Association of Lamotrigine Plasma Concentrations with Efficacy and Toxicity in Patients with Epilepsy: A Retrospective Study.

BACKGROUND: There is limited evidence to support the currently suggested lamotrigine (LTG) therapeutic reference range of 2.5-15 mg/L for the treatment of seizures. The objective of this study was to evaluate the association of LTG plasma concentrations with the efficacy and toxicity of the treatment in patients with epilepsy. METHODS: Patients whose LTG plasma concentration was measured between January 2013 and February 2022 were included. Efficacy was defined as seizure freedom for at least 6 months around the time of measured LTG concentration. Toxicity was defined as any LTG-related adverse drug effect documented in each patient's health record or when the reason for measuring the LTG concentration was toxicity. In addition, the dose-concentration relationship of LTG was assessed. RESULTS: In total, 549 concentrations from 259 patients with epilepsy were included. The most common reasons for therapeutic drug monitoring were suspected inefficacy (39%) and pregnancy (21%). The LTG plasma concentration was not associated with efficacy (adjusted odds ratio = 0.94; 95% confidence interval, 0.85-1.04). The LTG plasma concentration was positively associated with the incidence of toxicity after adjusting for age, sex, and number of antiepileptic drugs (odds ratio = 1.11; 95% confidence interval, 1.04-1.19). The daily dose had a significant linear correlation with the LTG plasma concentration ( P < 0.001). CONCLUSIONS: The LTG plasma concentration was associated with toxicity, whereas no association with efficacy was found. A reference range of 2.5-10 mg/L may be considered to decrease the risk of toxicity while maintaining similar efficacy. Therapeutic drug monitoring may be useful when LTG-related toxicity is suspected and in cases of pharmacokinetic changes (eg, pregnancy and concomitant use of interacting drugs) that can influence the LTG plasma concentration.

Obligate biotroph downy mildew consistently induces near-identical protective microbiomes in Arabidopsis thaliana.

Hyaloperonospora arabidopsidis (Hpa) is an obligately biotrophic downy mildew that is routinely cultured on Arabidopsis thaliana hosts that harbour complex microbiomes. We hypothesized that the culturing procedure proliferates Hpa-associated microbiota (HAM) in addition to the pathogen and exploited this model system to investigate which microorganisms consistently associate with Hpa. Using amplicon sequencing, we found nine bacterial sequence variants that are shared between at least three out of four Hpa cultures in the Netherlands and Germany and comprise 34% of the phyllosphere community of the infected plants. Whole-genome sequencing showed that representative HAM bacterial isolates from these distinct Hpa cultures are isogenic and that an additional seven published Hpa metagenomes contain numerous sequences of the HAM. Although we showed that HAM benefit from Hpa infection, HAM negatively affect Hpa spore formation. Moreover, we show that pathogen-infected plants can selectively recruit HAM to both their roots and shoots and form a soil-borne infection-associated microbiome that helps resist the pathogen. Understanding the mechanisms by which infection-associated microbiomes are formed might enable breeding of crop varieties that select for protective microbiomes.

Pharmacokinetic investigations of isavuconazole in paediatric cancer patients show reduced exposure of isavuconazole after opening capsules for administration via a nasogastric tube.

OBJECTIVES: To study the isavuconazole pharmacokinetics in a real-life paediatric cohort and confirm whether the isavuconazole exposures are within the adult exposure range. Furthermore, we are the first to describe unbound isavuconazole pharmacokinetics. METHODS: In this prospective, observational study, the isavuconazole dosing regimen was as follows (IV/oral/nasogastric tube): 5.4 mg/kg isavuconazole (maximum 200 mg/dose) three times daily on Days 1 and 2, followed by 5.4 mg/kg isavuconazole (maximum 200 mg/dose) once daily. At least one pharmacokinetic curve was assessed. Non-linear mixed effects modelling was used for analysis. Monte Carlo simulations were performed with the above mentioned maintenance dose for IV administrations and a weight band dosing regimen for oral/nasogastric tube administrations: I) <18 kg (100 mg daily); II) 18-37 kg (150 mg daily); III)>37 kg (200 mg daily). RESULTS: Seventeen paediatric patients with a median age of 9 years (range 1-17) and median weight of 26.0 kg (range 8.4-78.5) were evaluated. A two-compartment model describing linear pharmacokinetics of the unbound concentrations and saturable protein binding fitted the isavuconazole concentrations best. The absolute bioavailability of isavuconazole was 41.0% (95% CI: 32.4%-50.8%). The median (IQR) simulated exposures (AUC0-24h, SS) of the total isavuconazole concentrations after IV and oral/nasogastric tube administration were 87.7 mg·h/L (70.5-105.1) and 50.3 mg·h/L (39.0-62.4), respectively. The unbound isavuconazole fraction (unbound/total) ranged from 0.5% to 2.3%. CONCLUSIONS: This study revealed low bioavailability after nasogastric tube administration with opened capsules. Isavuconazole exposures were in the expected range following IV administration. Total and unbound isavuconazole pharmacokinetics were reported with a 5-fold range in the unbound fraction.

Sub-therapeutic trimethoprim and sulfamethoxazole plasma concentrations during continuous venovenous hemofiltration in a patient with COVID-19 and pulmonary Pneumocystis jirovecii co-infection: A case report.

OBJECTIVE: To investigate drug concentration of trimethoprim-sulfamethoxazole (TMP-SMX) using therapeutic drug monitoring (TDM) for severe Pneumocystis jirovecii (PJP) infection in a critically ill patient with COVID-19 receiving continuous venovenous hemofiltration treatment and regional citrate anticoagulation (RCA-CCVH). MATERIALS AND METHODS: A 72-year-old man with hypoxemic respiratory failure due to COVID-19 infection was admitted to the intensive care unit for invasive mechanical ventilation. The patient developed acute renal failure that required RCA-CVVH. Pulmonary co-infection with PJP was diagnosed, and a high TMP-SMX dose was initiated according to (inter)national guidelines with dose reduction after 3 days because of renal failure. Population pharmacokinetics were assessed for TMP and SMX as well as clearance by RCA-CVVH, volume of distribution, and time above threshold levels for measured plasma concentrations. RESULTS: During renal failure requiring RCA-CVVH, a corresponding dose reduction of TMP-SMX to 320/1,600 mg twice a day, according to current Dutch SWAB and Dutch Association of Hospital Pharmacists guidelines, resulted in unintended under-dosing with sub-therapeutic TMP-SMX concentrations. Pharmacokinetic modeling and dose adjustment of TMP-SMX to 640/3,200 mg 3 times daily resulted in steady-state TMP-SMX peak concentrations associated with efficacy against PJP. Hence, the patient was successfully weaned from the ventilator and discharged. CONCLUSION: We hypothesize that our new dose recommendation of 640/3,200 mg TMP-SMX 3 times daily is associated with an increased probability of critical patients being successfully liberated from mechanical weaning following PJP pneumonia and COVID-19 infection.

Preclinical anti-tumour activity of HexaBody-CD38, a next-generation CD38 antibody with superior complement-dependent cytotoxic activity.

BACKGROUND: HexaBody®-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC). METHODS: Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38. HexaBody-CD38-induced CDC, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), trogocytosis, and apoptosis were assessed using flow cytometry assays using tumour cell lines, and MM patient samples (CDC). CD38 enzymatic activity was measured using fluorescence spectroscopy. Anti-tumour activity of HexaBody-CD38 was assessed in patient-derived xenograft mouse models in vivo. FINDINGS: HexaBody-CD38 binds a unique epitope on CD38 and induced potent CDC in multiple myeloma (MM), acute myeloid leukaemia (AML), and B-cell non-Hodgkin lymphoma (B-NHL) cells. Anti-tumour activity was confirmed in patient-derived xenograft models in vivo. Sensitivity to HexaBody-CD38 correlated with CD38 expression level and was inversely correlated with expression of complement regulatory proteins. Compared to daratumumab, HexaBody-CD38 showed enhanced CDC in cell lines with lower levels of CD38 expression, without increasing lysis of healthy leukocytes. More effective CDC was also confirmed in primary MM cells. Furthermore, HexaBody-CD38 efficiently induced ADCC, ADCP, trogocytosis, and apoptosis after Fc-crosslinking. Moreover, HexaBody-CD38 strongly inhibited CD38 cyclase activity, which is hypothesized to relieve immune suppression in the tumour microenvironment. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of HexaBody-CD38 in patients with MM. FUNDING: Genmab.

Optimization of a Quantitative Anti-Drug Antibodies against Infliximab Assay with the Liquid Chromatography-Tandem Mass Spectrometry: A Method Validation Study and Future Perspectives.

Monoclonal antibodies (mAbs), such as infliximab, are important treatment options for different diseases. Immunogenicity is a major risk, resulting in anti-drug antibodies (ADAs), being associated with adverse events and loss of response, influencing long-term outcomes. The development of ADAs against infliximab is primarily measured by immunoassays like radioimmunoassay (RIA). Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized across different fields, this technique is currently not used for ADAs against infliximab measurements. Therefore, we developed the first LC-MS/MS method. Stable isotopically labeled infliximab antigen-binding fragments (SIL IFX F(ab')2) were used to bind and measure ADAs indirectly. Protein A magnetic beads were used to capture IgG, including ADAs, whereafter SIL IFX F(ab')2 was added for labeling. After washing, internal standard addition, elution, denaturation and digestion samples were measured by LC-MS/MS. Internal validation showed good linearity between 0.1 and 16 mg/L (R2 > 0.998). Sixty samples were used for cross-validation with RIA, and no significant difference between ADA concentrations was found. The methods had high correlation (R = 0.94, p < 0.001) and excellent agreement, intraclass correlation coefficient = 0.912 (95% confidence interval 0.858-0.947, p < 0.001). We present the first ADA against the infliximab LC-MS/MS method. The method is amendable for quantifying other ADAs, making it applicable as a template for future ADA methods.

Ion suppression, reduced long-term robustness and leakage current of the spray voltage during the ionization of trichloroacetic acid; a case study with a methylmalonic acid assay.

Sample clean-up with the protein precipitation solvent trichloroacetic acid (TCA), combined with a stable isotope labeled internal standard, is widely used for the analysis of endogenous and exogenous compounds in serum and plasma with liquid chromatography-tandem mass spectrometry (LC-MS/MS). During the application of an assay for methylmalonic acid (MMA), used for routine analysis in patient care, negative long-term side effects of TCA on assay performance were observed. Step-by-step extensive troubleshooting disclosed the limitations of using TCA in MS. After running over 2000 samples with the MMA assay over a course of one year, a black coating formed between the probe and the heater that was traced to the use of TCA. The MMA assay used a C18 column with an isocratic eluent of 95% water (0.1% formic acid) as starting condition, on which TCA was more retained than MMA. Next, concentrations of 2.2% TCA in the prepared serum or plasma sample caused a drop in spray voltage during ionization into the MS. This was caused by the strong acid properties of TCA, resulting in current loss of the spray voltage between the heated electrospray ionization (HESI) needle and the union holder, which had also a grounding function. Replacing the original metal HESI needle with a custom made fussed silica HESI needle or detaching the union from the union holder, eliminated the effect of the drop in spray voltage. In conclusion, TCA can seriously affect the long-term robustness by affecting the source of the MS. We recommend the use of a very low sample injection volume, and/or shifting the mobile phase to waste when TCA is eluting, when using TCA in LC-MS/MS analysis.

Can eHealth applications improve renal transplant outcomes for adolescents and young adults? A systematic review.

BACKGROUND AND OBJECTIVES: Adherence to medical treatment following a kidney transplant is particularly challenging during adolescence and young adulthood. There is increasing evidence of the benefits of the use of computer and mobile technology (labelled as eHealth hereafter) including serious gaming and gamification in many clinical areas. We aimed to conduct a systematic review of such interventions designed to improve self-management skills, treatment adherence and clinical outcomes in young kidney transplant recipients aged 16 to 30 years. METHOD: The Cochrane Library, MEDLINE, EMBASE, PsychINFO, SCOPUS and CINAHL databases were searched for studies published between 01 January 1990 and 20 October 2020. Articles were short-listed by two independent reviewers based on pre-defined inclusion/exclusion criteria. Reference lists were screened and authors of published conference abstracts contacted. Two reviewers independently appraised selected articles, systematically extracted data and assessed the quality of individual studies (CASP and SORT). Thematic analysis was used for evidence synthesis; quantitative meta-analysis was not possible. RESULTS: A total of 1098 unique records were identified. Short-listing identified four eligible studies, all randomized controlled trials (n = 266 participants). Trials mainly focused on mHealth applications or electronic pill dispensers (mostly for patients >18 years old). Most studies reported on clinical outcome measures. All showed improved adherence but there were no differences in the number of rejections. Study quality was low for all four studies. CONCLUSIONS: The findings of this review suggest that eHealth interventions can improve treatment adherence and clinical outcomes for young kidney transplant patients. More robust and high-quality studies are now needed to validate these findings. Future studies should also extend beyond short-term outcomes, and consider cost of implementation. The review was registered with PROSPERO (CRD42017062469).

Perceived Support from Best Friends and Depressive Symptoms During Adolescence: Disentangling Personal from Dyadic Level Effects.

Support from best friends is an important interpersonal factor in adolescent depression development but is often studied from an individual perspective in which dyadic effects are overlooked. This study aims to a) test whether differences in support vary at the individual level and are related to individual differences in the development of depressive symptoms, whether these differences vary at the dyadic level and are related to dyadic depression symptom development, or both, b) explore whether these associations are moderated by initial levels of depressive symptoms on the individual and/or dyadic level. Data from 452 adolescents (Mage = 13.03), nested in 226 same-gender friendship dyads (60.6% boy-dyads) who participated in the RADAR-Y project were included. Best friends self-reported annually (2006-2008; 3 waves) on their own depressive symptoms and perceived support from their friend. Multilevel models showed no direct association between support and depression development on the individual or dyadic level. However, the initial level of dyads' depressive symptoms moderated the association between dyadic support and dyads' subsequent depression symptom development. When dyads experienced relatively more initial depressive symptoms, higher levels of dyadic support were associated with relative increasing dyadic depressive symptoms. When dyads experienced relatively few initial depressive symptoms, higher levels of dyadic support were associated with relative decreasing dyadic depressive symptoms. Findings suggest that support from best friends can either protect against or exacerbate the development of depressive symptoms for friends, depending on the initial level of depressive symptoms of the dyad.

The efficacy of immune checkpoint inhibitors in elderly patients: a meta-analysis and meta-regression.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapy has improved patient survival in advanced cancers; however, the efficacy of ICIs in elderly patients is still elusive. This study assessed the efficacy of ICIs in elderly patients with advanced cancer in terms of overall survival (OS) and progression-free survival (PFS). MATERIALS AND METHODS: We carried out a systematic review and identified 30 head-to-head phase II/III randomized controlled trials that compared immunotherapy with the standard of care in advanced solid tumor patients. The data on patients younger or over 65 years of age were indexed from PubMed-Medline, Embase, and Scopus and obtained for meta-analysis. The subgroup analyses were stratified by primary tumor type, line of treatment, or type of immunotherapy, and a meta-regression analysis was carried out after adjusting for all other variables. RESULTS: The study included 17 476 patients, comprising 58% (10 119) younger (<65 years old) and 42% (7357) elderly (≥65 years old) patients. The hazard ratio (HR) for OS was 0.77 [95% confidence interval (CI) 0.70-0.85] and 0.77 (95% CI 0.70-0.85) in the younger and elderly groups, respectively, suggesting similar efficacies of ICIs in these two age groups. The subgroup analyses revealed no significant relationship between age and treatment outcomes, except for the PFS benefit in younger patients with melanoma than in elderly patients (HR 0.44 in younger patients versus 0.65 in elderly patients, P = 0.04). These results were further supported by meta-regression analysis, which showed no statistically significant difference in OS (P = 0.954) and PFS (P = 0.555) between the two age groups. CONCLUSIONS: The findings suggest that age-associated impairments of the immune system did not affect the efficacy of ICIs in elderly patients compared to younger patients. Therefore, the choice of ICIs for elderly patients can be considered, regardless of chronological age.

Clinical characteristics do not reliably identify non-adherence in patients with uncontrolled hypertension.

PURPOSE: Chemical adherence testing is a reliable method to assess adherence to antihypertensive drugs. However, it is expensive and has limited availability in clinical practice. To reduce the number and costs of chemical adherence tests, we aimed to develop and validate a clinical screening tool to identify patients with a low probability of non-adherence in patients with uncontrolled hypertension. MATERIALS AND METHODS: In 495 patients with uncontrolled hypertension referred to the University Medical Centre Utrecht (UMCU), the Netherlands, a penalised logistic regression model including seven pre-specified easy-to-measure clinical variables was derived to estimate the probability of non-adherence. Non-adherence was defined as not detecting at least one of the prescribed antihypertensive drugs in plasma or urine. Model performance and test characteristics were evaluated in 240 patients with uncontrolled hypertension referred to the Heartlands Hospital, United Kingdom. RESULTS: Prevalence of non-adherence to antihypertensive drugs was 19% in the UMCU and 44% in the Heartlands Hospital population. After recalibration of the model's intercept, predicted probabilities agreed well with observed frequencies. The c-statistic of the model was 0.63 (95%CI 0.53-0.72). Predicted probability cut-off values of 15%-22.5% prevented testing in 5%-15% of the patients, carrying sensitivities between 97% (64-100) and 90% (80-95), and negative predictive values between 74% (10-99) and 70% (50-85). CONCLUSION: The combination of seven clinical variables is not sufficient to reliably discriminate adherent from non-adherent individuals to safely reduce the number of chemical adherence tests. This emphasises the complex nature of non-adherence behaviour and thus the need for objective chemical adherence tests in patients with uncontrolled hypertension.

Quantification of emicizumab by mass spectrometry in plasma of people with hemophilia A: A method validation study.

Background: Emicizumab is a new treatment option for people with hemophilia A. Emicizumab was approved with a body-weight-based dosage regimen, without laboratory monitoring requirements. Guidelines, however, recommend measuring emicizumab concentrations when the presence of antidrug antibodies is suspected. Furthermore, drug monitoring can be useful in clinical decision making, in adherence checking, and for research purposes. Therefore, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying emicizumab. We performed a validation study on this LC-MS/MS method quantifying emicizumab in the plasma of people with hemophilia A. Methods: Sample preparation for LC-MS/MS analysis included ammonium sulfate protein precipitation and trypsin digestion. A signature peptide of emicizumab and a matching stable isotope-labeled internal standard were used to quantify emicizumab by LC-MS/MS analysis. Validation was performed in accordance with the "Guideline on Bioanalytical Method Validation" of the European Medicines Agency (EMA). The LC-MS/MS method was cross validated against a modified and calibrated (r 2 Diagnostics) one-stage clotting assay (OSA). Conclusions: The LC-MS/MS method demonstrated linearity over a wide range of emicizumab concentrations, far exceeding the concentrations observed in people with hemophilia A. Precision and accuracy were excellent, and all other validation parameters were also within the acceptance EMA criteria. Cross validation showed that the LC-MS/MS method and the OSA-based method can be used interchangeably for drug monitoring of emicizumab without the application of a correction factor.

Differential Contribution of NF-κB Signaling Pathways to CD4+ Memory T Cell Induced Activation of Endothelial Cells.

Endothelial cells (ECs) are important contributors to inflammation in immune-mediated inflammatory diseases (IMIDs). In this study, we examined whether CD4+ memory T (Tm) cells can drive EC inflammatory responses. Human Tm cells produced ligands that induced inflammatory responses in human umbilical vein EC as exemplified by increased expression of inflammatory mediators including chemokines and adhesion molecules. NF-κB, a key regulator of EC activation, was induced by Tm cell ligands. We dissected the relative contribution of canonical and non-canonical NF-κB signaling to Tm induced EC responses using pharmacological small molecule inhibitors of IKKß (iIKKß) or NF-κB inducing kinase (iNIK). RNA sequencing revealed substantial overlap in IKKß and NIK regulated genes (n=549) that were involved in inflammatory and immune responses, including cytokines (IL-1ß, IL-6, GM-CSF) and chemokines (CXCL5, CXCL1). NIK regulated genes were more restricted, as 332 genes were uniquely affected by iNIK versus 749 genes by iIKKß, the latter including genes involved in metabolism, proliferation and leukocyte adhesion (VCAM-1, ICAM-1). The functional importance of NIK and IKKß in EC activation was confirmed by transendothelial migration assays with neutrophils, demonstrating stronger inhibitory effects of iIKKß compared to iNIK. Importantly, iIKKß - and to some extent iNIK - potentiated the effects of currently employed therapies for IMIDs, like JAK inhibitors and anti-IL-17 antibodies, on EC inflammatory responses. These data demonstrate that inhibition of NF-κB signaling results in modulation of Tm cell-induced EC responses and highlight the potential of small molecule NF-κB inhibitors as a novel treatment strategy to target EC inflammatory responses in IMIDs.

Antiviral treatment with fluoxetine for rituximab-associated chronic echovirus 13 meningoencephalitis and myofasciitis.

BACKGROUND AND PURPOSE: Enterovirus infections pose a serious threat for patients with humoral deficiencies and may be lethal, whilst the efficacy of proposed treatment options such as corticosteroids, intravenous immunoglobulins and fluoxetine remains debated. METHODS: Viral clearance was investigated in a patient with rituximab-induced B-cell depletion and chronic echovirus 13 (E13) meningoencephalitis/myofasciitis in response to intravenous immunoglobulins and fluoxetine using sequential semi-quantitative E13 viral load measurements by real-time reverse transcription polymerase chain reaction. Fluoxetine concentrations in plasma and cerebrospinal fluid were determined by liquid chromatography mass spectrometry. RESULTS: Intravenous immunoglobulins appeared ineffective in this case of E13 infection, whereas virus clearance in cerebrospinal fluid was obtained after 167 days of oral fluoxetine. Since treatment with corticosteroids resulted in a flare of symptoms, rechallenge with viral load measurements was not attempted. CONCLUSION: In this report of a patient with rituximab-associated chronic echovirus 13 meningoencephalitis, viral clearance in response to single treatment options is assessed for the first time. Our observations further support the in vivo efficacy of fluoxetine against enteroviral infections. More research is needed to establish its efficacy in different enterovirus strains.

Fludarabine exposure predicts outcome after CD19 CAR T-cell therapy in children and young adults with acute leukemia.

The addition of fludarabine to cyclophosphamide as a lymphodepleting regimen prior to CD19 chimeric antigen receptor (CAR) T-cell therapy significantly improved outcomes in patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Fludarabine exposure, previously shown to be highly variable when dosing is based on body surface area (BSA), is a predictor for survival in allogeneic hematopoietic cell transplantation (allo-HCT). Hence, we hypothesized that an optimal exposure of fludarabine might be of clinical importance in CD19 CAR T-cell treatment. We examined the effect of cumulative fludarabine exposure during lymphodepletion, defined as concentration-time curve (AUC), on clinical outcome and lymphocyte kinetics. A retrospective analysis was conducted with data from 26 patients receiving tisagenlecleucel for r/r B-ALL. Exposure of fludarabine was shown to be a predictor for leukemia-free survival (LFS), B-cell aplasia, and CD19-positive relapse following CAR T-cell infusion. Minimal event probability was observed at a cumulative fludarabine AUCT0-∞ ≥14 mg*h/L, and underexposure was defined as an AUCT0-∞ <14 mg*h/L. In the underexposed group, the median LFS was 1.8 months, and the occurrence of CD19-positive relapse within 1 year was 100%, which was higher compared with the group with an AUCT0-∞ ≥14 mg*h/L (12.9 months; P < .001; and 27.4%; P = .0001, respectively). Furthermore, the duration of B-cell aplasia within 6 months was shorter in the underexposed group (77.3% vs 37.3%; P = .009). These results suggest that optimizing fludarabine exposure may have a relevant impact on LFS following CAR T-cell therapy, which needs to be validated in a prospective clinical trial.

Middle-up quantification of therapeutic monoclonal antibodies in human plasma with two dimensional liquid chromatography high resolution mass spectrometry: Adalimumab as a proof of principle.

Next generation human therapeutic monoclonal antibodies (t-mAbs) are harder to quantify with the widely used bottom-up tryptic digestion method. Due to their homology with endogenous immunoglobulins, there is a lack of unique and stable 'signature' peptides that can be targeted. Middle-up two dimensional liquid chromatography high resolution mass spectrometry (2D-LC-HRMS), targeting the entire light chain, was examined as an alternative. Adalimumab (ADM) was successfully quantified in human plasma after Melon® Gel sample purification, followed by orthogonal separation on a weak cation exchange (WCX) and reversed phase column. Charge and hydrophobicity were used to separate ADM from the polyclonal immunoglobulin background. HRMS with its high resolution and exact mass was able to isotopically resolve the ADM light chain and to provide another separation dimension on the basis of mass to charge ratio. Using the targeted single ion monitoring (T-SIM) with multiplex (MSX) option, three ADM light chain precursors, 2341.80, 2129.00, and 1951.68 m/z, and one internal standard precursor 2146.39 m/z, were measured simultaneously. The Melon® Gel sample purification was found to be very efficient in removing plasma proteins that would otherwise interfere with chromatographic separation and ionization. The linearity of the method for the analysis of ADM was excellent (R2=0.999) between 1 - 128 mg/L with an LLOQ signal to noise ratio (S/N) of 10. Within-run and between-run precision and accuracy were in concordance with the EMA guideline. Cross-validation of the 2D-LC-HRM method with the standard peptide LC-MS/MS method showed a good agreement (R2 = 0.86) between the methods. However, there was a bias present, possibly due to charge variant ADM formation over time. Since the presented 2D-LC-HRMS method is able to measure only the native form of ADM, it is able to provide a measure of the active form of ADM in patients.

Splenic Architecture and Function Requires Tight Control of Transmembrane TNF Expression.

Soluble tumor necrosis factor (sTNF) is an important inflammatory mediator and essential for secondary lymphoid organ (SLO) development and function. However, the role of its transmembrane counterpart (tmTNF) in these processes is less well established. Here, the effects of tmTNF overxpression on SLO architecture and function were investigated using tmTNF-transgenic (tmTNF-tg) mice. tmTNF overexpression resulted in enlarged peripheral lymph nodes (PLNs) and spleen, accompanied by an increase in small splenic lymphoid follicles, with less well-defined primary B cell follicles and T cell zones. In tmTNF-tg mice, the spleen, but not PLNs, contained reduced germinal center (GC) B cell fractions, with low Ki67 expression and reduced dark zone characteristics. In line with this, smaller fractions of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells were observed with a decreased Tfh:Tfr ratio. Moreover, plasma cell (PC) formation in the spleen of tmTNF-tg mice decreased and skewed towards IgA and IgM expression. Genetic deletion of TNFRI or -II resulted in a normalization of follicle morphology in the spleen of tmTNF-tg mice, but GC B cell and PC fractions remained abnormal. These findings demonstrate that tightly regulated tmTNF is important for proper SLO development and function, and that aberrations induced by tmTNF overexpression are site-specific and mediated via TNFRI and/or TNFRII signaling.

Overexpression of Transmembrane TNF Drives Development of Ectopic Lymphoid Structures in the Bone Marrow and B Cell Lineage Alterations in Experimental Spondyloarthritis.

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

A generic sample preparation method for the multiplex analysis of seven therapeutic monoclonal antibodies in human plasma or serum with liquid chromatography-tandem mass spectrometry.

Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total mAb concentrations in human plasma for clinical studies and therapeutic drug monitoring. We developed an easy, rapid, and robust sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated for infliximab (IFX), rituximab (RTX), cetuximab (CTX), dupilumab (DPL), dinutuximab (DNX), vedolizumab (VDZ), and emicizumab (EMZ). Saturated ammonium sulfate (AS) was used to precipitate immunoglobulins in human plasma. After centrifugation, supernatant containing albumin was decanted, and the precipitated immunoglobulin fraction was re-dissolved in buffer containing 6M guanidine. This fraction was then completely denatured, reduced, alkylated, and trypsin digested. Finally, signature peptides from the seven mAbs were simultaneously quantified on LC-MS/MS together with their internal standards stable isotopically labeled peptide counterparts. The linear dynamic ranges (1 - 512 mg/L) of IFX, CTX, RTX, and EMZ showed excellent (R2 > 0.999) linearity and those of DPL, DNX, and VDZ showed good (R2 > 0.995) linearity. The method was validated in accordance with the EMA guidelines. EDTA plasma, sodium citrate plasma, heparin plasma, and serum yielded similar results. Prepared samples were stable at room temperature (20°C) and at 5°C for 3 days, and showed no decline in concentration for all tested mAbs. This described method, which has the advantage of an easy, rapid, and robust pre-analytical sample preparation, can be used as a template to quantify other mAbs in human plasma or serum.

A Straightforward Method for 3D Visualization of B Cell Clusters and High Endothelial Venules in Lymph Nodes Highlights Differential Roles of TNFRI and -II.

Whole mount tissue immunolabeling and imaging of complete organs has tremendous benefits in characterizing organ morphology. Here, we present a straightforward method for immunostaining, clearing and imaging of whole murine peripheral lymph nodes (PLNs) for detailed analysis of their architecture and discuss all procedures in detail in a step-by-step approach. Given the importance of tumor necrosis factor receptor (TNFR) signaling in development of PLNs we used TNFRI-/- and TNFRII-/- mice models as proof-of-concept for this technique by visualizing and analyzing structural changes in PLN B cell clusters and high endothelial venules (HEVs). Samples were subjected to de- and rehydration with methanol, labeled with antibodies for B cells, T cells and high endothelial venules (HEVs) and optically cleared using benzyl alcohol-benzyl benzoate. Imaging was done using LaVision light sheet microscope and analysis with Imaris software. Using these techniques, we confirmed previous findings that TNFRI signaling is essential for formation of individual B cell clusters. In addition, Our data suggest that TNFRII signaling is also to some extent involved in this process as TNFRII-/- PLNs had a B cell cluster morphology reminiscent of TNFRI-/- PLNs. Moreover, visualization and objective quantification of the complete PLN high endothelial vasculature unveiled reduced volume, length and branching points of HEVs in TNFRI-/- PLNs, revealing an earlier unrecognized contribution of TNFRI signaling in HEV morphology. Together, these results underline the potential of whole mount tissue staining and advanced imaging techniques to unravel even subtle changes in lymphoid tissue architecture.