One of the factors that may impact drug levels of therapeutic antibodies in patients is immunogenicity, with potential loss of efficacy. Nowadays, many immunogenicity assays are available for testing ...antidrug antibodies (ADA). In this article, we discuss different types of immunogenicity assays and their clinical relevance in terms of drug tolerance, relation with pharmacokinetics (PK), neutralizing antibodies, potential adverse events associated with ADA, and prediction of ADA production. Drug-tolerant assays can provide insight into the process of immunogenicity, but for clinical management, these assays do not necessarily outperform drug-sensitive assays. The usefulness of any ADA assay for clinical decision making will be larger when drug concentrations are also measured, and this is true, in particular, for drug-tolerant assays.
Patients with rheumatoid arthritis (RA) can be successfully treated with tumor necrosis factor (TNF) inhibitors, including the monoclonal antibody adalimumab. Once in remission, a proportion of ...patients can successfully discontinue treatment, indicating that blocking TNF is no longer required for disease control. To explore the dynamics of circulating TNF during adalimumab treatment, we developed a competition enzyme-linked immunosorbent assay that can quantify TNF in the presence of large amounts of TNF inhibitor, i.e., a "drug-tolerant" assay. In 193 consecutive adalimumab-treated patients with RA, we demonstrated that circulating TNF increased in average of >50-fold upon treatment and reached a stable concentration in time for most patients. A similar increase in TNF was found in 30 healthy volunteers after one dose of adalimumab. This implies that TNF in circulation during anti-TNF treatment is not primarily associated with disease activity. During treatment, TNF was in complex with adalimumab and could be recovered as inactive 3:1 adalimumab-TNF complexes. No quantitative association was found between TNF and adalimumab concentrations. Low TNF concentrations at week 4 were associated with a higher frequency of antidrug antibodies (ADAs) at subsequent time points, less frequent methotrexate use at baseline, and less frequent remission after 52 weeks. Also in healthy volunteers, early low TNF concentrations are associated with ADAs. In conclusion, longitudinal TNF concentrations are mostly stable during adalimumab treatment and may therefore not predict successful treatment discontinuation. However, early low TNF is strongly associated with ADA formation and may be used as timely predictor of nonresponse toward adalimumab treatment.
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
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.
C-type lectins are innate receptors expressed on antigen-presenting cells that are involved in the recognition of glycosylated pathogens and self-glycoproteins. Upon ligand binding, internalization ...and/or signaling often occur. Little is known on the glycan specificity and ligands of the Dendritic Cell Immunoreceptor (DCIR), the only classical C-type lectin that contains an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). Here we show that purified DCIR binds the glycan structures Lewis(b) and Man3. Interestingly, binding could not be detected when DCIR was expressed on cells. Since DCIR has an N-glycosylation site inside its carbohydrate recognition domain (CRD), we investigated the effect of this glycan in ligand recognition. Removing or truncating the glycans present on purified DCIR increased the affinity for DCIR-binding glycans. Nevertheless, altering the glycosylation status of the DCIR expressing cell or mutating the N-glycosylation site of DCIR itself did not increase glycan binding. In contrast, cis and trans interactions with glycans induced DCIR mediated signaling, resulting in a decreased phosphorylation of the ITIM sequence. These results show that glycan binding to DCIR is influenced by the glycosylation of the CRD region in DCIR and that interaction with its ligands result in signaling via its ITIM motif.
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.
The Infliximab, has proven effective in treating rheumatoid arthritis (RA). A good clinical response is usually associated with high serum drug levels. Development of antibodies toward Infliximab ...(ATI) can increase drug clearance, leading to treatment failure.
To analyze whether serum Infliximab trough levels (ITL) at the induction phase are associated with Infliximab clearance and clinical outcomes at week(W) 54 and to investigate the association with immunogenicity development.
Observational retrospective study in which ITL from 66 RA patients were measured by capture ELISA at W0, W2, W6, W14 and 22. Patients were classified as ITLpos if Infliximab was detectable at W54 and ITLneg otherwise. ATI were assayed by bridging ELISA and by two drug-tolerant assays. ITL cut-off values were established by ROC curves. The association between ITL at early-stage and clearance of Infliximab at W54 was analyzed by univariable and multivariable logistic regression.
ITLneg patients (n=25) always had significantly lower Infliximab levels than ITLpos (n=41). An ITL value of 4.4 μg/mL at W6 best predicted W54 Infliximab absence. In the multivariable analysis, only ITL below the cut-off at W6 (OR: 86.6; 95%CI: 6.58-1139.99) and non-use of methotrexate (OR: 6.9; 95%CI: 1.04-45.84) remained significantly associated with W54 Infliximab absence. ATI were more frequent in patients with ITL below the cut-off at W6.
In RA, ITL at induction phase are inversely associated with Infliximab clearance and clinical outcomes at W54. ATI was the main reason for low early ITL. A predictive value of ITL at W6 was found as a useful prognostic measure of treatment efficacy.
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.
•Accurate anti-drug antibody (ADA) detection requires dissociation from the drug.•Anti-idiotype/Fc bridging facilitates detection of IgG4 and low-affinity ADAs.•Europium labeling enables fluorescence-based detection of ADAs immobilized on beads.•Eu-chelate fluorescence allows assays to be performed in a 96-well plate format.
Biologics have transformed management of inflammatory diseases. To optimize outcomes and reduce costs, dose adjustment informed by circulating drug levels has been proposed. We aimed to determine the ...real-world clinical utility of therapeutic drug monitoring in psoriasis. Within a multicenter (n = 60) prospective observational cohort, 544 psoriasis patients were included who were receiving adalimumab monotherapy and had at least one serum sample and Psoriasis Area and Severity Index (PASI) score available within the first year. We present models giving individualized probabilities of response for any given drug level: a minimally effective drug level of 3.2 μg/ml discriminates responders (PASI75 indicates 75% improvement in baseline PASI) from nonresponders, and gives an estimated PASI75 probability of 65% (95% confidence interval = 60–71). At 7 μg/ml, PASI75 probability is 81% (95% CI = 76–86); beyond 7 μg/ml, the drug level/response curve plateaus. Crucially, drug levels are predictive of response 6 months later, whether sampled early or at steady state. We confirm serum drug level to be the most important factor determining treatment response, highlighting the need to take drug levels into account when searching for biomarkers of response. This real-world study with pragmatic drug level sampling provides evidence to support the proactive measurement of adalimumab levels in psoriasis to direct treatment strategy, and is relevant to other inflammatory diseases.
One reason for a lack of response to rituximab as well as infusion-related anaphylactic adverse events is the development of antidrug Abs to rituximab. Besides rituximab, a number of other ...therapeutic Abs targeting CD20 are nowadays available as alternatives. In this study, we investigated the potential cross-reactivity of (human) anti-rituximab Abs to three other anti-CD20 mAbs: ofatumumab, obinutuzumab, and ocrelizumab. In 25 cases of anti-rituximab Abs, cross-reactivity was examined using both direct binding assays and inhibition immunoassays. Although no cross-reactivity was observed to ofatumumab or obinutuzumab, 8 of 25 samples also showed reactivity toward ocrelizumab in at least one of the two assays. Furthermore, in three cases of anti-ocrelizumab Abs, cross-reactivity to rituximab was observed in an inhibition immunoassay, albeit not in a direct binding assay. Our results suggest that obinutuzumab or ofatumumab are safe anti-CD20 alternatives in case of the presence of anti-rituximab Abs. It is advisable to proceed cautiously if switching from rituximab to ocrelizumab (or vice versa) is considered in case these alternatives may not be available.
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. 100AU/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 ADA.
•We developed three new drug tolerant assays for the detection of anti-drug antibodies.•In a major portion of the patient samples anti-drug antibodies are detected.•The four drug tolerant assays tested correlate well.•The consistent results of the assays break down at the lower end of the detectable range.•This highlights that anti-drug antibodies are best reported quantitatively.
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