Using Real-World Data to Guide Ustekinumab Dosing Strategies for Psoriasis: A Prospective Pharmacokinetic-Pharmacodynamic Study.

Variation in response to biologic therapy for inflammatory diseases, such as psoriasis, is partly driven by variation in drug exposure. Real-world psoriasis data were used to develop a pharmacokinetic/pharmacodynamic (PK/PD) model for the first-line therapeutic antibody ustekinumab. The impact of differing dosing strategies on response was explored. Data were collected from a UK prospective multicenter observational cohort (491 patients on ustekinumab monotherapy, drug levels, and anti-drug antibody measurements on 797 serum samples, 1,590 measurements of Psoriasis Area Severity Index (PASI)). Ustekinumab PKs were described with a linear one-compartment model. A maximum effect (Emax ) model inhibited progression of psoriatic skin lesions in the turnover PD mechanism describing PASI evolution while on treatment. A mixture model on half-maximal effective concentration identified a potential nonresponder group, with simulations suggesting that, in future, the model could be incorporated into a Bayesian therapeutic drug monitoring "dashboard" to individualize dosing and improve treatment outcomes

Toll-Like Receptor 4 Triggering Promotes Cytosolic Routing of DC-SIGN-Targeted Antigens for Presentation on MHC Class I

DC-SIGN is an antigen uptake receptor expressed on dendritic cells (DCs) with specificity for glycans present on a broad variety of pathogens and is capable of directing its cargo to MHC-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Therefore, DC-SIGN is a very promising target for the delivery of antigen for anti-cancer vaccination. Although the endocytic route leading to MHC-II presentation is characterized to a large extent, the mechanisms controlling DC-SIGN targeted cross-presentation of exogenous peptides on MHC-I, are not completely resolved yet. In this paper, we used imaging flow cytometry and antigen-specific CD8+ T cells to investigate the intracellular fate of DC-SIGN and its cargo in human DCs. Our data demonstrates that immature DCs and toll-like receptor 4 (TLR4) stimulated DCs had similar internalization capacity and were both able to cross-present antigen targeted via DC-SIGN. Interestingly, simultaneous triggering of TLR4 and DC-SIGN on DCs resulted in the translocation of cargo to the cytosol, leading to proteasome-dependent processing and increased CD8+ T cell activation. Understanding the dynamics of DC-SIGN-mediated uptake and processing is essential for the design of optimal DC-SIGN-targeting vaccination strategies aimed at enhancing CD8+ T cell responses

Capillary blood microsampling to determine serum biopharmaceutical concentration: Mitra® microsampler vs dried blood spot

Aim: For assessment of concentrations of biopharmaceuticals, for example, therapeutic drug monitoring, dried blood sampling of capillary blood is a convenient alternative to traditional venepuncture sampling. We investigated an alternative to dried blood spot collection on filter paper: sampling capillary blood using the Mitra® microsampler. Materials and Methods: Therapeutic monoclonal antibodies were spiked in whole blood, sampled using filter paper and Mitra microsampler and concentrations measured using specific ELISAs. Results: Good recoveries of adalimumab, infliximab, ustekinumab, vedolizumab, tocilizumab, natalizumab and rituximab were found up to 1 month of storage at room temperature, averaging 95.2% for the Mitra microsampler and 92.9% for Whatman® paper. Both hemoglobin and potassium yield satisfactory estimates for the volume of the cellular fraction of blood samples in combination with the Mitra microsampler. Conclusion: We established practical protocols for the estimation of serum/plasma concentrations of therapeutic antibodies via capillary blood microsampling

Drug-tolerant detection of anti-drug antibodies in an antigen-binding assay using europium chelate fluorescence

Accurate anti-drug antibody (ADA) measurements in patient sera requires dissociation of ADA-drug complexes combined with sensitive and specific ADA detection. Bridging type immunoassays are often used despite several disadvantages associated with this approach. A good drug-tolerant alternative is the acid-dissociation radioimmunoassay (ARIA), but this method is not easily implemented in most labs as specialized facilities are required for working with radioactive materials. We describe an innovative method for ADA detection that combines the advantages of antigen binding tests like the ARIA with the convenience of regular immunoassays. This acid-dissociation lanthanide-fluorescence immunoassay (ALFIA) involves dissociation of ADA-drug complexes, followed by binding to an europium-labeled drug derivative and subsequently an IgG pulldown on Sepharose beads. After europium elution, detection is achieved by measuring time-resolved fluorescence originating from europium chelate complexes. We measured anti-adalimumab ADA levels in sera of 94 rheumatoid arthritis patients using the ALFIA and showed this method to be highly drug tolerant, sensitive and specific for anti-adalimumab ADAs

Systematic comparison of drug-tolerant assays for anti-drug antibodies in a cohort of adalimumab-treated rheumatoid arthritis patients

Drug interference complicates assessment of immunogenicity of biologicals and results in an underestimation of anti-drug antibody (ADA) formation. Drug-tolerant assays have the potential to overcome such limitations. However, to which extent drug-tolerant assays provide an unbiased picture of the antibody response to a biological is unknown. In this study, we compared the measurement of ADA to adalimumab in 94 consecutive adalimumab-treated rheumatoid arthritis patients using the traditional antigen binding test (ABT) and four different drug-tolerant assays, the Ph-shift anti-Idiotype Antigen binding test (PIA) and three newly developed assays for this study: an acid-dissociation radioimmunoassay (ARIA), a temperature-shift radioimmunoassay (TRIA) and an electrochemoluminescence-based assay (ECL). Our results indicate that drug-tolerant assays provide a fairly consistent view on the antibody formation: quantitatively, the results from all four assays correlate well (Spearman r > 0.9). However, the percentage of ADA-positive patients ranges from 51 to 66% between assays, with the ARIA identifying the highest number of patients as positive. These differences are largely due to patients making low amounts of ADA; if ADA levels were above ca. 100 AU/ml, a patient was identified as positive in all four assays. Adalimumab concentrations were significantly lower in ADA-positive samples. Taken together, the results indicate that these different drug-tolerant assays provide a similar and reasonably consistent view on ADA responses, which however, breaks down at the lower end of the detectable range, and highlight that ADA is best reported quantitatively. Furthermore, if an even more sensitive drug-tolerant assay could be developed, one would probably find additional positive samples that will predominantly contain very low levels of AD

Dried blood spots from finger prick facilitate therapeutic drug monitoring of adalimumab and anti-adalimumab in patients with inflammatory diseases

Aims: Development of a self-sampling method for therapeutic drug monitoring (TDM) of biologicals will enhance TDM implementation in routine care and pharmacokinetic knowledge. The aim of this study was to compare adalimumab and anti-adalimumab antibody (ADA) concentration measurements in dried blood spots (DBS) obtained from finger prick with measurements in serum obtained via venepuncture, from patients with rheumatic inflammatory diseases. Methods: In this cross-sectional study, 161 consecutive patients were included. For clinical validation, DBS from finger prick and serum from venepuncture were collected simultaneously and adalimumab and ADA concentration were assessed by ELISA and antigen binding test (ABT), respectively. To convert DBS eluate results to values which can be compared to serum concentrations, five different methods were investigated, using a marker protein or a volumetric approach. Results: Adalimumab and ADA concentrations obtained from the finger prick/DBS method correlated well with serum results from the same patient (correlation coefficient > 0.87). Interestingly, antibody concentrations (either adalimumab, ADA or total immunoglobulin G) in DBS from finger prick, but not albumin, were systematically lower compared to serum. Spike experiments demonstrated a quantitative recovery for all tested proteins in DBS, suggesting a slightly different protein composition of blood collected via finger prick vs. venepuncture. We established a correction factor to relate finger prick/DBS values with serum values (approximately 1.2). Conclusions: We show here for the first time that adalimumab and ADA serum concentrations can be satisfactorily estimated by measuring concentrations in DBS eluates, collected by finger prick. This method offers great opportunity to simplify TDM of adalimumab

The Consequences of Multiple Simultaneous C-Type Lectin-Ligand Interactions: DCIR Alters the Endo-Lysosomal Routing of DC-SIGN

Antigen-presenting cells (APCs) are equipped with multiple receptors to allow proper pathogen recognition and capture. C-type lectin receptors (CLRs) recognize glycan structures on pathogens and endogenous glycoproteins for internalization and antigen processing and presentation. Often, the glycan specificity of these receptors is overlapping and/or pathogens are decorated with ligands for multiple CLRs, posing the question whether interference or cooperativity within the CLR family exists. Here, we used imaging flow cytometry to investigate the internalization properties of four different CLRs [mannose receptor, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), macrophage galactose-type lectin, and dendritic cell immunoreceptor (DCIR)] on different APCs, as well as their intracellular routing. Although the internalization score of the investigated CLRs was similar on monocytes, macrophages, and dendritic cells (DCs), DCIR internalization rates were lower compared to the other CLRs. Upon triggering, DCIR routed to intracellular compartments outside of the classical endo-lysosomal pathway, resulting in poor CD4(+) T-cell stimulation. Although DC maturation reduced CLR expression levels, it did not affect their internalization rates. Although CLR internalization appeared to be independently regulated, DC-SIGN routing was affected when DCIR was triggered simultaneously. In conclusion, our results provide new insights for the design of DC-based immunotherapeutic strategies and suggest that DCIR is an inferior target in this respect