend. The procedural and technical details of MPS imaging reveal a high level of guideline conformity.

Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins.

Bispecific antibodies have recently attracted intense interest. CrossMab technology was described in 2011 as novel approach enabling correct antibody light-chain association with their respective heavy chain in bispecific antibodies, together with methods enabling correct heavy-chain association using existing pairs of antibodies. Since the original description, CrossMab technology has evolved in the past decade into one of the most mature, versatile, and broadly applied technologies in the field, and nearly 20 bispecific antibodies based on CrossMab technology developed by Roche and others have entered clinical trials. The most advanced of these are the Ang-2/VEGF bispecific antibody faricimab, currently undergoing regulatory review, and the CD20/CD3 T cell bispecific antibody glofitamab, currently in pivotal Phase 3 trials. In this review, we introduce the principles of CrossMab technology, including its application for the generation of bi-/multispecific antibodies with different geometries and mechanisms of action, and provide an overview of CrossMab-based therapeutics in clinical trials.

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity.

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the 'Fc-load' on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Engineering therapeutic bispecific antibodies using CrossMab technology.

Bispecific antibodies have recently gained major interest as they allow novel mechanisms-of-action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. A major issue in engineering IgG-based bispecific antibodies has been to enable the correct association of heavy and light chains resulting in correct assembly of the desired bispecific antibody in sufficient yield. Various approaches have been described during recent years to tackle this challenge. We have developed the so-called CrossMab technology that enforces correct light chain association based on the domain crossover of immunoglobulin domains in the Fab region of the bispecific antibody. This versatile technology allows the generation of different bispecific antibody formats including asymmetric heterodimeric monovalent 1 + 1 bispecific antibodies and asymmetric heterodimeric bispecific antibodies with 2 + 1 valency in combination with approaches enabling Fc-hetermodimerization like knob-into-hole technology as well as the generation of tetravalent symmetric bispecific antibodies with 2 + 2 valency, also known as Tandem-Fab based IgG antibodies, using processes suitable for the large scale production of therapeutic bispecific antibodies. Notably, as of now, at least eight different bispecific antibodies using CrossMab technology entered clinical development, and additional CrossMabs are in late preclinical development. This review provides a summary of the status and progress with the engineering and generation of CrossMab technology based bispecific antibodies as well as their therapeutic application.

Variable heavy-variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly.

Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers. The CrossMabCH1-CL exchange does not lead to the formation of unexpected side products, whereas the CrossMabFab and the CrossMabVH-VL formats result in the formation of typical side products. Thus, CrossMabCH1-CL was initially favored for therapeutic antibody development. Here, we report a novel improved CrossMab design principle making use of site-specific positional exchanges of charged amino acid pairs in the constant domain of these CrossMabs to enable the correct light chain assembly in the CrossMabVH-VL and improvements for the CrossMabFab design.

Volume/perfusion ratio from lung SPECT/CT.

AIM: In pulmonary emphysema lung volume reduction procedures (LVRP) can optimize respiratory pump function. Identification of the most affected lobe can be reached using relative lobar volume (relVol) from CT, but this approach disregards the corresponding lobar perfusion. The aim of the study was therefore to establish a new parameter combining relVol from CT and relative perfusion (relPerf) from perfusion SPECT as a single parameter (volume/perfusion ratio (VPR)) to optimize the identification procedure. METHODS: As a proof of principle VPR was calculated from hybrid V-/P-SPECT/CT scans from 20 patients with severe pulmonary emphysema (SPE) before LVRP. Lung V-/P-SPECT/CT (Siemens SymbiaT) was done with Technegas and 99mTc-MAA. Quantification of lobar perfusion from scintigraphy and volume from CT was performed using "HERMES Hybrid 3D - Lung Lobe Quantification". Using normal ranges - from 12 patients with suspected pulmonary embolism and normal lung structure and perfusion - all lobes were classified as normal or abnormal to identify targets for LVRP. RESULTS: Normal values for VPR: right upper lobe 1.09 ± 0.10, middle lobe 1.31 ± 0.31, right lower lobe 0.87 ± 0.08; left upper lobe 1.09 ± 0.11, left lower lobe 0.87 ± 0.12. In the 20 SPE patients there were only 7 lobes with pathological values for rel- Vol, 14 lobes with pathological values for rel- Perf but 31 lobes with pathological VPR. CONCLUSION: Estimation of VPR from lung SPECT/CT enables a combined view of lobar volume and perfusion with one parameter. In SPE patients VPR allows identifying possible target structures with much higher sensitivity than when using relPerf or relVol alone. The specificity and the prognostic value of this new parameter have to be tested in a clinical trial.

The use of CrossMAb technology for the generation of bi- and multispecific antibodies.

The major challenge in the generation of bispecific IgG antibodies is enforcement of the correct heavy and light chain association. The correct association of generic light chains can be enabled using immunoglobulin domain crossover, known as CrossMAb technology, which can be combined with approaches enabling correct heavy chain association such as knobs-into-holes (KiH) technology or electrostatic steering. Since its development, this technology has proven to be very versatile, allowing the generation of various bispecific antibody formats, not only heterodimeric/asymmetric bivalent 1+1 CrossMAbs, but also tri- (2+1), tetravalent (2+2) bispecific and multispecific antibodies. Numerous CrossMAbs have been evaluated in preclinical studies, and, so far, 4 different tailor-made bispecific antibodies based on the CrossMAb technology have entered clinical studies. Here, we review the properties and activities of bispecific CrossMAbs and give an overview of the variety of CrossMAb-enabled antibody formats that differ from heterodimeric 1+1 bispecific IgG antibodies.

Cytokines in noninvasively obtained amniotic fluid as predictors of fetal inflammatory response syndrome.

BACKGROUND: In patients with preterm premature rupture of membranes, intrauterine inflammation and/or infection is frequently present, can lead to fetal inflammatory response syndrome, and is associated with adverse neonatal outcome. Clinical decision making requires balancing the potential benefits of pregnancy prolongation against the risk of intrauterine infection. Diagnostic tests in maternal serum are of moderate prediction value and amniocentesis is an invasive procedure. Therefore, markers obtained noninvasively would be helpful in patients with expectant management. OBJECTIVES: To determine the predictive values of amniotic fluid interleukin-6 and tumor necrosis factor-α in vaginal secretions for fetal inflammatory response syndrome and/or histologic funisitis and for adverse neonatal outcome in patients with preterm premature rupture of membranes. STUDY DESIGN: In this prospective multicenter case-control study, vaginal secretions were sampled daily with a noninvasive method from 99 women with preterm premature rupture of membranes and expectant management. Amniotic fluid interleukin-6 and tumor necrosis factor-α were measured by 2 different immunoassays (an automated chemiluminescent enzyme immunoassay and a lateral flow immunoassay). After delivery, patients were divided into a control or a fetal inflammatory response syndrome group according to neonatal interleukin-6 in cord plasma and/or the presence of funisitis. Univariate and multivariate regression analyses were performed and prediction models were developed by calculating receiver operating characteristic curves. RESULTS: Gestational age at delivery was lower and latency period was longer in the fetal inflammatory response syndrome group compared to the control group. The strongest risk factor for composite adverse neonatal outcome was fetal inflammatory response syndrome (odds ratio, 2.48; confidence interval, 1.40-4.38). The median concentrations of amniotic fluid interleukin-6 and tumor necrosis factor-α in vaginal secretions were significantly higher in the fetal inflammatory response group compared to the control group in both immunoassays (P < .001). The area under the curve of the clinical reference model (including common clinical parameters) was 0.66. Adding interleukin-6 and tumor necrosis factor-α into the model improved the area under the curve to 0.92 (in both assays, interleukin-6 IMMULITE and QuickLine); 0.87 (tumor necrosis factor-α IMMULITE) and 0.94 (tumor necrosis factor-α QuickLine), respectively. CONCLUSION: The strongest risk factor for worse neonatal outcome (composite neonatal outcome) was fetal inflammatory response syndrome. Amniotic fluid interleukin-6 and tumor necrosis factor-α seem to be good predictors for fetal inflammatory response syndrome and for histologic funisitis and may improve the clinical management of patients with preterm premature rupture of membranes. The noninvasive technique of sampling amniotic fluid from vaginal secretions facilitates daily measurements and bedside assessment of cytokines and is in this respect preferable to invasive amniocentesis.

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry.

The quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

In vitro-assessment of putative antiprogestin activities of phytochemicals and synthetic UV absorbers in human endometrial Ishikawa cells.

Critical steps of embryo implantation are controlled by progesterone. These processes can be interrupted by progesterone receptor (PR) antagonists, e.g. drugs used for abortion. Antiprogestin effects induced by natural compounds and environmental chemicals have been rarely addressed. In our in vitro study, we investigated putative antiprogestin activities of the plant compounds apigenin (API) and trans-ferulic acid (t-FA) as well as the UV absorbers octyl methoxycinnamate (OMC) and 4-methylbenzylidene camphor (4-MBC). They were compared with the selective progesterone receptor modulators (SPRMs) mifepristone (RU486) and ulipristal acetate (UPA) as well as the full PR-antagonist ZK137316. Effects of test compounds in combination with progesterone on the progesterone-sensitive target gene estrogen sulfotransferase (SULT1E1) were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The agonistic effect of progesterone on SULT1E1 mRNA levels was concentration-dependently antagonized by RU486, UPA and ZK137316 as well as, with lower potency, apigenin. t-FA, OMC and 4-MBC had no effect on SULT1E1 mRNA levels. We demonstrated that apigenin, although at higher concentrations, exerts a similar effect as the well-characterized SPRMs RU486 and UPA or the progesterone antagonist ZK137316 in this model. Our endometrium-specific Ishikawa cell assay is a useful complement to artificial transactivation assays for the identification of environmental substances with antiprogestin activities.

The current and future status of nuclear cardiology: a consensus report.

Cardiac imaging now provides a range of anatomical and functional information with some overlap in the ability of individual techniques to guide diagnosis and management. This report summarizes the conclusions of a panel of cardiac imagers who assembled to discuss the current state of the field. It focuses principally on options for nuclear cardiology, the choice between individual techniques, and areas where further advances would benefit patient management.

Crystal structure of an anti-Ang2 CrossFab demonstrates complete structural and functional integrity of the variable domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.

Vulnerability to psychotogenic effects of ketamine is associated with elevated D2/3-receptor availability.

Previous positron emission tomography (PET) studies employing competition paradigms have shown either no change or substantial declines in striatal [(11)C]-raclopride binding after challenge with psychotogenic doses of the N-methyl-D-aspartate antagonist ketamine. We sought to probe the relationship between the severity of ketamine-induced psychotic symptoms and altered dopamine D(2/3) receptor availability throughout brain using the high affinity ligand [(18)F]-fallypride (FP). PET recordings were obtained in a group of 10 healthy, young male volunteers, in a placebo condition, and in the course of an infusion with ketamine at a psychotomimetic dose. Administration of the Positive and Negative Syndrome Scale and the Thought and Language Index in both conditions revealed a substantial emergence of mainly negative symptoms of schizophrenia, persisting until the end of the 3 h PET recordings. The baseline FP binding in cortex, caudate nucleus and other brain regions was highly predictive of the individual severity of psychotic symptoms in the ketamine condition. However, there was no evidence of ketamine-evoked reductions in FP binding. In the context of earlier findings, we speculate that high baseline D(2/3)-receptor availability may impart benefits with regard to cognitive flexibility, but increases the risk of maladaptive information processing in the face of environmental stresses and challenges.

Quantitative myocardial perfusion-SPECT: algorithm-specific influence of reorientation on calculation of summed stress score.

PURPOSE: Myocardial perfusion SPECT (MPS) with software-assisted determination of summed stress score (SSS) is of established importance for diagnosis/therapy planning in coronary artery disease. Differences in contour finding suggest algorithm-specific influence on quantification if heart axes are chosen incorrectly. Thus, this study quantified the influence of heart-axis tilt on SSS calculation using Quantitative Perfusion SPECT and 4D-MSPECT. PATIENTS AND METHODS: Stress MPS of 50 men acquired on a triple-head gamma camera were correctly reoriented by experienced technologists (R0) and then tilted by 5 degrees/10 degrees/15 degrees/20 degrees/30 degrees/45 degrees along both long axes (R5-R45). SPECT images were quantified for SSS using QPS and 4D-MSPECT. SSS values for R0 and R5-R45 were analyzed using correlation analysis. Weighted kappa values (κ) were calculated to measure agreement regarding perfusion abnormality severity (4-step SSS rating: 0-3, 4-8, 9-13, and ≥14). RESULTS: For QPS SSS correlation, R0 vs. tilted datasets remained very high (R > 0.97) up to 20 degrees, but degraded for higher tilts (R = 0.895/0.780 for 30 degrees/45 degrees). 4D-MSPECT showed comparable SSS correlation only up to 10 degrees (R > 0.95) and strong deterioration thereafter (R = 0.863-0.347 for 15-45 degrees). Deviation in severity class from R0 increased from 6/50 (R5; κ = 0.914) to 25/50 (R45; κ = 0.252) using QPS and from 7/50 (R5; κ = 0.899) to 33/50 (R45; κ = 0.065) using 4D-MSPECT. CONCLUSION: For tilted MPS datasets, considerable differences in SSS calculation emerge using QPS and 4D-MSPECT. QPS showed more stable results than 4D-MSPECT.

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies.

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.

Bispecific antibody derivatives based on full-length IgG formats.

Monoclonal antibodies have emerged as an effective therapeutic modality, and numerous antibodies have been approved for the treatment of several severe diseases or are currently in clinical development. To improve their therapeutic potential, monoclonal antibodies are constantly evolved by protein engineering. Particularly, the generation of bispecific antibodies raised special interest because of their ability to bind two different antigens at the same time, and the efficiency of these formats has been demonstrated in several clinical and preclinical studies. Up to now, the major drawbacks in using bispecific antibodies as a therapeutic agent have been difficult design and low-yield expression of homogeneous antibody populations. However, major technological improvements were made in protein engineering during the last years. This allows the design of several new IgG-based bispecific antibody formats that can be prepared in high yields and high homogeneity using conventional expression and purification techniques. Especially, recent development of IgG-fusions with disulfide-stabilized Fv fragments and of CrossMab-technologies facilitates the generation of bispecific antibodies with IgG-like architectures. Here we describe design principles and methods to express and purify different bispecific antibody formats derived from full-length IgGs.

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies.

We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.