Characterization and root cause analysis of immunogenicity to pasotuxizumab (AMG 212), a prostate-specific membrane antigen-targeting bispecific T-cell engager therapy.

Introduction: In oncology, anti-drug antibody (ADA) development that significantly curtails response durability has not historically risen to a level of concern. The relevance and attention ascribed to ADAs in oncology clinical studies have therefore been limited, and the extant literature on this subject scarce. In recent years, T cell engagers have gained preeminence within the prolific field of cancer immunotherapy. These drugs whose mode of action is expected to potently stimulate anti-tumor immunity, may potentially induce ADAs as an unintended corollary due to an overall augmentation of the immune response. ADA formation is therefore emerging as an important determinant in the successful clinical development of such biologics. Methods: Here we describe the immunogenicity and its impact observed to pasotuxizumab (AMG 212), a prostate-specific membrane antigen (PSMA)-targeting bispecific T cell engager (BiTE®) molecule in NCT01723475, a first-in-human (FIH), multicenter, dose-escalation study in patients with metastatic castration-resistant prostate cancer (mCRPC). To explain the disparity in ADA incidence observed between the SC and CIV arms of the study, we interrogated other patient and product-specific factors that may have explained the difference beyond the route of administration. Results: Treatment-emergent ADAs (TE-ADA) developed in all subjects treated with at least 1 cycle of AMG 212 in the subcutaneous (SC) arm. These ADAs were neutralizing and resulted in profound exposure loss that was associated with contemporaneous reversal of initial Prostate Surface Antigen (PSA) responses, curtailing durability of PSA response in patients. Pivoting from SC to a continuous intravenous (CIV) administration route remarkably yielded no subjects developing ADA to AMG 212. Through a series of stepwise functional assays, our investigation revealed that alongside a more historically immunogenic route of administration, non-tolerant T cell epitopes within the AMG 212 amino acid sequence were likely driving the high-titer, sustained ADA response observed in the SC arm. Discussion: These mechanistic insights into the AMG 212 ADA response underscore the importance of performing preclinical immunogenicity risk evaluation as well as advocate for continuous iteration to better our biologics.

Antidepressant treatment effects on hippocampal protein turnover: Molecular and spatial insights from mass spectrometry.

A major challenge in managing depression is that antidepressant drugs take a long time to exert their therapeutic effects. For the development of faster-acting treatments, it is crucial to get an improved understanding of the molecular mechanisms underlying antidepressant mode of action. Here, we used a novel mass spectrometry-based workflow to investigate how antidepressant treatment affects brain protein turnover at single-cell and subcellular resolution. We combined stable isotope metabolic labeling, quantitative Tandem Mass Spectrometry (qTMS) and Multi-isotope Imaging Mass Spectrometry (MIMS) to simultaneously quantify and image protein synthesis and turnover in hippocampi of mice treated with the antidepressant paroxetine. We identified changes in turnover of individual hippocampal proteins that reveal altered metabolism-mitochondrial processes and report on subregion-specific turnover patterns upon paroxetine treatment. This workflow can be used to investigate brain protein turnover changes in vivo upon pharmacological interventions at a resolution and precision that has not been possible with other methods to date. Our results reveal acute paroxetine effects on brain protein turnover and shed light on antidepressant mode of action.

Ketamine's antidepressant effect is mediated by energy metabolism and antioxidant defense system.

Fewer than 50% of all patients with major depressive disorder (MDD) treated with currently available antidepressants (ADs) show full remission. Moreover, about one third of the patients suffering from MDD does not respond to conventional ADs and develop treatment-resistant depression (TRD). Ketamine, a non-competitive, voltage-dependent N-Methyl-D-aspartate receptor (NMDAR) antagonist, has been shown to have a rapid antidepressant effect, especially in patients suffering from TRD. Hippocampi of ketamine-treated mice were analysed by metabolome and proteome profiling to delineate ketamine treatment-affected molecular pathways and biosignatures. Our data implicate mitochondrial energy metabolism and the antioxidant defense system as downstream effectors of the ketamine response. Specifically, ketamine tended to downregulate the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) metabolite ratio which strongly correlated with forced swim test (FST) floating time. Furthermore, we found increased levels of enzymes that are part of the 'oxidative phosphorylation' (OXPHOS) pathway. Our study also suggests that ketamine causes less protein damage by rapidly decreasing reactive oxygen species (ROS) production and lend further support to the hypothesis that mitochondria have a critical role for mediating antidepressant action including the rapid ketamine response.

Tau Deletion Prevents Stress-Induced Dendritic Atrophy in Prefrontal Cortex: Role of Synaptic Mitochondria.

Tau protein in dendrites and synapses has been recently implicated in synaptic degeneration and neuronal malfunction. Chronic stress, a well-known inducer of neuronal/synaptic atrophy, triggers hyperphosphorylation of Tau protein and cognitive deficits. However, the cause-effect relationship between these events remains to be established. To test the involvement of Tau in stress-induced impairments of cognition, we investigated the impact of stress on cognitive behavior, neuronal structure, and the synaptic proteome in the prefrontal cortex (PFC) of Tau knock-out (Tau-KO) and wild-type (WT) mice. Whereas exposure to chronic stress resulted in atrophy of apical dendrites and spine loss in PFC neurons as well as significant impairments in working memory in WT mice, such changes were absent in Tau-KO animals. Quantitative proteomic analysis of PFC synaptosomal fractions, combined with transmission electron microscopy analysis, suggested a prominent role for mitochondria in the regulation of the effects of stress. Specifically, chronically stressed animals exhibit Tau-dependent alterations in the levels of proteins involved in mitochondrial transport and oxidative phosphorylation as well as in the synaptic localization of mitochondria in PFC. These findings provide evidence for a causal role of Tau in mediating stress-elicited neuronal atrophy and cognitive impairment and indicate that Tau may exert its effects through synaptic mitochondria.

Purine and pyrimidine metabolism: Convergent evidence on chronic antidepressant treatment response in mice and humans.

Selective Serotonin Reuptake Inhibitors (SSRIs) are commonly used drugs for the treatment of psychiatric diseases including major depressive disorder (MDD). For unknown reasons a substantial number of patients do not show any improvement during or after SSRI treatment. We treated DBA/2J mice for 28 days with paroxetine and assessed their behavioral response with the forced swim test (FST). Paroxetine-treated long-time floating (PLF) and paroxetine-treated short-time floating (PSF) groups were stratified as proxies for drug non-responder and responder mice, respectively. Proteomics and metabolomics profiles of PLF and PSF groups were acquired for the hippocampus and plasma to identify molecular pathways and biosignatures that stratify paroxetine-treated mouse sub-groups. The critical role of purine and pyrimidine metabolisms for chronic paroxetine treatment response in the mouse was further corroborated by pathway protein expression differences in both mice and patients that underwent chronic antidepressant treatment. The integrated -omics data indicate purine and pyrimidine metabolism pathway activity differences between PLF and PSF mice. Furthermore, the pathway protein levels in peripheral specimens strongly correlated with the antidepressant treatment response in patients. Our results suggest that chronic SSRI treatment differentially affects purine and pyrimidine metabolisms, which may explain the heterogeneous antidepressant treatment response and represents a potential biosignature.

Stable isotope metabolic labeling suggests differential turnover of the DPYSL protein family.

PURPOSE: In this work, we discuss how in vivo 15 N metabolic labeling in combination with MS simultaneously provides information on protein expression and protein turnover. EXPERIMENTAL DESIGN: We metabolically labeled mice with the stable nitrogen isotope 15 N using a 15 N-enriched diet and analyzed unlabeled (14 N) versus 15 N-labeled brain tissue with LC-MS/MS. We then compared the 14 N versus 15 N peptide isotopologue clusters of 14 N and 15 N-labeled dihydropyrimidinase-related (DPYSL) proteins. RESULTS: We present a workflow assessing protein expression and turnover at different time points of mouse brain development. Our data demonstrate distinct protein turnover patterns of DPYSL3 and DPYSL5 compared to other quantified proteins. We report the presence of two DPYSL3 and DPYSL5 populations with different 15 N incorporation rates, indicating altered protein turnover during development. CONCLUSIONS AND CLINICAL RELEVANCE: In vivo 15 N metabolic labeling allows the simultaneous investigation of protein expression and turnover, enabling detailed protein dynamics studies. We report for the first time protein turnover data for the DPYSL2, DPYSL3, and DPYSL5 protein family members. As DPYSL proteins have important functions for nervous system maturation, our data provide useful information on their molecular fate during brain development.

Fluoxetine Treatment Rescues Energy Metabolism Pathway Alterations in a Posttraumatic Stress Disorder Mouse Model.

Posttraumatic stress disorder (PTSD) is a prevalent psychiatric disorder. Several studies have attempted to characterize molecular alterations associated with PTSD, but most findings were limited to the investigation of specific cellular markers in the periphery or defined brain regions. In the current study, we aimed to unravel affected molecular pathways/mechanisms in the fear circuitry associated with PTSD. We interrogated a foot shock-induced PTSD mouse model by integrating proteomics and metabolomics profiling data. Alterations at the proteome level were analyzed using in vivo (15)N metabolic labeling combined with mass spectrometry in the prelimbic cortex (PrL), anterior cingulate cortex (ACC), basolateral amygdala, central nucleus of the amygdala and CA1 of the hippocampus between shocked and nonshocked (control) mice, with and without fluoxetine treatment. In silico pathway analyses revealed an upregulation of the citric acid cycle pathway in PrL, and downregulation in ACC and nucleus accumbens (NAc). Chronic fluoxetine treatment prevented decreased citric acid cycle activity in NAc and ACC and ameliorated conditioned fear response in shocked mice. Our results shed light on the role of energy metabolism in PTSD pathogenesis and suggest potential therapy through mitochondrial targeting.

Variability assessment of (15)N metabolic labeling-based proteomics workflow in mouse plasma and brain.

(15)N metabolic labeling-based quantitative proteomics is used for the identification of disease- and phenotype-related alterations in live organisms. The variability of (15)N metabolic labeling proteomics workflows has been assessed in plants and bacteria. However, no study has addressed this topic in mice. We have investigated the repeatability of a quantitative in vivo(15)N metabolic labeling proteomics workflow in mice by assessing LC variability, peptide and protein profiling characteristics and overall (15)N/(14)N protein quantification accuracy in technical replicates of plasma and brain specimens. We furthermore examined how sample preparation affects these parameters in plasma and brain. We found that specimen type (i.e. plasma or brain) influences the variability of the (15)N metabolic labeling workflow in an LC-independent manner.

Homer1/mGluR5 activity moderates vulnerability to chronic social stress.

Stress-induced psychiatric disorders, such as depression, have recently been linked to changes in glutamate transmission in the central nervous system. Glutamate signaling is mediated by a range of receptors, including metabotropic glutamate receptors (mGluRs). In particular, mGluR subtype 5 (mGluR5) is highly implicated in stress-induced psychopathology. The major scaffold protein Homer1 critically interacts with mGluR5 and has also been linked to several psychopathologies. Yet, the specific role of Homer1 in this context remains poorly understood. We used chronic social defeat stress as an established animal model of depression and investigated changes in transcription of Homer1a and Homer1b/c isoforms and functional coupling of Homer1 to mGluR5. Next, we investigated the consequences of Homer1 deletion, overexpression of Homer1a, and chronic administration of the mGluR5 inverse agonist CTEP (2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine) on the effects of chronic stress. In mice exposed to chronic stress, Homer1b/c, but not Homer1a, mRNA was upregulated and, accordingly, Homer1/mGluR5 coupling was disrupted. We found a marked hyperactivity behavior as well as a dysregulated hypothalamic-pituitary-adrenal axis activity in chronically stressed Homer1 knockout (KO) mice. Chronic administration of the selective and orally bioavailable mGluR5 inverse agonist, CTEP, was able to recover behavioral alterations induced by chronic stress, whereas overexpression of Homer1a in the hippocampus led to an increased vulnerability to chronic stress, reflected in an increased physiological response to stress as well as enhanced depression-like behavior. Overall, our results implicate the glutamatergic system in the emergence of stress-induced psychiatric disorders, and support the Homer1/mGluR5 complex as a target for the development of novel antidepressant agents.

Analysis of individual protein turnover in live animals on a proteome-wide scale.

Classical quantitative proteomics studies focus on the relative or absolute concentration of proteins at a given time. In contrast, the investigation of protein turnover reveals the dynamics leading to these states. Analyzing the balance between synthesis and degradation of individual proteins provides insights into the regulation of protein concentration and helps understanding underlying biological processes. Comparing the half-lives of proteins allows detecting functional relationships and common regulation mechanisms. Moreover, comparing turnover of individual brain and plasma proteins between control- and treatment-groups indicates turnover changes induced by the treatment.Here, we describe a procedure for determining turnover information of individual proteins in mice on a proteome-wide scale based on partial (15)N metabolic labeling. We will outline the complete experimental workflow starting from (15)N labeling the animals over sample preparation and mass spectrometric measurement up to the analysis of the data.

¹5N metabolic labeling: evidence for a stable isotope effect on plasma protein levels and peptide chromatographic retention times.

Many quantitative proteomics methods rely on protein and peptide labeling with stable isotopes. We have recently found that the introduction of ¹5N into organisms via in vivo metabolic labeling affects protein expression levels as well as metabolic pathways and behavioral phenotypes. Here, we present further evidence for a stable isotope effect based on the plasma proteome analysis of ¹5N-labeled mice. We compared plasma proteomes of ¹5N-labeled and unlabeled (¹4N) mice by quantitative MS. We found a number of protein level differences, some of which were verified immunochemically. In addition, we observed divergent chromatographic retention time and peak full width at half maximum (FWHM) between ¹5N-labeled and ¹4N tryptic peptides. Our data point toward a systemic effect of the introduction of heavy isotopes in vivo.

Proteomic and metabolomic profiling reveals time-dependent changes in hippocampal metabolism upon paroxetine treatment and biomarker candidates.

Most of the commonly used antidepressants block monoamine reuptake transporters to enhance serotonergic or noradrenergic neurotransmission. Effects besides or downstream of monoamine reuptake inhibition are poorly understood and yet presumably important for the drugs' mode of action. In the present study we aimed at identifying hippocampal cellular pathway alterations in DBA/2 mice using paroxetine as a representative Selective Serotonin Reuptake Inhibitor (SSRI). Furthermore we identified biomarker candidates for the assessment of antidepressant treatment effects in plasma. Hippocampal protein levels were compared between chronic paroxetine- and vehicle-treated animals using in vivo(15)N metabolic labeling combined with mass spectrometry. We also studied the time course of metabolite level changes in hippocampus and plasma using a targeted polar metabolomics profiling platform. In silico pathway analyses revealed profound alterations related to hippocampal energy metabolism. Glycolytic metabolite levels acutely increased while Krebs cycle metabolite levels decreased upon chronic treatment. Changes in energy metabolism were influenced by altered glycogen metabolism rather than by altered glycolytic or Krebs cycle enzyme levels. Increased energy levels were reflected by an increased ATP/ADP ratio and by increased ratios of high-to-low energy purines and pyrimidines. In the course of our analyses we also identified myo-inositol as a biomarker candidate for the assessment of antidepressant treatment effects in the periphery. This study defines the cellular response to paroxetine treatment at the proteome and metabolome levels in the hippocampus of DBA/2 mice and suggests novel SSRI modes of action that warrant consideration in antidepressant development efforts.

The (15)N isotope effect as a means for correlating phenotypic alterations and affected pathways in a trait anxiety mouse model.

Stable isotope labeling techniques hold great potential for accurate quantitative proteomics comparisons by MS. To investigate the effect of stable isotopes in vivo, we metabolically labeled high anxiety-related behavior (HAB) mice with the heavy nitrogen isotope (15)N. (15)N-labeled HAB mice exhibited behavioral alterations compared to unlabeled ((14)N) HAB mice in their depression-like phenotype. To correlate behavioral alterations with changes on the molecular level, we explored the (15)N isotope effect on the brain proteome by comparing protein expression levels between (15)N-labeled and (14)N HAB mouse brains using quantitative MS. By implementing two complementary in silico pathway analysis approaches, we were able to identify altered networks in (15)N-labeled HAB mice, including major metabolic pathways such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Here, we discuss the affected pathways with regard to their relevance for the behavioral phenotype and critically assess the utility of exploiting the (15)N isotope effect for correlating phenotypic and molecular alterations.

Proteome scale turnover analysis in live animals using stable isotope metabolic labeling.

At present most quantitative proteomics investigations are focused on the analysis of protein expression differences between two or more sample specimens. With each analysis a static snapshot of a cellular state is captured with regard to protein expression. However, any information on protein turnover cannot be obtained using classic methodologies. Protein turnover, the result of protein synthesis and degradation, represents a dynamic process, which is of equal importance to understanding physiological processes. Methods employing isotopic tracers have been developed to measure protein turnover. However, applying these methods to live animals is often complicated by the fact that an assessment of precursor pool relative isotope abundance is required. Also, data analysis becomes difficult in case of low label incorporation, which results in a complex convolution of labeled and unlabeled peptide mass spectrometry signals. Here we present a protein turnover analysis method that circumvents this problem using a (15)N-labeled diet as an isotopic tracer. Mice were fed with the labeled diet for limited time periods and the resulting partially labeled proteins digested and subjected to tandem mass spectrometry. For the interpretation of the mass spectrometry data, we have developed the ProTurnyzer software that allows the determination of protein fractional synthesis rates without the need of precursor relative isotope abundance information. We present results validating ProTurnyzer with Escherichia coli protein data and apply the method to mouse brain and plasma proteomes for automated turnover studies.

A MS data search method for improved 15N-labeled protein identification.

Quantitative proteomics using stable isotope labeling strategies combined with MS is an important tool for biomarker discovery. Methods involving stable isotope metabolic labeling result in optimal quantitative accuracy, since they allow the immediate combination of two or more samples. Unfortunately, stable isotope incorporation rates in metabolic labeling experiments using mammalian organisms usually do not reach 100%. As a consequence, protein identifications in (15)N database searches have poor success rates. We report on a strategy that significantly improves the number of (15)N-labeled protein identifications and results in a more comprehensive and accurate relative peptide quantification workflow.

QuantiSpec--Quantitative mass spectrometry data analysis of (15)N-metabolically labeled proteins.

For relative protein quantitation by mass spectrometry we metabolically labeled E. coli bacteria with (15)N-enriched diets. Proteins extracted from (15)N-labeled and unlabeled E. coli bacteria were mixed, separated by two-dimensional gel electrophoresis and enzymatically digested. The resulting tryptic peptides were analyzed by MALDI mass spectrometry. For the relative protein quantitation we developed fully automated software, QuantiSpec (Quantitative Mass Spectrometry Analysis Software), which uses data from MALDI TOF mass spectrometry and the Mascot database search engine. QuantiSpec detects natural as well as partially or fully labeled peptide isotope distributions. For each identified peptide the (15)N incorporation rate is determined by comparing the experimental to a set of theoretical isotope patterns based on the peptide sequence. Relative quantitation is accomplished by calculating the signal intensity ratios for each (14)N/(15)N peptide pair.