https://orcid.org/0000-0003-0735-4648
Abstract
We investigated the impact of concomitant MTX on ustekinumab (UST) levels and antidrug antibody (ADA) formation in PsA and evaluated consequences in pharmacodynamics and pharmacokinetics.
We conducted a post-hoc analysis on 112 PsA serum samples of subjects treated with open-label UST and either concomitant MTX (UST/MTX, n = 58) or placebo (UST/pbo, n = 54) obtained in a randomized (1:1), double-blind, multicentre trial. A validated antibody-binding-based multitiered testing was used to detect ADA and ADA with neutralizing capacity (nADA). The impact of MTX on UST immunogenicity was analysed by comparison of UST/pbo with UST/MTX cohorts at different time points. Patient- and disease-related predispositions for ADA formation were investigated with multiple linear regression analysis. Immunogenicity impact on pharmacokinetics, safety and efficacy was determined by cohort comparison between patients with and without ADA formation.
Over 52 weeks, 11 UST/pbo- and 19 UST/MTX-treated patients developed ADA (P > 0.05). In the UST/pbo cohort, the visit-dependent UST levels were in the range of 0.047 (0.05) –0.110 (0.07) µg/ml overall, and 0.037 (0.04)–0.091 (0.08) µg/ml in ADA-confirmed subjects. In UST/MTX-treated patients, the UST levels exhibited an intervisit variation in the range of 0.0502 (0.04)–0.106 (0.07) µg/ml overall and 0.029 (0.03)–0.097 (0.07) µg/ml in ADA positive subjects (P > 0.05). At week 52, ADA-confirmed patients did not differ significantly (P > 0.05) in safety or clinical outcomes from ADA-negative patients.
Concomitant MTX had no significant impact on UST immunogenicity. Furthermore, ADA formation was not associated with impairments in UST safety, efficacy or trough levels.
ClinicalTrials.gov, https://clinicaltrials.gov, NCT03148860.
Concomitant methotrexate treatment had no impact on ustekinumab immunogenicity in psoriatic arthritis.
Antidrug antibody formation was not associated with impairments in ustekinumab safety, efficacy or trough levels.
Introduction
The introduction of biopharmaceuticals in targeted treatment strategies for rheumatic diseases has considerably improved patient outcomes [1]. One obstacle of biologics, however, is their potential immunogenicity, which might limit sustained efficacy. Despite improved manufacturing procedures including the production of genetically humanized or even fully human recombinant antibodies [2], patients may still elicit immune responses towards their biologic DMARDs (bDMARDs) during prolonged periods of repetitive therapeutic application. This drug-related immunogenicity includes the formation of antidrug antibodies (ADA) that can impair the biotherapeutics’ mode of action through alterations in pharmacodynamics and pharmacokinetics [3].
As the number of biotherapeutics introduced into the market is expected to further increase over the next few years, immunogenicity and its implications on drug safety and treatment outcome will remain a relevant parameter for drug evaluation and treatment recommendations. In this regard, previous studies have already demonstrated the significance of drug-associated but also the impact of disease- and patient-related factors [4]. While some studies report increased safety hazards or impaired pharmacokinetic properties due to immunogenicity, other data do not support any negative impact of ADA formation on biologic treatment [5–8].
Thus, the broad usage of biotherapeutics in different disease entities with the growing availability of biosimilars [9] will remain an increasing challenge for integration of data on immunogenicity, interpretation of clinical information and demands for a personalized treatment approach.
Most of the present knowledge on the clinical impact of ADA formation has been obtained from studies in RA and accordingly extrapolated from these well-established findings [10, 11] to other disease entities in different therapeutic contexts. However, the validity of this approach is limited since it neglects disease- and drug-specific predispositions for ADA formation. This is illustrated by contradicting data for the impact of add-on conventional synthetic DMARD treatment to bDMARDs in the prevention of ADA formation in different disease entities. While a meta-analysis of 936 patients with RA or inflammatory bowel disease by Garcês et al. [12] revealed that ADA formation was mitigated by concomitant immunomodulators such as azathioprine, MTX or mercaptopurine, another study in psoriasis patients illustrated that add-on MTX remained completely ineffective in reducing the immunogenicity of the TNF inhibitor adalimumab [13].
Preliminary data on the immunogenicity of ustekinumab (UST) are available from randomized trials in PsA treatment. UST is an anti-IL-12/23 p40 monoclonal antibody that is, among others, approved for the treatment of PsA, inflammatory bowel disease and psoriasis [14]. In PHOENIX 1 and 2 [15, 16] only a small proportion (4.4%) of PsA patients developed UST-ADA. Here, patients with lower UST doses were reported to have higher ADA rates and a poorer psoriasis improvement, without stating causality. There are also inconclusive data regarding the clinical relevance of concomitant MTX as a mitigation tool in immunogenicity. While PsA patients in the PSUMMIT treated with UST and concomitant MTX showed reduced immunogenicity compared with subjects with monotherapy, data on safety and efficacy did not differ between the two cohorts [17].
To date, no study has specifically addressed the impact of MTX on UST immunogenicity. Consequently, it is of high clinical relevance to determine whether concomitant MTX has a clinically relevant impact on UST immunogenicity in PsA patients. The present study is the first to investigate this question by applying a self-developed validated multitiered UST immunogenicity testing approach in patients with PsA [8, 18]. Our methods were shown to be sensitive, reproducible and valid in detecting and quantifying UST, UST-specific ADA and neutralizing ADA (nADA), respectively. We also demonstrated that MTX did not disturb measurements and that the methods fulfilled the requirements for immunogenicity testing set by regulatory agencies [18–20].
In this post-hoc analysis of UST-treated PsA patients, we transferred a validated multitiered approach to investigate the impact of UST immunogenicity on levels, efficacy, risk and treatment tolerance. Our research aims to identify patient-, treatment- or disease-related characteristics that facilitate ADA formation and identifies patient groups at higher risk for clinically relevant immunogenicity. One of the major aims of this work was also to investigate whether concomitant MTX treatment or pre-treatment has an impact on the initiation or the degree of UST immunogenicity in PsA patients. Moreover, our analysis also provides the groundwork for future risk detection and mitigation.
Methods
Patients and ethical considerations
The study protocol of the MUST study was approved by the ethics committee of Goethe University (Ethikkommission des Fachbereichs Medizin der Goethe Universität, approval number: 199/15), and by each local ethics committee at participating sites. Preliminarily data of the MUST (Clinicaltrials.gov identifier: NCT03148860) study including patient eligibility and study design have been reported previously [21]. All patients gave written informed consent prior to participation. Briefly, patients naïve to UST and with active PsA, defined as ≥4 tender and swollen joints, and a 28-joint DAS (DAS28) ≥ 3.2 at screening were randomly assigned to receive open-label UST and either concomitant 15 mg weekly MTX (UST/MTX cohort) or concomitant placebo (UST/pbo cohort). UST 45 mg or 90 mg, in patients with a body weight >100 mg, was administered subcutaneously at weeks 0, 4, and then every 12 weeks.
Samples
In total, we analysed samples of 112 patients. Blood samples were obtained prior to UST application on the same day as scheduled injection. Samples for baseline analysis were taken at enrolment, before the first UST administration (blank probes). Serum was separated by centrifugation at 4°C and 1000 g for 15 min. Samples were then immediately stored at −20°C until analysis.
Serum samples for surface plasmon resonance spectroscopy (SPR) measurements were diluted 1:10 with HISPEC assay diluent (Bio‐Rad Laboratories Inc., Hercules, CA, USA) and 10% non‐specific binding (NSB)‐Reducer (GE Healthcare, Chicago, IL, USA). ELISA measurements were conducted with samples diluted 1:5 with PBS and 0.05% Tween 20.
Immunogenicity testing
The immunogenicity testing used was fully validated and described before [18]. The approach included detection and confirmation of UST, UST-specific ADA, and nADA in a multitiered manner as recommended by the FDA guidance for immunogenicity testing [19]. UST (Stelara) for calibration and coating was provided by Janssen-Cilag GmbH (Neuss, Germany). The human anti‐UST antibody (HCA210) used for standardization and UST detection was obtained from Bio‐Rad Laboratories.
Samples were screened for ADA detection (limit of detection [LoD] = 0.0034 µg/ml) in a sandwich-ELISA. Plates were coated with UST at 1 µg/ml to screen for ADA. Fluorescence signals were measured with the EnSpire Multilabel Plate Reader (Perkin‐Elmer, Waltham, MA, USA) with excitation and emission wavelength set at 325 and 420 nm, respectively. Positively screened samples were ADA confirmed in a SPR analysis with the Biacore T200 (GE Healthcare). UST binding in SPR (limit of quantification [LoQ] = 0.144 µg/ml) was assessed with IL-12 (50 µg/ml) immobilized for 840 s at a 5 µl/min flow rate on a CM5-Chip (GE Healthcare). Confirmation for ADA (LoQ = 0.08 µg/ml) was performed on a CM5‐Chip (GE Healthcare) with UST (180 µg/ml) immobilized for 840 s at a 5 µl/min flow rate. Confirmed ADA were then classified for non-neutralizing ADA and nADA in an ELISA.
Impact of ADA on efficacy and safety
To determine the impact of ADA formation on UST efficacy and through levels, patients were stratified by Disease Activity in Psoriatic Arthritis (DAPSA) score at week 52. Here, a DAPSA <4 was defined as remission, while a score >4 was considered as active disease [22]. Cohorts of patients with confirmed ADA at any time point and ADA negative patients were further compared regarding DAS28 [23] and safety data. A DAS28 ≥ 3.2 indicated active disease.
Statistical analysis
For statistical analysis, GraphPad Prism software version 9 (GraphPad Software Inc., San Diego, CA, USA) was used. The Kolmogorov–Smirnov test was used for normality of distribution. Samples were screened positive for ADA if the measured signal was above the 90% confidence interval of the upper 95th percentile of the blank probes [19].
We compared mean UST and ADA levels with 95% CI in patients receiving UST and MTX with the reference group at weeks 0, 4, 16, 40 and 52 with a two-way ANOVA with Šidák correction. Patients detected and confirmed with ADA at any time point during the 52-analysis period and patients without confirmed ADA were compared regarding cohort characteristics. Differences in continuous variables were calculated with a two-sided independent Student’s t-test or Fisher’s exact test for categorical data. Odds ratios (OR) and corresponding 95% CI were calculated to estimate the risk of categorical cohort characteristics associated with ADA detection. Significance for testing was set at P < 0.05. Multiple linear regression was used to identify independent predictors that could be associated with ADA formation. To analyse clinical impact, UST through levels were compared between patients with a DAPSA ≤ 4 and a DAPSA > 4 at week 52 for patients with confirmed ADA at any time and ADA negative patients.
Results
Incidence of ADA, non-neutralizing ADA and nADA
One of the main objectives of this study was to analyse the impact of concomitant MTX on UST immunogenicity. We therefore analysed UST and ADA levels in samples from patients treated with UST over 52 weeks and compared those together with clinical parameters between the UST/MTX and UST/pbo cohort.
Samples were screened for ADA with ELISA, as it allowed detection at lower ADA levels (LoD = 0.0034 µg/ml) compared with SPR analysis (LoD = 0.086 µg/ml). Our selection of the confirmation assay was based on previous work that showed more precise and accurate measurements especially for low-affinity analytes with SPR than in ELISA [18]. To take the UST dosing interval that influences serum levels into consideration, ADA rates were compared for each visit separately.
In total, we analysed samples of 112 participants; 58 patients received open-label UST and concomitant MTX, and 54 patients in the placebo cohort were treated with UST and placebo. Samples ADA positive in the screening ELISA and quantifiable in the SPR assay were considered confirmed and were classified in a functional ELISA for nADA and non-neutralizing ADA.
First, we investigated whether the treatment of MTX affects the formation of ADA and/or nADA. Over the course of 52 weeks, 19 patients (32.7%) of the UST/MTX cohort developed ADA, of which two patients had non-neutralizing ADA (3.4%) while the other 17 were classified as nADA. In the UST/pbo cohort, 11 patients (20.3%) developed ADA, of which 10 were positive for nADA. Comparison in ADA prevalence showed no significant difference (Fig. 1) between UST/MTX- and UST/pbo-treated patients (P > 0.05).
Prevalence of ADA. Prevalence (%) of confirmed ADA in weeks (w) 4, 16, 40 and 52 in patients treated with open label ustekinumab (UST) and double-blind placebo (UST/pbo, n = 54) or MTX (UST/MTX, n = 58). ADA: antidrug antibodies
Multiple linear regression analysis and concomitant MTX
The multiple linear regression analysis of concomitant MTX, sex, UST dosage, age, weight at baseline and weight change at week 52 were not significant predictor variables using ADA detection until week 52 as the dependent variable at a 5% significance level (Supplementary Table S1, available at Rheumatology online). The multiple linear regression analysis showed a predictive R2 of 0.031. Our analysis showed no increased risk of ADA detection in patients with concomitant or pre-treatment with MTX (Table 1).
Differences in cohort characteristics and odds ratios (OR) in patients with and without confirmed ADA
| Cohort characteristic | No ADA detection (n = 82) | Confirmed ADA (n = 30) | All patients (n = 112) | 95% CI | P-value |
|---|---|---|---|---|---|
| Age, mean (s.d.), years | 49.18 (14.15) | 47.03 (12.63) | 48.6 (13.79) | 43.74, 50.32 | 0.19 |
| Weight, mean (s.d.), kg | 89.99 (17.12) | 89.39 (16.6) | 89.83 (16.99) | 85.07, 93.72 | 0.86 |
| Weight change until w52, mean (s.d.), kg | −0.45 (4.61) | 1.51 (15.76) | 0.06 (9.12) | 0.528, 2.18 | 0.51 |
| UST dose, mean (s.d.), mg | 58.71 (20.71) | 53.8 (18.9) | 57.45 (20.13) | 49.8, 57.8 | 0.24 |
| DAS28-CRP, mean (s.d.) | 4.49 (0.87) | 4.5 (0.72) | 4.49 (0.83) | 4.31, 4.69 | 0.93 |
| DAS28-ESR, mean (s.d.) | 4.62 (1.125) | 4.8 (0.96) | 4.67 (1.08) | 4.55, 5.05 | 0.39 |
| DAPSA, mean (s.d.) | 36.29 (14.59) | 33.75 (11.79) | 35.6 (13.93) | 30.68, 36.82 | 0.35 |
| Cohort characteristic | No ADA detection (n = 82) | Confirmed ADA (n = 30) | All patients (n = 112) | 95% CI | P-value |
|---|---|---|---|---|---|
| Age, mean (s.d.), years | 49.18 (14.15) | 47.03 (12.63) | 48.6 (13.79) | 43.74, 50.32 | 0.19 |
| Weight, mean (s.d.), kg | 89.99 (17.12) | 89.39 (16.6) | 89.83 (16.99) | 85.07, 93.72 | 0.86 |
| Weight change until w52, mean (s.d.), kg | −0.45 (4.61) | 1.51 (15.76) | 0.06 (9.12) | 0.528, 2.18 | 0.51 |
| UST dose, mean (s.d.), mg | 58.71 (20.71) | 53.8 (18.9) | 57.45 (20.13) | 49.8, 57.8 | 0.24 |
| DAS28-CRP, mean (s.d.) | 4.49 (0.87) | 4.5 (0.72) | 4.49 (0.83) | 4.31, 4.69 | 0.93 |
| DAS28-ESR, mean (s.d.) | 4.62 (1.125) | 4.8 (0.96) | 4.67 (1.08) | 4.55, 5.05 | 0.39 |
| DAPSA, mean (s.d.) | 36.29 (14.59) | 33.75 (11.79) | 35.6 (13.93) | 30.68, 36.82 | 0.35 |
| Cohort characteristic | No ADA detection (n = 82) | Confirmed ADA (n = 30) | OR | 95% CI | P-value |
|---|---|---|---|---|---|
| Gender, male/female, n | 46/36 | 14/16 | 0.68 | 0.29, 1.56 | 0.39 |
| Pretreatment with MTX/No MTX pretreatment, n | 39/43 | 13/17 | 0.84 | 0.34, 1.92 | 0.83 |
| Concomitant MTX/concomitant placebo, n | 38/44 | 20/10 | 2.31 | 0.99, 5.66 | 0.08 |
| Adverse events/no adverse events, n | 4/78 | 0/30 | 0.28 | 0.01, 5.47 | 0.4 |
| Allergic reactions/no allergic reactions, n | 0/82 | 0/30 | +infinity | 0, 5.56 × 10−309 | >0.99 |
| Severe adverse events/no severe adverse events, n | 0/82 | 0/30 | +infinity | 0, 5.56 × 10−309 | >0.99 |
| Drop-out before w24/full study completion, n | 4/78 | 2/28 | 1.39 | 0.25, 6.23 | 0.65 |
| Cohort characteristic | No ADA detection (n = 82) | Confirmed ADA (n = 30) | OR | 95% CI | P-value |
|---|---|---|---|---|---|
| Gender, male/female, n | 46/36 | 14/16 | 0.68 | 0.29, 1.56 | 0.39 |
| Pretreatment with MTX/No MTX pretreatment, n | 39/43 | 13/17 | 0.84 | 0.34, 1.92 | 0.83 |
| Concomitant MTX/concomitant placebo, n | 38/44 | 20/10 | 2.31 | 0.99, 5.66 | 0.08 |
| Adverse events/no adverse events, n | 4/78 | 0/30 | 0.28 | 0.01, 5.47 | 0.4 |
| Allergic reactions/no allergic reactions, n | 0/82 | 0/30 | +infinity | 0, 5.56 × 10−309 | >0.99 |
| Severe adverse events/no severe adverse events, n | 0/82 | 0/30 | +infinity | 0, 5.56 × 10−309 | >0.99 |
| Drop-out before w24/full study completion, n | 4/78 | 2/28 | 1.39 | 0.25, 6.23 | 0.65 |
ADA: antidrug antibodies; w: weeks; UST: ustekinumab.
Differences in cohort characteristics and odds ratios (OR) in patients with and without confirmed ADA
| Cohort characteristic | No ADA detection (n = 82) | Confirmed ADA (n = 30) | All patients (n = 112) | 95% CI | P-value |
|---|---|---|---|---|---|
| Age, mean (s.d.), years | 49.18 (14.15) | 47.03 (12.63) | 48.6 (13.79) | 43.74, 50.32 | 0.19 |
| Weight, mean (s.d.), kg | 89.99 (17.12) | 89.39 (16.6) | 89.83 (16.99) | 85.07, 93.72 | 0.86 |
| Weight change until w52, mean (s.d.), kg | −0.45 (4.61) | 1.51 (15.76) | 0.06 (9.12) | 0.528, 2.18 | 0.51 |
| UST dose, mean (s.d.), mg | 58.71 (20.71) | 53.8 (18.9) | 57.45 (20.13) | 49.8, 57.8 | 0.24 |
| DAS28-CRP, mean (s.d.) | 4.49 (0.87) | 4.5 (0.72) | 4.49 (0.83) | 4.31, 4.69 | 0.93 |
| DAS28-ESR, mean (s.d.) | 4.62 (1.125) | 4.8 (0.96) | 4.67 (1.08) | 4.55, 5.05 | 0.39 |
| DAPSA, mean (s.d.) | 36.29 (14.59) | 33.75 (11.79) | 35.6 (13.93) | 30.68, 36.82 | 0.35 |
| Cohort characteristic | No ADA detection (n = 82) | Confirmed ADA (n = 30) | All patients (n = 112) | 95% CI | P-value |
|---|---|---|---|---|---|
| Age, mean (s.d.), years | 49.18 (14.15) | 47.03 (12.63) | 48.6 (13.79) | 43.74, 50.32 | 0.19 |
| Weight, mean (s.d.), kg | 89.99 (17.12) | 89.39 (16.6) | 89.83 (16.99) | 85.07, 93.72 | 0.86 |
| Weight change until w52, mean (s.d.), kg | −0.45 (4.61) | 1.51 (15.76) | 0.06 (9.12) | 0.528, 2.18 | 0.51 |
| UST dose, mean (s.d.), mg | 58.71 (20.71) | 53.8 (18.9) | 57.45 (20.13) | 49.8, 57.8 | 0.24 |
| DAS28-CRP, mean (s.d.) | 4.49 (0.87) | 4.5 (0.72) | 4.49 (0.83) | 4.31, 4.69 | 0.93 |
| DAS28-ESR, mean (s.d.) | 4.62 (1.125) | 4.8 (0.96) | 4.67 (1.08) | 4.55, 5.05 | 0.39 |
| DAPSA, mean (s.d.) | 36.29 (14.59) | 33.75 (11.79) | 35.6 (13.93) | 30.68, 36.82 | 0.35 |
| Cohort characteristic | No ADA detection (n = 82) | Confirmed ADA (n = 30) | OR | 95% CI | P-value |
|---|---|---|---|---|---|
| Gender, male/female, n | 46/36 | 14/16 | 0.68 | 0.29, 1.56 | 0.39 |
| Pretreatment with MTX/No MTX pretreatment, n | 39/43 | 13/17 | 0.84 | 0.34, 1.92 | 0.83 |
| Concomitant MTX/concomitant placebo, n | 38/44 | 20/10 | 2.31 | 0.99, 5.66 | 0.08 |
| Adverse events/no adverse events, n | 4/78 | 0/30 | 0.28 | 0.01, 5.47 | 0.4 |
| Allergic reactions/no allergic reactions, n | 0/82 | 0/30 | +infinity | 0, 5.56 × 10−309 | >0.99 |
| Severe adverse events/no severe adverse events, n | 0/82 | 0/30 | +infinity | 0, 5.56 × 10−309 | >0.99 |
| Drop-out before w24/full study completion, n | 4/78 | 2/28 | 1.39 | 0.25, 6.23 | 0.65 |
| Cohort characteristic | No ADA detection (n = 82) | Confirmed ADA (n = 30) | OR | 95% CI | P-value |
|---|---|---|---|---|---|
| Gender, male/female, n | 46/36 | 14/16 | 0.68 | 0.29, 1.56 | 0.39 |
| Pretreatment with MTX/No MTX pretreatment, n | 39/43 | 13/17 | 0.84 | 0.34, 1.92 | 0.83 |
| Concomitant MTX/concomitant placebo, n | 38/44 | 20/10 | 2.31 | 0.99, 5.66 | 0.08 |
| Adverse events/no adverse events, n | 4/78 | 0/30 | 0.28 | 0.01, 5.47 | 0.4 |
| Allergic reactions/no allergic reactions, n | 0/82 | 0/30 | +infinity | 0, 5.56 × 10−309 | >0.99 |
| Severe adverse events/no severe adverse events, n | 0/82 | 0/30 | +infinity | 0, 5.56 × 10−309 | >0.99 |
| Drop-out before w24/full study completion, n | 4/78 | 2/28 | 1.39 | 0.25, 6.23 | 0.65 |
ADA: antidrug antibodies; w: weeks; UST: ustekinumab.
To assess whether concomitant MTX had an impact on immunogenicity rates, we compared patients who had confirmed ADA over the period of 52 weeks in the UST/MTX with the UST/pbo cohort. Our results showed no significant difference (P > 0.05) in mean concentration of confirmed ADA between patients with concomitant MTX or placebo (Fig. 2). We did not detect pre-existing ADA in patients naïve to UST treatment. Our findings indicate that treatment with MTX influences neither the concentration nor the frequency of formation of ADA.
Concentration of confirmed ADA. Mean concentration of confirmed ADA with 95% confidence intervals in patients treated with ustekinumab and placebo (UST/pbo, n = 54) or MTX (UST/MTX, n = 58), respectively, in weeks (w) 0, 4, 15, 40 and 52. ADA: antidrug antibodies
Impact of ADA on pharmacokinetics and pharmacodynamics
To evaluate the impact of ADA on UST pharmacokinetics, we assigned patients within the UST/MTX and UST/pbo cohort into two subgroups, those with and those without ADA formation over the course of 52 weeks. Our findings showed that the visit-dependent mean (s.d.) UST concentration (Fig. 3) in the overall UST/MTX cohort was 0.052 (0.04)–0.106 (0.07) µg/ml and 0.029 (0.03)–0.097 (0.07) µg/ml in patients with confirmed ADA. Mean (s.d.) UST levels in the UST/pbo cohort were in the range of 0.047 (0.05)–0.119 (0.07) µg/ml and 0.037 (0.04)–0.091 (0.08) µg/ml in the subset of ADA positive subjects, respectively. In both cohorts, the UST levels showed no significant differences between ADA positive subjects and the whole cohort (P > 0.05). These data indicate that neither concomitant MTX nor ADA formation had an impact on UST through levels.
UST levels in the various groups. Mean ustekinumab (UST) levels and 95% confidence intervals in patients treated with UST and concomitant placebo (pbo) or methotrexate (MTX) compared with serum samples of patients with confirmed antidrug antibodies (ADA+). ADA: antidrug antibodies
To analyse clinical implications for ADA formation, we conducted a multiple linear regression analysis that included gender (female), UST dosage, mean UST concentration, age, weight and weight change after 52 weeks. Data analysis showed a positive linear relation (Fig. 4) between UST concentration and ADA detection (P = 0.02). The coefficient of determination (R2) of the model was 0.031, and the variance inflation factor of all tested variables was <1.6.
Linear relationship between UST and ADA levels (P = 0.02). ADA: antidrug antibodies; UST: ustekinumab
Impact of ADA on efficacy and safety
Impairments in therapeutic efficacy along with safety are the most common causes for treatment discontinuation. To analyse the impact of ADA on efficacy and safety, we compared cohort characteristics between patients with and without UST-specific ADA (Table 1). Our analysis showed no differences (P > 0.05) in demographic parameters, disease activity, treatment response, dropout rates or a pre-treatment with MTX. Furthermore, patients did not differ significantly regarding safety signals (Table 1).
We ascertained possible ADA-associated impairments in UST efficacy by comparing patients in remission, defined as DAPSA ≤ 4, and patients with active disease (DAPSA > 4). Mean UST levels were compared between patients that had confirmed ADA within 52 weeks and subjects that were ADA negative. No significant difference between patients in remission (DAPSA ≤ 4) or patients with active PsA (DAPSA > 4) at week 52 was observed (Fig. 5). These findings indicate that ADA formation had no impact on therapeutic efficacy in patients with PsA.
UST levels and dependence on DAPSA. Comparison of mean UST levels in ADA positive and negative patients with DAPSA ≤ 4 (A) and DAPSA > 4 (B) in week 52. Values are shown with 95% CIs. ADA: antidrug antibodies; DAPSA: Disease Activity in Psoriatic Arthritis; UST: ustekinumab
Discussion
To the best of our knowledge, this is the first post-hoc analysis of samples from a placebo-controlled randomized trial to investigate the role of MTX on UST immunogenicity in patients with PsA. We further analysed the effects of ADA formation on the UST serum concentration, clinical efficacy and safety of UST.
Over the course of 52 weeks, UST/MTX- and UST/pbo-treated cohorts showed stable immunogenicity rates at a comparable level. The vast majority of ADA was detected 4 weeks after treatment initiation. Overall, MTX seemed not to alter UST through levels or efficacy of UST. Importantly, our results further provide evidence that clinical outcomes and safety data, particularly allergic side effects, were not altered by UST-specific ADA.
Concomitant MTX is used in PsA with the premise of reducing immunogenicity and therefore improving drug efficacy [24]. While the PSUMMIT trial [25] contradicted this assumption, no randomized controlled studies looked at this hypothesis. Accordingly, our research is the first to provide unequivocal study data demonstrating no mitigating impact of MTX on the UST immunogenicity. Moreover, MTX-associated toxicity [26] might lead to treatment discontinuation or impair the patient’s adherence to treatment. Supporting the non-inferiority of a UST monotherapy might also improve quality of life while reducing costs.
A major strength of this study is the longitudinal impact analysis on UST immunogenicity by using validated and highly sensitive assays. Especially the use of SPR enables a direct mass and thus concentration determination without the need for labelling of affinity analysis [27]. However, the approach utilized suffers from the limitation that complexed ADA is undetectable with the methods used. This restriction might explain the dosage-dependent ADA detection observed in the PHOENIX 2 trial [15]. To detect complexed ADA a dissociation step, for instance through an acidification assay [18], is required. Unfortunately, the induced pH shift disturbs further analysis with a second method and thus does not allow a confirmatory assay. In conclusion, small amounts of bound nADA could stay undetected with our method. Nonetheless, we assume that nADA at these concentrations might be clinically irrelevant as even higher concentrations did not show a measurable impact. This issue may constitute an area for future study.
Furthermore, there are potential limitations due to the small number of individuals developing ADA. Although the sample size of patients with confirmed ADA is comparable to [17] or is even bigger than [28] in other groups, small numbers yield higher data variability and might cause misinterpretation. This might include the higher prevalence of ADA formation within the UST/MTX cohort compared with the UST/pbo group. Although the difference was not significant, it contradicts the assumption of an MTX-driven immunogenicity reduction [29, 30] reported by several other groups. However, a non-significant P-value does not prove an absence of association but implies that the null hypothesis ‘MTX prevents ADA formation’ cannot be rejected. A non-significant P-value could indicate either a lack of true association or a type 2 error. Since our study revealed only a small number of patients with ADA formation, a type 2 error is possible and the rejection of the null hypothesis might be false. A higher patient number would be required to overcome this issue.
According to its drug approval, UST was dosed based on body weight. Although dose and efficacy dependency is generally well characterized, our data analysis showed no dose-dependent effects on immunogenicity responses. This observation is also supported by previous studies [17]. Our results showed a weakly positive linear relationship between UST and ADA levels (Fig. 4), which is surprising since ADAs are broadly associated with lower drug levels in PsA [4, 31]. In addition, low levels of UST facilitate ADA detection since the binding sites are less likely to be occupied. Consequently, complexed ADAs are impaired in binding to the coated UST and do not induce an assay signal. Finally, given the small R2 (0.031) of the regression model, our results should be interpreted with caution. Nevertheless, some studies indicate an ADA-induced prolongation of the biotherapeutics’ half-life through altered clearance [32], which must also be taken into account here. However, we found no difference in adverse events between patients with or without ADA formation. Our results support the previous notion that the safety data are comparable to placebo even at higher UST doses [33].
We detected higher immunogenicity rates in UST therapy compared with other groups [17, 28, 34]. Unfortunately, a lack of transparency regarding validation parameters such as assay sensitivity, selectivity or LoD prevents direct comparison between assays used in different studies.
In conclusion, this post-hoc analysis of serum samples from a multicentre, randomized and placebo-controlled study demonstrates that MTX had no significant impact on UST-ADA formation. The data further suggest that presence of ADA could be associated with modified UST clearance. However, statistical analysis revealed that neither MTX nor ADA affected UST through levels, safety or efficacy.
It is a matter for future research to investigate whether a subset of patients might be at higher risk for developing clinically relevant ADA, as our data suggest that changes in drug clearance may occur. This effect, however, seems to be rather quantitively limited and could be related to individual patient disease characteristics or concomitant treatment other than MTX. In this context, we advise immunogenicity testing as a personalized approach to explore the underlying causes of drug-related adverse events and derive mitigation strategies from the findings obtained.
Supplementary material
Supplementary material is available at Rheumatology online.
Data availability
Data will be made available upon request.
Funding
This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No. 853988. The JU receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA and JDRF INTERNATIONAL. The project was supported by the Landesoffensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz (LOEWE) Centre Novel Drug Targets against Poverty-Related and Neglected Tropical Infectious Diseases (DRUID), the LOEWE Centre Translational Biodiversity Genomics (TBG), the LOEWE Centre for Personalized Translational Epilepsy Research (Cepter), the Fraunhofer Cluster of Excellence Immune mediated diseases (CIMD), the Leistungszentrum innovative Therapeutics (TheraNova) and the DGRh Forschungsinitiative 2020.
Disclosure statement: F.B., M.K. and H.B. received a research grant from Janssen Cilag. The other authors report no conflicts of interest.
Ethics: The MUST (Clinicaltrials.gov identifier: NCT03148860) study complies with the Declaration of Helsinki, the locally appointed ethics committee (Ethikkommission des Fachbereichs Medizin der Goethe Universität, approval number: 199/15) has approved the research protocol and written informed consent has been obtained from the subjects.
References
Stelara. Ustekinumab – Summary of product characteristics. Janssen-Cilag
Food and Drug Administration. Immunogenicity Testing of Therapeutic Protein Products—Developing and Validating Assays for Anti-Drug Antibody Detection.
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