Bovine ultralong CDR-H3 derived knob paratopes elicit potent TNF-α neutralization and enable the generation of novel adalimumab-based antibody architectures with augmented features.

In this work we have generated cattle-derived chimeric ultralong CDR-H3 antibodies targeting tumor necrosis factor α (TNF-α) via immunization and yeast surface display. We identified one particular ultralong CDR-H3 paratope that potently neutralized TNF-α. Interestingly, grafting of the knob architecture onto a peripheral loop of the CH3 domain of the Fc part of an IgG1 resulted in the generation of a TNF-α neutralizing Fc (Fcknob) that did not show any potency loss compared with the parental chimeric IgG format. Eventually, grafting this knob onto the CH3 region of adalimumab enabled the engineering of a novel TNF-α targeting antibody architecture displaying augmented TNF-α inhibition.

Evaluation of the Homogenous Mobility Shift Assay for Infliximab and Adalimumab Anti-drug Antibody Detection in the Clinical Laboratory.

BACKGROUND: Detecting antidrug antibodies (ADAs) against infliximab or adalimumab is useful for therapeutic drug monitoring. Various ADA detection methods exist, and antibody titer is an output in some algorithms. Homogenous mobility shift assay (HMSA) measures relative ADA concentration and determines drug-ADA complex size in vitro. However, the relevance of complex size determination in drug monitoring remains unclear. Hence, the association between complex size, ADA concentration, and sample detectable neutralizing activity was evaluated. METHODS: Sera from infliximab-treated and adalimumab-treated patients who tested positive for ADA in the National Screening Service were analyzed using 3 ADA assays. HMSA determined the relative ADA concentrations and complex sizes, competitive ligand-binding assay evaluated the sample neutralizing capacity, and enzyme-linked immunosorbent assay detected immunoglobulin (Ig)G4 ADA. RESULTS: Most ADA-positive samples (>80%) formed drug-ADA dimer complexes, whereas 17% had dimer and multimer complexes, and 3% had multimeric complexes. Multimer presence had 100% positive predictive value for detectable neutralizing activity. ADA concentration and detectable neutralizing activity were moderately correlated (r = 0.65) in adalimumab-treated patients and strongly correlated (r = 0.81) in infliximab-treated patients. In adalimumab-treated patients, multimer presence was a stronger predictor of neutralizing activity than ADA concentration was, but not in infliximab-treated patients. However, in infliximab-treated patient samples, multimer presence revealed a distinct subset with high ADA concentrations, neutralizing activity, and IgG4 ADA. CONCLUSIONS: Multimers detected using HMSA had a strong positive predictive value for competitive ligand-binding assay detectable neutralizing activity. Multimeric IgG4-containing ADA-drug complexes revealed a distinct subset of infliximab-treated patient samples, whose clinical relevance merits further investigation.

Antibiotic use differentially affects the risk of anti-drug antibody formation during anti-TNFα therapy in inflammatory bowel disease patients: a report from the epi-IIRN.

OBJECTIVE: Anti-drug antibodies (ADA) to anti-tumour necrosis factor (anti-TNF) therapy drive treatment loss of response. An association between intestinal microbial composition and response to anti-TNF therapy was noted. We therefore aimed to assess the implications of antibiotic treatments on ADA formation in patients with inflammatory bowel disease (IBD). DESIGN: We analysed data from the epi-IIRN (epidemiology group of the Israeli IBD research nucleus), a nationwide registry of all patients with IBD in Israel. We included all patients treated with anti-TNF who had available ADA levels. Survival analysis with drug use as time varying covariates were used to assess the association between antibiotic use and ADA development. Next, specific pathogen and germ-free C57BL mice were treated with respective antibiotics and challenged with infliximab. ADA were assessed after 14 days. RESULTS: Among 1946 eligible patients, with a median follow-up of 651 days from initiation of therapy, 363 had positive ADA. Cox proportional hazard model demonstrated an increased risk of ADA development in patients who used cephalosporins (HR=1.97, 95% CI 1.58 to 2.44), or penicillins with ß-lactamase inhibitors (penicillin-BLI, HR=1.4, 95% CI 1.13 to 1.74), whereas a reduced risk was noted in patients treated with macrolides (HR=0.38, 95% CI 0.16 to 0.86) or fluoroquinolones (HR=0.20, 95% CI 0.12 to 0.35). In mice exposed to infliximab, significantly increased ADA production was observed in cephalosporin as compared with macrolide pretreated mice. Germ-free mice produced no ADA. CONCLUSION: ADA production is associated with the microbial composition. The risk of ADA development during anti-TNF therapy can possibly be reduced by avoidance of cephalosporins and penicillin-BLIs, or by treatment with fluoroquinolones or macrolides.

De-escalation from Dose-Intensified Anti-TNF Therapy Is Successful in the Majority of IBD Patients at 12 Months.

BACKGROUND: Data on outcomes following de-escalation of intensified anti-TNF therapy in inflammatory bowel disease (IBD) are limited and concerns about relapse limit willingness to de-escalate. AIMS: To evaluate rates of successful de-escalation at 12 months and to determine factors that may predict success. METHODS: Single-centre experience of IBD patients that were de-escalated following deep remission on dose-intensified infliximab (IFX) or adalimumab (ADA) for secondary loss of response. Patients were classified as 'successes' if remaining on reduced anti-TNF or 'failures' if requiring re-escalation, steroids, surgery or enrolment into a clinical trial at 12 months. Patient demographics, disease characteristics, biomarkers (faecal calprotectin, C-reactive protein, albumin) and anti-TNF drug levels were collected 6-monthly. RESULTS: Of 25 patients (20 CD, 5 UC), 16 (64%) were successes 12 months post-de-escalation. Median time to failure was 6 months. Six of the nine failures required anti-TNF re-escalation and three entered a clinical trial. Re-escalation recaptured response in all six patients. There was no significant difference in baseline biomarker activity between the two groups. There was no difference in infliximab levels between successes and failures at the time of de-escalation (5.5 vs. 5.3, p = 0.63) as well as 6 months (3.1 vs. 4.6, p = 0.95) and 12 months (3.2 vs. 4.5, p = 0.58) post-de-escalation. CONCLUSION: Nearly two-thirds of patients remained on reduced anti-TNF dosing 12 months after de-escalation. All patients who failed de-escalation were recaptured after dose re-escalation. De-escalation with close monitoring may be considered in patients on intensified anti-TNF therapy in sustained remission.

HLA-DQA1*05 Carriage Associated With Development of Anti-Drug Antibodies to Infliximab and Adalimumab in Patients With Crohn's Disease.

BACKGROUND & AIMS: Anti-tumor necrosis factor (anti-TNF) therapies are the most widely used biologic drugs for treating immune-mediated diseases, but repeated administration can induce the formation of anti-drug antibodies. The ability to identify patients at increased risk for development of anti-drug antibodies would facilitate selection of therapy and use of preventative strategies. METHODS: We performed a genome-wide association study to identify variants associated with time to development of anti-drug antibodies in a discovery cohort of 1240 biologic-naïve patients with Crohn's disease starting infliximab or adalimumab therapy. Immunogenicity was defined as an anti-drug antibody titer ≥10 AU/mL using a drug-tolerant enzyme-linked immunosorbent assay. Significant association signals were confirmed in a replication cohort of 178 patients with inflammatory bowel disease. RESULTS: The HLA-DQA1*05 allele, carried by approximately 40% of Europeans, significantly increased the rate of immunogenicity (hazard ratio [HR], 1.90; 95% confidence interval [CI], 1.60-2.25; P = 5.88 × 10-13). The highest rates of immunogenicity, 92% at 1 year, were observed in patients treated with infliximab monotherapy who carried HLA-DQA1*05; conversely the lowest rates of immunogenicity, 10% at 1 year, were observed in patients treated with adalimumab combination therapy who did not carry HLA-DQA1*05. We confirmed this finding in the replication cohort (HR, 2.00; 95% CI, 1.35-2.98; P = 6.60 × 10-4). This association was consistent for patients treated with adalimumab (HR, 1.89; 95% CI, 1.32-2.70) or infliximab (HR, 1.92; 95% CI, 1.57-2.33), and for patients treated with anti-TNF therapy alone (HR, 1.75; 95% CI, 1.37-2.22) or in combination with an immunomodulator (HR, 2.01; 95% CI, 1.57-2.58). CONCLUSIONS: In an observational study, we found a genome-wide significant association between HLA-DQA1*05 and the development of antibodies against anti-TNF agents. A randomized controlled biomarker trial is required to determine whether pretreatment testing for HLA-DQA1*05 improves patient outcomes by helping physicians select anti-TNF and combination therapies. ClinicalTrials.gov ID: NCT03088449.

Association of adalimumab trough concentrations and treatment response in patients with juvenile idiopathic arthritis.

OBJECTIVE: The objective of this study was to assess the relationship between adalimumab trough concentrations and treatment response in paediatric patients with JIA. METHODS: This was a monocentric cohort study of JIA patients treated with adalimumab. Clinical data and samples were collected during routine follow-up. Adalimumab trough concentrations were quantified by a novel liquid chromatography-tandem mass spectrometry assay. Anti-adalimumab antibodies were measured in samples with trough concentrations of ≤5mg/l. Disease activity was evaluated using the clinical Juvenile Arthritis DAS with 71-joint count (cJADAS71). Response to adalimumab was defined according to recent international treat-to-target guidelines. RESULTS: A total of 35 adalimumab trough samples were available from 34 paediatric patients with JIA. Although there was no significant difference in adalimumab dose, trough concentrations were significantly lower in patients with secondary failure [median 1.0 mg/l; interquartile range (IQR) 1.0-5.3] compared with patients with primary failure (median 13.97 mg/l; IQR 11.81-16.67) or an adequate response (median 14.94 mg/l; IQR 10.31-16.19) to adalimumab. CONCLUSION: Adalimumab trough concentrations were significantly lower in JIA patients with secondary failure compared with patients with primary failure or an adequate response to adalimumab. Our results suggest that trough concentration measurements could identify JIA patients who require increased adalimumab doses to achieve or maintain therapeutic drug concentrations.

Proactive Monitoring of Adalimumab Trough Concentration Associated With Increased Clinical Remission in Children With Crohn's Disease Compared With Reactive Monitoring.

BACKGROUND & AIMS: Proactive monitoring of drug trough concentrations and antibodies against drugs might help determine whether patients are likely to respond to treatment and increase efficacy. We investigated whether proactive drug monitoring is associated with higher rates of clinical remission in pediatric patients with Crohn's disease (CD). METHODS: We performed a nonblinded, randomized controlled trial of 78 children with CD (6-18 years old; 29% female; mean age, 14.3 ± 2.6 years) who had not received prior treatment with a biologic agent but had responded to adalimumab induction therapy, under scheduled monitoring of clinical and biologic measures (based on clinical factors and levels of C-reactive protein and fecal calprotectin), at pediatric gastroenterology units in Israel from July 2015 through December 2018. The patients were randomly assigned to groups that received proactive monitoring (trough concentrations measured at weeks 4 and 8 and then every 8 weeks until week 72, n = 38) or reactive monitoring (physicians were informed of trough concentrations after loss of response, n = 40). In both groups, doses and intervals of adalimumab were adjusted to achieve trough concentrations of 5 µg/mL. The primary endpoint was sustained corticosteroid-free clinical remission at all visits (week 8 through week 72). RESULTS: The primary endpoint was achieved by 31 children (82%) in the proactive group and 19 children (48%) in the reactive group (P = .002). Sixteen patients in the proactive monitoring group (42%) achieved a composite outcome of sustained corticosteroid-free remission, C-reactive protein ≤0.5 mg/dL, and level of fecal calprotectin ≤150 µg/g compared with 5 patients in the reactive monitoring group (12%) (P = .003). By week 72 of treatment, 33 patients in the proactive monitoring group had received adalimumab intensification (87%) compared with 24 patients in the reactive monitoring group (60%) (P = .001). CONCLUSIONS: In a randomized controlled trial of pediatric patients with CD, we found that proactive monitoring of adalimumab trough concentrations and adjustment of doses and intervals resulted in significantly higher rates corticosteroid-free clinical remission than reactive monitoring (measuring trough concentration after loss of response). Clinicaltrials.gov no.: NCT02256462.

Adalimumab Immunogenicity Is Negatively Correlated with Anti-Hinge Antibody Levels in Patients with Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA) are frequently treated with anti-tumor necrosis factor-α immunoglobulin therapy but develop neutralizing antibodies against these drugs, necessitating therapeutic monitoring of drug concentrations and anti-drug antibodies. Patients with RA have multiple factors related to their autoimmune disposition that interfere with conventionally used methods to detect anti-drug antibodies. Currently deployed analytical methods have significant limitations that hinder clinical interpretation and/or routine use, and no method can detect immunogenicity and drug levels simultaneously to provide clinically meaningful recommendations. Given these limitations, the objective of this study was to identify sources of and associations with assay interference in patients with RA. We designed a modular immunogenicity and drug concentration detection technology to identify the factors that interfere with the detection of adalimumab and anti-adalimumab antibodies in a cohort of 206 patients with RA. Patients were included from the University of Pittsburgh Rheumatoid Arthritis Comparative Effectiveness Research registry. In this cohort, we analyzed clinical and plasma factors associated with anti-adalimumab and anti-hinge antibodies. A novel flow cytometry-based assay was developed and validated that simultaneously measures adalimumab and anti-adalimumab antibody concentrations, overcoming many of the interference factors that are limitations of conventional assays, including anti-fragment crystallizable (Fc) and anti-hinge antibodies. C-reactive protein (P = 0.035), Disease Activity Score-28 (DAS28) score (P = 0.002), and disease activity category (P = 0.009) were significantly associated with anti-adalimumab antibodies but not with anti-hinge antibodies (P > 0.05). Anti-hinge antibodies were inversely associated with drug-neutralizing antibodies (P = 0.002). In patients with RA, anti-hinge antibodies may have a potential protective effect against the development of anti-adalimumab antibodies. SIGNIFICANCE STATEMENT: Using a novel cytometric assay that simultaneously measures drug and anti-drug antibodies, we overcame many interferences that hinder the clinical interpretation of adalimumab immunogenicity testing. Our investigation in patients with RA demonstrated that immunogenicity impaired the pharmacological action of adalimumab via analysis of RA disease severity markers. We also demonstrate that patients with anti-hinge antibodies had lower anti-adalimumab antibody levels and decreased drug neutralization. Our results suggest that anti-hinge antibodies can predict adalimumab immunogenicity before the start of therapy.

Therapeutic drug monitoring of adalimumab in RA: no predictive value of adalimumab serum levels and anti-adalimumab antibodies for prediction of response to the next bDMARD.

BACKGROUND: After adalimumab treatment failure, tumour necrosis factor inhibition (TNFi) and non-TNFi biological disease-modifying anti-rheumatic drugs (bDMARDs) are equally viable options on a group level as subsequent treatment in rheumatoid arthritis (RA) based on the current best evidence synthesis. However, preliminary data suggest that anti-adalimumab antibodies (anti-drug antibodies, ADA) and adalimumab serum levels (ADL) during treatment predict response to a TNFi as subsequent treatment. OBJECTIVE: To validate the association of presence of ADA and/or low ADL with response to a subsequent TNFi bDMARD or non-TNFi bDMARD. Sub-analyses were performed for primary and secondary non-responders. METHODS: A diagnostic test accuracy retrospective cohort study was done in consenting RA patients who discontinued adalimumab after >3 months of treatment due to inefficacy and started another bDMARD. Inclusion criteria included the availability of (random timed) serum samples between ≥8 weeks after start and ≤2 weeks after discontinuation of adalimumab, and clinical outcome measurements Disease Activity Score in 28 joints - C-reactive protein (DAS28-CRP) between 3 to 6 months after treatment switch. Test characteristics for EULAR (European League Against Rheumatism) good response (DAS28-CRP based) after treatment with the next (non-)TNFi bDMARD were assessed using area under the receiver operating characteristic and sensitivity/specificity. RESULTS: 137 patients were included. ADA presence was not predictive for response in switchers to a TNFi (sensitivity/specificity 18%/75%) or a non-TNFi (sensitivity/specificity 33%/70%). The same was true for ADL levels in patients that switched to a TNFi (sensitivity/specificity 50%/52%) and patients that switched to a non-TNFi (sensitivity/specificity 32%/69%). Predictive value of ADA and ADL were similar for both primary and secondary non-responders to adalimumab. CONCLUSIONS: In contrast to earlier research, we could not find predictive value for response to a second TNFi or non-TNFi for either ADA or random timed ADL.

Development of Label-Free Immunoassays as Novel Solutions for the Measurement of Monoclonal Antibody Drugs and Antidrug Antibodies.

BACKGROUND: Immunoassays based on label-free technologies (label-free immunoassay [LFIA]) offer an innovative approach to clinical diagnostics and demonstrate great promise for therapeutic drug monitoring (TDM) of monoclonal antibody (mAb) drugs. An LFIA measures immunocomplex formation in real time and allows for quantification on initial binding rate, which facilitates fast measurement within a few minutes. METHODS: Based on thin-film interferometry (TFI) technology, open-access LFIAs were developed for the quantification of the mAb drugs adalimumab (ADL) and infliximab (IFX) and for the detection of the antidrug antibodies (ADAs) to the mAb drugs (ADL-ADAs and IFX-ADAs). RESULTS: The LFIAs for active mAb drugs (ADL and IFX) and for ADAs (ADL-ADAs and IFX-ADAs) were validated. The analytical measurement range (AMR) for both ADL and IFX was from 2 to 100 µg/mL. The AMR for ADL-ADAs was from 5 to 100 µg/mL and for IFX-ADAs was 10 to 100 µg/mL. In the comparison of LFIAs and reporter gene assays, the correlation coefficient was 0.972 for the quantification of ADL and 0.940 for the quantification of IFX. The concordance rate was 90% for the detection of ADL-ADAs and 76% for the detection of IFX-ADAs. CONCLUSIONS: The LFIAs for active mAb drugs and ADAs were appropriate for the TDM of ADL and IFX. The TFI technology has unique advantages compared with other technologies used for the measurement of mAb drugs. Label-free technologies, especially those allowing for open-access LFIAs, have great potential for clinical diagnostics.

Development of anti-drug monoclonal antibody panels against adalimumab and infliximab.

The generation of anti-drug antibodies (ADAs) is one of the most serious problems in therapy using monoclonal antibodies (mAbs), because ADAs can impact the pharmacokinetics, efficacy, and safety of mAbs. It is therefore important to detect the generated ADAs in patients. For the appropriate detection of ADAs, methods that detect various types of ADAs (e.g., low- and high-affinity ADAs) are needed, but since there are no adequate reference preparations of ADAs relevant to human ADAs in most cases, it is difficult to determine whether or not the developed methods have enough analytical performance. Here, we developed human-rat chimeric ADA panels against the anti-TNF-α therapeutic antibodies infliximab and adalimumab. The developed ADA panels consist of 7 (for infliximab) and 11 (for adalimumab) ADAs with various binding characters, and most of the ADAs are neutralizing antibodies. Using these ADA panels, we compared the detectability of model methods, i.e., binding assays using SPR, BLI, and ECL, and a cell-based assay to detect neutralization activity. Since we obtained ADAs showing low and high responses with the various methods, the ADA panels we developed were shown to be useful for the development of ADA assays.

Validation of a de-immunization strategy for monoclonal antibodies using cynomolgus macaque as a surrogate for human.

The immunogenicity of biotherapeutics presents a major challenge during the clinical development of new protein drugs including monoclonal antibodies. To address this, multiple humanization and de-immunization techniques that employ in silico algorithms and in vitro test systems have been proposed and implemented. However, the success of these approaches has been variable and to date, the ability of these techniques to predict immunogenicity has not been systematically tested in humans or other primates. This study tested whether antibody humanization and de-immunization strategies reduce the risk of anti-drug antibody (ADA) development using cynomolgus macaque as a surrogate for human. First human-cyno chimeric antibodies were constructed by grafting the variable domains of the adalimumab and golimumab monoclonal antibodies onto cynomolgus macaque IgG1 and Igκ constant domains followed by framework germlining to cyno to reduce the xenogenic content. Next, B and T cell epitopes and aggregation-prone regions were identified using common in silico methods to select domains with an ADA risk for additional modification. The resultant engineered antibodies had a comparable affinity for TNFα, demonstrated similar biophysical properties, and exhibited significantly reduced ADA levels in cynomolgus macaque compared with the parental antibodies, with a corresponding improvement in the pharmacokinetic profile. Notably, plasma concentrations of the engineered antibodies were quantifiable through 504 hours (chimeric) and 840 hours (germlined/de-immunized), compared with only 336 hours (adalimumab) or 336-672 hours (golimumab). The results point to the significant value in the investment in these engineering strategies as an important guide for monoclonal antibody optimization that can contribute to improved clinical outcomes.

Immunogenicity of biologic agents in juvenile idiopathic arthritis: a systematic review and meta-analysis.

OBJECTIVE: The clinical impact of anti-drug antibodies (ADAbs) in paediatric patients with JIA remains unknown. This systematic review and meta-analysis aimed to summarize the prevalence of ADAbs in JIA studies; investigate the effect of ADAbs on treatment efficacy and adverse events; and explore the effect of immunosuppressive therapy on antibody formation. METHODS: PubMed, Embase and the Cochrane Library were systematically searched to identify relevant clinical trials and observational studies that reported prevalence of ADAbs. Studies were systematically reviewed in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses and appropriate proportional and pairwise meta-analyses were performed. RESULTS: A total of 5183 references were screened; 28 articles, involving 26 studies and 2354 JIA patients, met eligibility criteria. Prevalence of ADAbs ranged from 0% to 82% across nine biologic agents. Overall pooled prevalence of ADAbs was 16.9% (95% CI, 9.5, 25.9). Qualitative analysis of included studies indicated that antibodies to infliximab, adalimumab, anakinra and tocilizumab were associated with treatment failure and/or hypersensitivity reactions. Concomitant MTX uniformly reduced the risk of antibody formation during adalimumab treatment (risk ratio 0.33; 95% CI 0.21, 0.52). CONCLUSION: The association of ADAbs with treatment failure and hypersensitivity reactions indicates their clinical relevance in paediatric patients with JIA. Based on our findings, we recommend a preliminary course of action regarding immunogenicity of biologic agents in patients with JIA. Further strategies to predict, prevent, detect and manage immunogenicity could optimize treatment outcomes and personalize treatment with biologic therapies.

Detection of anti-adalimumab antibodies in a RA responsive cohort of patients using three different techniques.

Reliable monitoring of clinical relevant anti-drug antibodies is fundamental in the follow-up of patients under adalimumab treatment. The aim of this study is to compare anti-adalimumab antibodies by using three methods based on different technologies. A cross-sectional study was performed in 50 patients with rheumatoid arthritis (RA) treated with adalimumab. Anti-adalimumab antibodies were detected in patients' sera by different techniques: bridging ELISA, reporter gene assay (RGA), and surface plasmon resonance (SPR). Results showed that all methods recognized anti-adalimumab antibodies and the percentage of positives fluctuated among the assays. Five (10%) of the 50 patients were positive in ELISA, 4 (8%) in RGA, and 6 (12%) in SPR. Among positive patients, 4 were positive in the three assays, one patient uniquely in ELISA, and two in SPR. Spearman correlation between ELISA and RGA showed good agreement (Spearman r = 0.800). No correlation between RGA and SPR was observed (Spearman r = 0.108). Similar results were obtained between ELISA and SPR (Spearman r = - 0.241). Summarizing, ELISA, RGA and SPR recognized anti-adalimumab antibodies in few RA patients, showing good agreement among the methodology employed. On the other hand, differences observed between SPR and ELISA or RGA highlight the relevance of the employed technologies in anti-drug antibody identification.

The transient production of anti-TNF-α antibody Adalimumab and a comparison of its characterization to the biosimilar Cinorra.

Recombinant antibodies have emerged over the last few decades as the fastest growing class of therapeutic proteins for autoimmune diseases. Post-translation modifications of antibodies produced by human cell lines are highly consistent with those existing in natural human proteins and this is a major advantage of utilizing these cell lines. Cinorra is a biosimilar form of the antibody Adalimumab, which is an antagonist of TNF-α used for the treatment of autoimmune diseases. Adalimumab and Cinorra were produced by stable expression from CHO cells. The aim of this study was to select HEK cells as a host for producing Adalimumab to reveal whether the antibody produced by this human-derived cell line has similar characterization to Cinorra. Adalimumab was transiently produced in HEK-293T cells, characterized and analyzed for its properties. Circular dichroism spectroscopy confirmed a strong structural similarity of the expressed antibody with Cinorra. Likewise its binding activity and kinetic affinity to TNF-α (EC50 = 416.5 ng/ml, KD = 3.89 E-10 M,) were highly similar to that of Cinorra (EC50 = 421.2 ng/ml and KD = 3.34 E-10 M,). Additionally there was near identical neutralization of TNF-α-mediated cellular cytotoxicity (IC50 of the expressed = 4.93 nM; IC50 of Cinorra = 4.5 nM). Results indicate that Adalimumab produced by HEK-293T cells possesses a similarly efficient function and biological activity to Cinorra. Consequently, human-derived host cells with human post-translational modifications might potentially provide a basis for the development of Adalimumab with pharmaceutical properties for research and therapeutic use.

Antidrug antibody detection for adalimumab depends on the type of assay used: an experimental approach to help clinicians interpret diagnostic data.

OBJECTIVES: To compare different methods of antidrug antibody (ADA) against adalimumab detection in ankylosing spondylitis (AS) patients and the impact of ADA on adalimumab drug levels and mean ASDAS-CRP. METHODS: We used the acid-dissociation-radioimmunoassay (ARIA), antidrug-binding-test (ABT) and a bridging Enzyme-linked Immunosorbent Assay (ELISA) to detect ADA at 4, 12 and 24 weeks of treatment. Patients were divided into groups; all assays negative (All-neg), only ARIA positive (ARIA-only-pos), ARIA and ABT positive, bridging ELISA negative (ARIA/ABT-double-pos) and all assays positive (All-pos). RESULTS: Eighty-three consecutive AS patient were included. At week 4, 18% compared to 11% and 0% of the patients tested positive for ADA in the ARIA, ABT and bridging ELISA, respectively. At week 12 and 24, cumulative 52% and 69% patients tested positive in the ARIA, compared to 27% and 30% patients in the ABT and 2% patients in the bridging ELISA. Adalimumab levels between All-neg and ARIA-only-pos were 9.1 (5.5-12.5) and 8.5 (5.7-12.3). Drug levels differed between ARIA/ABT-double-pos (2.7 (1.3-4.4)) and All-neg (9.1 (5.5-12.5)). All-pos patients had undetectable drug levels. Mean ASDAS-CRP at week 24 differs between All-neg (1.9 (±1.2)), and All-pos (3.8 (±1.9)) and ARIA/ABT-double-pos (2.0 (±1.1)) and All-pos. CONCLUSIONS: The majority of AS patients had detectable ADA against adalimumab in the ARIA. The ARIA detects more ADA compared to the less drug tolerant ABT and bridging ELISA. The clinical relevance depends on the impact on the bio-availability of the drug. A drug level measurement therefore helps to interpret ADA data regardless of type of assay used.

Crucial Role for Immune Complexes but Not FcRn in Immunization against Anti-TNF-α Antibodies after a Single Injection in Mice.

The immunogenicity of infliximab and adalimumab is a major concern because patients may develop Abs also called antidrug Abs (ADA), directed against these anti-TNF-α Abs after just a few weeks of treatment. These ADAs can lead to a decrease in biologic concentration, which is associated with lower treatment efficacy. Our aim was to study the involvement of immune complexes and neonatal Fc receptor (FcRn) in the emergence of ADAs in the case of anti-TNF-α Abs. Wild type and FcRn knockout mice were injected once with either infliximab or adalimumab, alone or preincubated with TNF-α. Adalimumab cross-reacts with murine TNF-α whereas infliximab is species specific. When injected alone, only adalimumab elicited a humoral response. By preforming immune complexes with TNF-α, an anti-infliximab response was elicited. Surprisingly, both wild type and FcRn knockout mice were able to mount an immune response against anti-TNF-α Abs, suggesting that immune complexes are a major determinant of this immunization.

Management of Anti-drug Antibodies to Biologic Medications in Children With Inflammatory Bowel Disease.

BACKGROUND: Treatment of pediatric inflammatory bowel disease (IBD) with monoclonal anti- tumor necrosis factor-alpha (TNFα) can result in immunogenicity and formation of anti-drug antibodies (ADAs). ADAs are associated with loss of clinical response and worsening disease progression. Data examining treatment interventions to overcome ADA in pediatric patients with IBD are lacking. RESULTS: Medical records were reviewed from 234 children and adolescents with IBD treated with infliximab or adalimumab who underwent therapeutic drug monitoring (626 tests). All patients who had detectable antibodies were further analyzed. A total 58 patients (24.8%) developed ADA while being treated with infliximab or adalimumab. The incidence of antibody development was 12.9 per 100 person-years of anti-TNF treatment. Twenty-eight patients underwent dose optimization and 54% had undetectable ADA on follow-up monitoring. The mean duration of antibody suppression was 16.8 ±â€Š10.9 months in those who were successfully suppressed with optimization. Patients who switched to a second anti-TNF medication were not more likely to develop antibodies to the second agent. CONCLUSIONS: With limited therapies for IBD and the chronicity of the disease, we advocate salvage of the current anti-TNF through dose optimization in pediatric patients with antibody level <10 U/mL.

Generic immune complex assay for detection of murine anti-drug-antibodies in complex with human IgG.

Rapid and versatile methods are needed for evaluation of immunogenicity in early safety studies. The present work presents a generic, simple and easy to use sandwich enzyme-linked immunosorbent assay for quasi-quantitative measurement of circulating immune complexes (CICs) formed by anti-drug antibodies (ADAs) in complex with human IgG in mouse plasma. The assay is suitable for evaluating the presence of in vivo formed CICs in mice exposed to human IgG antibodies independent of target and IgG subtype. The assay is established using commercially available antibodies, and calibrated using CIC mimics based on bis(sulfosuccinimidyl)suberate conjugated human and mouse IgG. The development and qualification process of the generic methodology is described and include acceptance criteria, stability, sensitivity, drug tolerance, spike recovery, precision and cut point determination. In order to demonstrate assay performance, its use is exemplified by quantifying CICs in mice administered with a fully human anti-TNF-α IgG1 antibody (adalimumab) or a humanized anti-trinitrophenol (TNP) IgG4 antibody. Results show a well-qualified reproducible assay set-up with adequate sensitivity, easy discrimination between positive and negatives and quasi-quantitative measurement of ADA-human IgG CICs in mice administered with each of two different human/humanized IgG antibodies.

Clinical relevance of monitoring serum adalimumab levels in axial spondyloarthritis.

Our aim was to assess the relationship between serum adalimumab levels, anti-drug antibodies (ADA) and disease activity in patients with axial spondylarthritis (SpA). We have carried out a single-centre cross-sectional study. adalimumab and ADA levels were analysed with ELISA and correlated with SpA activity using BASDAI and ASDAS scores. Adalimumab cut-off value was calculated to discriminate inactive disease/low disease activity (BASDAI < 4; ASDAS < 2.1) from moderate/high disease activity (BASDAI ≥ 4; ASDAS ≥ 2.1), using a receiver operating characteristic (ROC) curve. Up to January 2016, 51 consecutive patients were included. The median (range) age was 46.6 (18-68) and 47.1% were women. ADA prevalence was 27.5%, with none detected in the 21.6% receiving concomitant disease-modifying antirheumatic drugs (DMARDs) (p = 0.021). Adalimumab level was normal (> 3 mg/l) in 36 patients (70.6%), all without ADA. Fifteen patients (29.4%) had subtherapeutic adalimumab levels (< 3 mg/l), with ADA in 14 (93%). Median adalimumab (mg/l) was significantly higher in patients with inactive disease/low disease activity: BASDAI < 4 vs ≥ 4: 9.5 vs 2.6 (p < 0.01); ASDAS-CRP < 2.1 vs ≥ 2.1: 9.3 vs 0.3 (p < 0.001); ASDAS-ESR < 2.1 vs ≥ 2.1: 9.9 vs 3.0 (p < 0.001), and this finding was consistent with the result of the multivariate model. Patients with inactive disease/low disease activity presented significantly lower ADA levels. The adalimumab level cut-offs and area under the curve (AUC) obtained in the ROC curves were: ASDAS-CRP (< 2.1) 4.6 mg/l (AUC 81.2%; 95% CI 67.5-94.9; p < 0.001); ASDAS-ESR (< 2.1) 7.7 mg/l (AUC 82.4%; 95% CI 69.3-95.5; p < 0.001); BASDAI (< 4) 6.4 mg/l (AUC 73.5%; 95% CI 58.6-88.3; p < 0.01). In conclusion, presence of ADA in axial SpA patients treated with adalimumab was associated with lower serum drug levels. ADA levels were lower and adalimumab levels were higher in patients with inactive disease/low disease activity based on BASDAI and ASDAS indices. Concomitant treatment with MTX reduces de likelihood of finding ADA. Serum adalimumab levels above 4.6 mg/l are recommended to avoid compromising efficacy.