Low-affinity Binders Research Articles

T cell recognition of novel shared breast cancer antigens is frequently observed in peripheral blood of breast cancer patients

ABSTRACTAdvances within cancer immunotherapy have fueled a paradigm shift in cancer treatment, resulting in increasing numbers of cancer types benefitting from novel treatment options. Despite originally being considered an immunologically silent malignancy, recent studies encourage the research of breast cancer immunogenicity to evaluate immunotherapy as a treatment strategy. However, the epitope landscape in breast cancer is minimally described, limiting the options for antigen-specific, targeted strategies. Aromatase, never in mitosis A-related kinase 3 (NEK3), protein inhibitor of activated STAT3 (PIAS3), and prolactin are known as upregulated proteins in breast cancer. In the present study, these four proteins are identified as novel T cell targets in breast cancer.From the four proteins, 147 peptides were determined to bind HLA-A*0201 and -B*0702 using a combined in silico/in vitro affinity screening. T cell recognition of all 147 peptide-HLA-A*0201/-B*0702 combinations was assessed through the use of a novel high-throughput method utilizing DNA barcode labeled multimers.T cell recognition of sequences within all four proteins was demonstrated in peripheral blood of patients, and significantly more T cell responses were detected in patients compared to healthy donors for both HLA-A*0201 and -B*0702. Notably, several of the identified responses were directed toward peptides, with a predicted low or intermediate binding affinity. This demonstrates the importance of including low-affinity binders in the search for epitopes within shared tumor associated antigens (TAAs), as these might be less subject to immune tolerance mechanisms.The study presents four novel TAAs containing multiple possible targets for immunotherapy of breast cancer.

A theoretical analysis of single molecule protein sequencing via weak binding spectra.

We propose and theoretically study an approach to massively parallel single molecule peptide sequencing, based on single molecule measurement of the kinetics of probe binding (Havranek, et al., 2013) to the N-termini of immobilized peptides. Unlike previous proposals, this method is robust to both weak and non-specific probe-target affinities, which we demonstrate by applying the method to a range of randomized affinity matrices consisting of relatively low-quality binders. This suggests a novel principle for proteomic measurement whereby highly non-optimized sets of low-affinity binders could be applicable for protein sequencing, thus shifting the burden of amino acid identification from biomolecular design to readout. Measurement of probe occupancy times, or of time-averaged fluorescence, should allow high-accuracy determination of N-terminal amino acid identity for realistic probe sets. The time-averaged fluorescence method scales well to weakly-binding probes with dissociation constants of tens or hundreds of micromolar, and bypasses photobleaching limitations associated with other fluorescence-based approaches to protein sequencing. We argue that this method could lead to an approach with single amino acid resolution and the ability to distinguish many canonical and modified amino acids, even using highly non-optimized probe sets. This readout method should expand the design space for single molecule peptide sequencing by removing constraints on the properties of the fluorescent binding probes.

In vivo assessment of neuroinflammation in progressive multiple sclerosis: a proof of concept study with [18F]DPA714 PET

BackgroundOver the past decades, positron emission tomography (PET) imaging has become an increasingly useful research modality in the field of multiple sclerosis (MS) research, as PET can visualise molecular processes, such as neuroinflammation, in vivo. The second generation PET radioligand [18F]DPA714 binds with high affinity to the 18-kDa translocator-protein (TSPO), which is mainly expressed on activated microglia. The aim of this proof of concept study was to evaluate this in vivo marker of neuroinflammation in primary and secondary progressive MS.MethodsAll subjects were genotyped for the rs6971 polymorphism within the TSPO gene, and low-affinity binders were excluded from participation in this study. Eight patients with progressive MS and seven age and genetic binding status matched healthy controls underwent a 60 min dynamic PET scan using [18F]DPA714, including both continuous on-line and manual arterial blood sampling to obtain metabolite-corrected arterial plasma input functions.ResultsThe optimal model for quantification of [18F]DPA714 kinetics was a reversible two-tissue compartment model with additional blood volume parameter. For genetic high-affinity binders, a clear increase in binding potential was observed in patients with MS compared with age-matched controls. For both high and medium affinity binders, a further increase in binding potential was observed in T2 white matter lesions compared with non-lesional white matter. Volume of distribution, however, did not differentiate patients from healthy controls, as the large non-displaceable compartment of [18F]DPA714 masks its relatively small specific signal.ConclusionThe TSPO radioligand [18F]DPA714 can reliably identify increased focal and diffuse neuroinflammation in progressive MS when using plasma input-derived binding potential, but observed differences were predominantly visible in high-affinity binders.

High-Throughput, Biosensor-Based Approach to Examine Bone Morphogenetic Protein (BMP)-Receptor Interactions.

Binding of a BMP to its cognate cell surface receptors is the initiating step in the BMP signaling cascade. Thus, knowing which BMP-receptor complexes form is vital for understanding the physiological activities of a particular BMP. Here, we describe a surface plasmon resonance (SPR)-based, high-throughput approach that allows fast identification and evaluation of BMP-receptor complexes. Briefly, the extracellular, BMP-binding domains of receptors are produced as human IgG1-Fc-fusion proteins. The Fc moiety enables simple capture of the Fc-receptor-fusion protein on the sensor chip, supports a highly reproducible, uniform approach of surface regeneration, and ensures full activity of the receptor moiety. BMPs are injected over the captured receptors at one concentration (approximately 60-100nM), permitting stratification of high-affinity, medium-affinity, and low-affinity binders. Using this concentration range, equilibrium dissociation constants for high-affinity and medium-affinity binders can be estimated with good accuracy and with great precision from the single injection binding curves.

Abstract 5757: Preclinical development and testing of a therapeutic antibody against LRG1

Abstract Introduction: The purpose of this study was to generate a therapeutic function-blocking antibody against leucine-rich alpha-2-glycoprotein 1 (LRG1), a novel regulator of pathological angiogenesis that is up-regulated in many cancers, and demonstrate efficacy in proof of principle experiments in mouse models. Experimental Procedures: Mice were immunized with recombinant human LRG1 and hybridomas generated. More than 100 monoclonal antibodies (mAbs) were first screened by surface plasmon resonance (SPR) to measure affinity for target, and then in a co-culture angiogenesis assay to evaluate functional activity. The most promising candidates were then tested in vivo in the mouse model of laser-induced choroidal neovascularization, and from this study a lead was humanized and deimmunized. The resulting antibody is a hinge-stabilized IgG4 that is now undergoing manufacturing and pre-clinical safety and toxicology testing. Results: Of the original set of mouse mAbs, an initial screen to eliminate low affinity (KD greater than 1nM) binders resulted in 25 progressing to functional testing in angiogenesis assays. The seven most effective blocking mAbs were then tested in vivo in the mouse model of laser-induced choroidal neovascularization. Intraocular injections of mAb yielded two that exhibited equivalent inhibition of neovascularization to a therapeutic VEGF-blocker, and of these, the higher affinity mAb (15C4) was selected for humanization. Epitope mapping identified an amino-acid sequence within human LRG1, that in the form of a synthetic peptide was able to compete the binding of 15C4 to LRG1. Multiple species sequence alignment revealed the epitope to be partially conserved with the corresponding mouse sequence, and SPR analysis showed that 15C4 binding to recombinant mouse LRG1 was several orders of magnitude lower than to human LRG1. Nevertheless, administration of 15C4 to tumor-bearing mice significantly inhibited tumor growth in a range of subcutaneous and genetic models. Furthermore, due to the vessel normalizing effects of 15C4, combinations of mAb with both immunotherapy and cytotoxics were significantly more effective at restricting tumor growth than either monotherapy. Conclusions: We have generated and characterized a novel vessel-normalizing, fully humanized function-blocking mAb against LRG1 that shows efficacy in mouse tumor models. GMP manufacture is on-going, and early stage clinical trials are planned in 2019. Financial Support: The work was supported by the Medical Research Council, the Rosetrees Trust and UCL Business. Citation Format: Stephen Moss, David Kallenberg, Vineeta Tripathi, Sterenn Davis, Jestin George, Marie O'Connor, Laura Dowsett, John Greenwood. Preclinical development and testing of a therapeutic antibody against LRG1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5757.

PR‐10 proteins as potential mediators of melatonin‐cytokinin cross‐talk in plants: crystallographic studies of LlPR‐10.2B isoform from yellow lupine

Atomic coordinates and processed structure factors corresponding to the final models of the LlPR-10.2B/melatonin and LlPR-10.2B/melatonin + trans-zeatin complexes have been deposited with the Protein Data Bank (PDB) under the accession codes 5MXB and 5MXW. The corresponding raw X-ray diffraction images have been deposited in the RepOD Repository at the Interdisciplinary Centre for Mathematical and Computational Modelling (ICM) of the University of Warsaw, Poland, and are available for download with the following Digital Object Identifiers (DOI): https://doi.org/10.18150/repod.9923638 and https://doi.org/10.18150/repod.6621013.

TSPO PET with [18F]GE-180 sensitively detects focal neuroinflammation in patients with relapsing–remitting multiple sclerosis

Expression of the translocator protein (TSPO) is upregulated in activated macrophages/microglia and is considered to be a marker of neuroinflammation. We investigated the novel TSPO ligand [18F]GE-180 in patients with relapsing-remitting multiple sclerosis (RRMS) to determine the feasibility of [18F]GE-180 PET imaging in RRMS patients and to assess its ability to detect active inflammatory lesions in comparison with the current gold standard, contrast-enhanced magnetic resonance imaging (MRI). Nineteen RRMS patients were prospectively included in this study. All patients underwent TSPO genotyping and were classified as high-affinity, medium-affinity or low-affinity binders (HAB/MAB/LAB). PET scans were performed after administration of 189 ± 12MBq [18F]GE-180, and 60-90min summation images were used for visual analysis and assessment of standardized uptake values (SUV). The frontal nonaffected cortex served as a pseudoreference region (PRR) for evaluation of SUV ratios (SUVR). PET data were correlated with MRI signal abnormalities, i.e. T2 hyperintensity or contrast enhancement (CE). When available, previous MRI data were used to follow the temporal evolution of individual lesions. Focal lesions were identified as hot spots by visual inspection. Such lesions were detected in 17 of the 19 patients and overall 89 [18F]GE-180-positive lesions were found. TSPO genotyping revealed 11 patients with HAB status, 5 with MAB status and 3 with LAB status. There were no associations between underlying binding status (HAB, MAB and LAB) and the signal intensity in either lesions (SUVR 1.87 ± 0.43, 1.95 ± 0.48 and 1.86 ± 0.80, respectively; p = 0.280) or the PRR (SUV 0.36 ± 0.03, 0.40 ± 0.06 and 0.37 ± 0.03, respectively; p = 0.990). Of the 89 [18F]GE-180-positive lesions, 70 showed CE on MRI, while the remainder presented as T2 lesions without CE. SUVR were significantly higher in lesions with CE than in those without (2.00 ± 0.53 vs. 1.60 ± 0.15; p = 0.001). Notably, of 19 [18F]GE-180-positive lesions without CE, 8 previously showed CE, indicating that [18F]GE-180 imaging may be able to detect lesional activity that is sustained beyond the blood-brain barrier breakdown. [18F]GE-180 PET can detect areas of focal macrophage/microglia activation in patients with RRMS in lesions with and without CE on MRI. Therefore, [18F]GE-180 PET imaging is a sensitive and quantitative approach to the detection of active MS lesions. It may provide information beyond contrast-enhanced MRI and is readily applicable to all patients. [18F]GE-180 PET imaging is therefore a promising new tool for the assessment of focal inflammatory activity in MS.

Understanding non-linear effects from Hill-type dynamics with application to decoding of p53 signaling

Analytical equations are derived depicting four possible scenarios resulting from pulsed signaling of a system subject to Hill-type dynamics. Pulsed Hill-type dynamics involves the binding of multiple signal molecules to a receptor and occurs e.g., when transcription factor p53 orchestrates cancer prevention, during calcium signaling, and during circadian rhythms. The scenarios involve: (i) enhancement of high-affinity binders compared to low-affinity ones, (ii) slowing reactions involving high-affinity binders, (iii) transfer of the clocking of low-affinity binders from the signal molecule to the products, and (iv) a unique clocking process that produces incremental increases in the activity of high-affinity binders with each signal pulse. In principle, these mostly non-linear effects could control cellular outcomes. An applications to p53 signaling is developed, with binding to most gene promoters identified as category (iii) responses. However, currently unexplained enhancement of high-affinity promoters such as CDKN1a (p21) by pulsed signaling could be an example of (i). In general, provision for all possible scenarios is required in the design of mathematical models incorporating pulsed Hill-type signaling as some aspect.

The NMR2 Method to Determine Rapidly the Structure of the Binding Pocket of a Protein–Ligand Complex with High Accuracy

Structural characterization of complexes is crucial for a better understanding of biological processes and structure-based drug design. However, many protein–ligand structures are not solvable by X-ray crystallography, for example those with low affinity binders or dynamic binding sites. Such complexes are usually targeted by solution-state NMR spectroscopy. Unfortunately, structure calculation by NMR is very time consuming since all atoms in the complex need to be assigned to their respective chemical shifts. To circumvent this problem, we recently developed the Nuclear Magnetic Resonance Molecular Replacement (NMR2) method. NMR2 very quickly provides the complex structure of a binding pocket as measured by solution-state NMR. NMR2 circumvents the assignment of the protein by using previously determined structures and therefore speeds up the whole process from a couple of months to a couple of days. Here, we recall the main aspects of the method, show how to apply it, discuss its advantages over other methods and outline its limitations and future directions.

Direct NMR Probing of Hydration Shells of Protein Ligand Interfaces and Its Application to Drug Design.

Fragment-based drug design exploits initial screening of low molecular weight compounds and their concomitant affinity improvement. The multitude of possible chemical modifications highlights the necessity to obtain structural information about the binding mode of a fragment. Herein we describe a novel NMR methodology (LOGSY titration) that allows the determination of binding modes of low affinity binders in the protein-ligand interface and reveals suitable ligand positions for the addition of functional groups that either address or substitute protein-bound water, information of utmost importance for drug design. The particular benefit of the methodology and in contrast to conventional ligand-based methods is the independence of the molecular weight of the protein under study. The validity of the novel approach is demonstrated on two ligands interacting with bromodomain 1 of bromodomain containing protein 4, a prominent cancer target in pharmaceutical industry.

Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low-affinity DNA binders.

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial-filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low-affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly-dT) of different lengths (from 5 to 60-mer) were investigated. It was shown that the size increase-dependent random coil-like conformation of Poly-dT favorably acted on the apparent selectivity and resolution. The base-unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single-stranded to double-stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single-stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers.

11C-ER176, a Radioligand for 18-kDa Translocator Protein, Has Adequate Sensitivity to Robustly Image All Three Affinity Genotypes in Human Brain.

Nine healthy volunteers-3 high-affinity binders (HABs), 3 mixed-affinity binders (MABs), and 3 low-affinity binders (LABs)-were studied with whole-body 11C-ER176 PET imaging. SUVs from 60 to 120 min after injection derived from each organ were compared between genotypes. Eight separate healthy volunteers-3 HABs, 3 MABs, and 2 LABs-underwent brain PET imaging. The 3 HABs underwent a repeated brain scan after TSPO blockade with XBD173 (N-benzyl-N-ethyl-2-(7-methyl-8-oxo-2-phenylpurin-9-yl)acetamide) to determine nondisplaceable distribution volume (VND) via Lassen occupancy plotting and thereby estimate BPND in brain. Regional SUV averaged from 60 to 120 min after injection in brain and peripheral organs with high TSPO densities such as lung and spleen were greater in HABs than in LABs. On the basis of VND determined via the occupancy plot, the whole-brain BPND for LABs was estimated to be 1.4 ± 0.8, which was much lower than that for HABs (4.2 ± 1.3) but about the same as that for HABs with 11C-PBR28 ([methyl-11C]N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine)) (∼1.2). Obvious in vivo sensitivity to rs6971 was observed in 11C-ER176 that had not been expected from in vitro studies, suggesting that the future development of any improved radioligand for TSPO should consider the possibility that in vitro properties will not be reflected in vivo. We also found that 11C-ER176 has adequately high BPND for all rs6971 genotypes. Thus, the new radioligand would likely have greater sensitivity in detecting abnormalities in patients.

Early and protective microglial activation in Alzheimer's disease: a prospective study using 18F-DPA-714 PET imaging.

While emerging evidence suggests that neuroinflammation plays a crucial role in Alzheimer's disease, the impact of the microglia response in Alzheimer's disease remains a matter of debate. We aimed to study microglial activation in early Alzheimer's disease and its impact on clinical progression using a second-generation 18-kDa translocator protein positron emission tomography radiotracer together with amyloid imaging using Pittsburgh compound B positron emission tomography. We enrolled 96 subjects, 64 patients with Alzheimer's disease and 32 controls, from the IMABio3 study, who had both (11)C-Pittsburgh compound B and (18)F-DPA-714 positron emission tomography imaging. Patients with Alzheimer's disease were classified as prodromal Alzheimer's disease (n = 38) and Alzheimer's disease dementia (n = 26). Translocator protein-binding was measured using a simple ratio method with cerebellar grey matter as reference tissue, taking into account regional atrophy. Images were analysed at the regional (volume of interest) and at the voxel level. Translocator protein genotyping allowed the classification of all subjects in high, mixed and low affinity binders. Thirty high+mixed affinity binders patients with Alzheimer's disease were dichotomized into slow decliners (n = 10) or fast decliners (n = 20) after 2 years of follow-up. All patients with Alzheimer's disease had an amyloid positive Pittsburgh compound B positron emission tomography. Among controls, eight had positive amyloid scans (n = 6 high+mixed affinity binders), defined as amyloidosis controls, and were analysed separately. By both volumes of interest and voxel-wise comparison, 18-kDa translocator protein-binding was higher in high affinity binders, mixed affinity binders and high+mixed affinity binders Alzheimer's disease groups compared to controls, especially at the prodromal stage, involving the temporo-parietal cortex. Translocator protein-binding was positively correlated with Mini-Mental State Examination scores and grey matter volume, as well as with Pittsburgh compound B binding. Amyloidosis controls displayed higher translocator protein-binding than controls, especially in the frontal cortex. We found higher translocator protein-binding in slow decliners than fast decliners, with no difference in Pittsburgh compound B binding. Microglial activation appears at the prodromal and possibly at the preclinical stage of Alzheimer's disease, and seems to play a protective role in the clinical progression of the disease at these early stages. The extent of microglial activation appears to differ between patients, and could explain the overlap in translocator protein binding values between patients with Alzheimer's disease and amyloidosis controls.

In vivo evidence of a functional association between immune cells in blood and brain in healthy human subjects

Microglia, the resident macrophages in the central nervous system, are thought to be maintained by a local self-renewal mechanism. Although preclinical and in vitro studies have suggested that the brain may contain immune cells also from peripheral origin, the functional association between immune cells in the periphery and brain at physiological conditions is poorly understood.We examined 32 healthy individuals using positron emission tomography (PET) and [11C]PBR28, a radioligand for the 18-kDa translocator protein (TSPO) which is expressed both in brain microglia and blood immune cells. In 26 individuals, two measurements were performed with varying time intervals. In a subgroup of 19 individuals, of which 12 had repeat examinations, leukocyte numbers in blood was measured on each day of PET measurements. All individuals were genotyped for TSPO polymorphism and categorized as high, mixed, and low affinity binders. We assessed TSPO binding expressed as total distribution volume of [11C]PBR28 in brain and in blood cells.TSPO binding in brain was strongly and positively correlated to binding in blood cells both at baseline and when analyzing change between two PET examinations. Furthermore, there was a significant correlation between change of leukocyte numbers and change in TSPO binding in brain, and a trend-level correlation to change in TSPO binding in blood cells. These in vivo findings indicate an association between immunological cells in blood and brain via intact BBB, suggesting a functional interaction between these two compartments, such as interchange of peripherally derived cells or a common regulatory mechanism. Measurement of radioligand binding in blood cells may be a way to control for peripheral immune function in PET studies using TSPO as a marker of brain immune activation.

Recent Advances in Multi-Task QSAR Modeling for Drug Design

Computer-aided drug design may provide a massive scope to reduce the overall duration as well as the expenditure associated with drug discovery. Although the concept of Quantitative Structure Activity Relationship (QSAR) is almost fifty years old, it is still considered as a very useful method for finding novel therapeutic agents. In the last decade, the field of QSAR has witnessed great advances in terms of techniques, ideas and approaches. 3 Multi-task QSAR is one such approach that has a huge opportunity to contribute in the discovery of novel therapeutic agents. Classical QSAR techniques focus on the classical idea that one drug is aimed to act on one target. However, due to the highly complex and multi-factorial nature of some diseases like cancer, microbial infections, neurodegenerative and metabolic disorders, the classical idea of drug development frequently produces less satisfying effects. The effect of the inhibition of one specific target is compensated by different back-up or fail-safe mechanisms. Polypharmacology focuses on the concept of simultaneous inhibition of multiple targets by single therapeutic agents. Contrary to single target drugs that bind with a specific target with high affinity, multi-target agents are low affinity binders. Therefore, management of therapy is easier for the multi-target agents. Multi-task QSAR techniques are able to incorporate the ideas of polypharmacology. While conventional QSAR techniques are based on the datasets of compounds with a single target and same or similar assay conditions, multi-task QSAR can consider data on multiple targets and various assay conditions at a time. In addition, multi-task QSAR techniques are also useful to address the problems of fewer datapoints. QSAR is a statistical technique and a higher amount of data-points generates more reliable models. However, for several diseases the availability of a data is less and developed models on the available data may not be able to provide detailed insight into structural requirements of the molecules for higher potency. As multi-task QSAR models may be developed on the data collected from different assay conditions, more information may be retrieved for the explanation of structural patterns. Therefore, multi-task QSAR has emerged as one of the most essential tools in the last few years and various groups of researchers participated in the development of multi-task QSAR models. Though conventional 2D QSAR techniques have found several alternatives such as 3D QSAR, pharmacophore mapping as well as molecular docking, 2D-QSAR is unique in handling problems of multi-task modeling. Multi-task QSAR has been utilized for the developing models for anti-cancer, anti-bacterial 17 and neuroprotective agents. In addition, reports on multi-target modeling on kinase and ubiquitineproteosome pathways are also found. Furthermore, multi-task agents are also useful for understanding structural patterns required for toxicities of nanoparticles, environmental pollutants etc. Although development of multi-task QSAR model is quite similar to conventional modeling as far as descriptor calculation and model generation are concerned, the construction and management of a dataset is quite different. In most of the cases, classification models are generated with the data by suitable machine learning tools such as Linear Discriminant Analyses (LDA), Artificial Neural Network (ANN), Support Vector Machine (SVM) etc. Generally, a specific cut-off value for each system is fixed by the user to classify the data into ‘sensitive, and ‘non-sensitive’. Sometimes special statistical treatments, like averaging the descriptor values for specific system and calculation of moving average, are required for handling the complicated data. Some attempts were taken to develop regression based multi-target models. However, there still is a debate about the actual effectiveness of multi-target QSAR modeling as the size of a data set is sometimes so increased that the model may not be properly validated by conventional approaches. Moreover, there are concerns over applicability domain of the multitask models. Nevertheless, multi-task QSAR models may provide opportunity to widen the area of QSAR studies as well as to contribute to the investigation of different emerging areas such as nanotoxicity, polypharmacology, drug repositioning, environmental safety management etc.

Ligand-inducible dimeric antibody for selecting antibodies against a membrane protein based on mammalian cell proliferation.

A method for selecting antibodies against a membrane protein is important for attaining a variety of antibody-based diagnostics and therapies. In this study, we propose a novel system to select specific antibodies against a membrane protein based on mammalian cell proliferation as a readout. The system employs a chimeric membrane protein in which a target membrane protein of interest is fused to the intracellular signaling domain of a cytokine receptor. The chimeric membrane protein transduces a cell proliferation signal through dimerization when co-expressed with a specific single-chain Fv fused with a mutant of FK-binding protein 12 (scFv-Fk) that can be conditionally dimerized by a synthetic ligand AP20187. To demonstrate this system, ErbB2 and gp130 were chosen as the target membrane protein and cytokine receptor, respectively. Consequently, co-expression of the ErbB2/gp130 chimera and ErbB2-specific scFv-Fk rendered the cells proliferative in response to AP20187. The system also allowed selection of high-affinity binders from a mixture composed of dominant low-affinity binders. This system may be extended to affinity maturation of scFvs by modulating AP20187 concentration in the selection process.

Imaging Striatal Microglial Activation in Patients with Parkinson's Disease.

This study investigated whether the second-generation translocator protein 18kDa (TSPO) radioligand, [18F]-FEPPA, could be used in neurodegenerative parkinsonian disorders as a biomarker for detecting neuroinflammation in the striatum. Neuroinflammation has been implicated as a potential mechanism for the progression of Parkinson’s disease (PD). Positron Emission Tomography (PET) radioligand targeting for TSPO allows for the quantification of neuroinflammation in vivo. Based on genotype of the rs6791 polymorphism in the TSPO gene, 16 mixed-affinity binders (MABs) (8 PD and age-matched 8 healthy controls (HCs)), 16 high-affinity binders (HABs) (8 PD and age-matched 8 HCs) and 4 low-affinity binders (LABs) (3 PD and 1 HCs) were identified. Total distribution volume (VT) values in the striatum were derived from a two-tissue compartment model with arterial plasma as an input function. There was a significant main effect of genotype on [18F]-FEPPA VT values in the caudate nucleus (p = 0.001) and putamen (p < 0.001), but no main effect of disease or disease x genotype interaction in either ROI. In the HAB group, the percentage difference between PD and HC was 16% in both caudate nucleus and putamen; in the MAB group, it was -8% and 3%, respectively. While this PET study showed no evidence of increased striatal TSPO expression in PD patients, the current findings provide some insights on the possible interactions between rs6791 polymorphism and neuroinflammation in PD.

Can Studies of Neuroinflammation in a TSPO Genetic Subgroup (HAB or MAB) Be Applied to the Entire AD Cohort?

Neuroinflammation plays a significant role in Alzheimer disease (AD), and translocator protein (TSPO) PET imaging allows us to quantify this process. However, the binding of second-generation TSPO tracers depends on the TSPO genotype coded by the rs6971 single-nucleotide polymorphism, with a 40%-50% increase in BP in high-affinity binders (HABs) compared with mixed-affinity binders (MABs), whereas low-affinity binders (LABs) are unsuitable for evaluation. Hence, several studies are using either HAB alone or HAB and MAB subjects. To translate the findings of neuroinflammation studies to the entire population, it is crucial to establish the influence of TSPO genotypes on AD. Here, we investigated whether different TSPO genotypes influence cognitive function, amyloid load, and disease progression over time. We evaluated 798 subjects (225 control, 388 with mild cognitive impairment [MCI], and 185 with AD) from the Alzheimer's Disease Neuroimaging Initiative database at baseline and during follow-up. All subjects were screened for TSPO genotype and underwent detailed clinical and neuropsychologic assessments yearly for 4 y. Of the 798 subjects, 255 also had T1- and T2-weighted MR imaging and amyloid PET with (11)C-Pittsburgh compound B or (18)F-florbetapir. We demonstrated that all TSPO binding groups (HAB, MAB, and LAB) have same level of amyloid load in AD and MCI subjects. We also demonstrated that the prevalence is 50.3% for HAB, 41.2% for MAB, and 8.5% for LAB, without a statistical difference among the AD, MCI, and control groups. During longitudinal follow-up, the mean change in neuropsychometric test scores on the Mini-Mental State Examination, the cognitive and modified Alzheimer Disease Assessment Scales (ADASs), and the Geriatric Depression Scale over time were similar in AD and MCI subjects among the 3 TSPO binding groups. Analysis of the covariates showed that diagnostic group (control, MCI, AD), apolipoprotein E4 status, and sex had a significant effect on decline on the modified Alzheimer Disease Assessment Scale (>3 points of the scale), but age and TSPO genotype did not. This study suggests that information obtained from evaluating a subgroup of AD or MCI subject using second-generation TSPO tracers can be translated to the entire AD and MCI population. Thus, we can study fewer AD subjects in evaluating new antineuroinflammatory and antimicroglial agents in intervention studies and in observational studies evaluating the role of neuroinflammation.

How to Perform Translocator Protein PET-CT Scanning for Microglial Activation in Schizophrenia Patients.

Introduction Activated microglia express translocator protein (TSPO) on the outer mitochondrial membrane. PET ligands targeting TSPO allow in vivo non-invasive visualization and quantification of neuroinflammation. Whereas inflammation in schizophrenia was previously studied using 11 C-PK11195, 18 F-PBR111 is a novel second-generation tracer, with high specific TSPO binding and longer half-life. Objective To establish a protocol for 18 F-PBR111 TSPO PET in schizophrenia. Methods A pilot study on a Siemens Biograph mCT PET-scanner in healthy controls and schizophrenia patients (n=9). Results Subjects underwent a 90-minute dynamic brain PET-CT, following i.v. bolus injection of 214±13 MBq 18 F-PBR111. An arterial input function was measured using continuous blood sampling (Twilite, Switzerland) with discrete samples for metabolite analysis. The metabolite corrected plasma input function (IPF) was calculated from the whole blood input function, individual plasma to whole blood and parent fraction data as determined by a SEP-PAK procedure. Dynamic PET data were reconstructed and a post-reconstruction motion correction was applied. Regional tissue time activity curves (TACs) were extracted from the PET images for regions of interest determined from individual MRI images. Total volume of distribution (V T ) was then calculated from fitting a reversible two-tissue compartmental model to the measured TACs using the individual IPF. Prior genotyping for TSPO receptor polymorphism (rs6971) allowed to exclude low-affinity binders (estimated 10% of European population). The procedure was well tolerated. Conclusions We established a protocol for 18 F-PBR111 TSPO PET in healthy subjects and schizophrenia patients, thereby providing useful information for others considering 18 F-PBR111 TSPO PET imaging for evaluation of neuroinflammation.

Quantitative imaging of neuroinflammation in human white matter: a positron emission tomography study with translocator protein 18 kDa radioligand, [18F]-FEPPA.

The ability to quantify translocator protein 18 kDa (TSPO) in white matter (WM) is important to understand the role of neuroinflammation in neurological disorders with WM involvement. This article aims to extend the utility of TSPO imaging in WM using a second-generation radioligand, [18F]-FEPPA, and high-resolution research tomograph (HRRT) positron emission tomography (PET) camera system. Four WM regions of interests (WM-ROI), relevant to the study of aging and neuroinflammatory diseases, were examined. The corpus callosum, cingulum bundle, superior longitudinal fasciculus, and posterior limb of internal capsule were delineated automatically onto subject's T1 -weighted magnetic resonance image using a diffusion tensor imaging-based WM template. The TSPO polymorphism (rs6971) stratified individuals to three genetic groups: high-affinity binders (HAB), mixed-affinity binders (MAB), and low-affinity binders. [18F]-FEPPA PET scans were acquired on 32 healthy subjects and analyzed using a full kinetic compartment analysis. The two-tissue compartment model showed moderate identifiability (coefficient of variation 15-19%) for [18F]-FEPPA total volume distribution (VT ) in WM-ROIs. Noise affects VT variability, although its effect on bias was small (6%). In a worst-case scenario, ≤6% of simulated data did not fit reliably. A simulation of increased TSPO density exposed minimal effect on variability and identifiability of [18F]-FEPPA VT in WM-ROIs. We found no association between age and [18F]-FEPPA VT in WM-ROIs. The VT values were 15% higher in HAB than in MAB, although the difference was not statistically significant. This study provides evidence for the utility and limitations of [18F]-FEPPA PET to measure TSPO expression in WM.