Introduction

In recent years, the use of biologic drugs, such as infliximab and adalimumab, has revolutionized the treatment of various autoimmune diseases. Infliximab and adalimumab are monoclonal antibody (mAb) drugs of human IgG1/kappa isotypes that specifically target the inflammatory cytokine tumor necrosis factor alpha (TNF) and have shown remarkable efficacy in managing chronic immune-mediated inflammatory diseases such as rheumatoid arthritis (1), inflammatory bowel disease (2), and psoriasis (3). However, the development of anti-drug antibodies (ADAs) against these biologics can significantly impact treatment outcomes, leading to reduced drug efficacy, loss of response, and potential adverse effects (4, 5). Immunogenicity studies of infliximab and adalimumab have revealed significant interindividual variations in different patient populations (6). Treatment failures are common in patients receiving TNF inhibitors; up to 55% of patients do not respond due to the development of ADAs (2, 6, 7). The development of ADAs is mostly associated with decreased levels of bioactive adalimumab and infliximab. It is invariably difficult to predict the development of ADAs in individual patients and the resultant loss of response. Traditionally, ADAs against TNF inhibitors have only been evaluated when lack of treatment responses were already observed. Circulating infliximab and adalimumab concentrations were found to be subtherapeutic in these cases (4, 8). Recently, proactive therapeutic monitoring of TNF blockers and their ADAs has been increasingly adopted to personalize treatment strategy and detect ADAs earlier in treatment (2, 9). In proactive monitoring scenarios, serum drug concentrations can still appear in the therapeutic range even though ADAs have already been developed (2, 10).

Different from evaluating immunogenicity during drug development and clinical trial phases, clinical evaluations of infliximab and adalimumab ADAs in patients generally do not adopt a screening to confirmation tiered approach (11). In routine clinical evaluation of immune responses to TNF inhibitors such as adalimumab and infliximab, it is recommended that the evaluation of ADAs be accompanied by the measurement of drug concentrations (5, 6). Drug concentrations of infliximab and adalimumab in patients can be measured by ligand binding immunoassays (12), mass spectrometry-based methodologies (13, 14), and TNF activity inhibition functional assays (15, 16). If the detection of adalimumab or infliximab is based on its ability to bind or inhibit TNF, the presence of neutralizing ADAs and their blocking of the TNF-binding sites may further interfere with the determination of infliximab or adalimumab drug concentrations in patients (17). The qualitative presence of ADAs, together with a sub-therapeutic level of drug, is evidence for immunogenicity and may warrant a change in therapeutic agent (9). ADA titers may also be monitored in patients receiving long-term therapy to evaluate treatment response or dose adjustments (18). The clinical utility of semi-quantitative ADA titers is yet to be fully explored (19).

Different analytical approaches, such as immunoassay and surface plasmon resonance (SPR), have been employed to detect and quantify titers of ADAs, providing valuable insights into the immunogenicity of these biologic agents (17, 20). However, the immunoassays are largely manual, utilizing a 96-well microplate format (16, 21, 22). To detect ADAs in serum that may also contain adalimumab or infliximab, an acid dissociation step may be utilized prior to the immunoassay. To address these challenges, automated immunoassays have emerged as a promising alternative, offering improved precision, reproducibility, and throughput using a random-access format. These advanced platforms use sensitive detection technologies, such as electrochemiluminescence (ECL) immunoassays or enzyme-linked immunosorbent assays (ELISAs), coupled with robotic sample handling and data analysis algorithms. By streamlining the assay workflow and minimizing operational error, automated immunoassays have the potential to enhance the accuracy and efficiency of ADA detection.

Here we describe and evaluate two new automated immunoassays for analysis of serum ADAs to infliximab and adalimumab. These two research prototype assays are based on the ImmunoCAP™ technology with a sponge-like cellulose solid phase creating a large protein-binding capacity (23) for binding ADA using the bridging assay format. The assays do not include an acid-dissociation step. We also compared these novel assays against existing clinically validated ADA immunoassays, assessing performance characteristics such as sensitivity, specificity, precision, linearity, and robustness. Additionally, we explored the drug tolerance of the assays and how different interferents, including high levels of rheumatoid factors (RF), may impact assay results.

Materials and Methods

Sample Collection

Residual de-identified patient serum samples were collected at Mayo Clinic Laboratories after routine testing of adalimumab and infliximab ADAs using the clinical assays. Adalimumab and infliximab ADAs results from routine clinical testing were collected for comparative analysis described below. The samples were stored at − 20 °C for up to 6 months until testing with the automated ImmunoCAP ADA assays. All studies involving human samples followed the research study protocol approved by the Institutional Review Board of Mayo Clinic (IRB ID: 22–008528). Infliximab or adalimumab drug concentrations and patients’ autoimmune diagnoses (when available) was also collected as part of the clinical information. Additional anonymous serum samples from apparently healthy blood donors with no known autoimmune disorders were used as negative control samples and provided through the internal sample unit at Thermo Fischer Scientific, Uppsala, Sweden.

Drug-specific mAbs

Two recombinant affinity-maturated phage display-derived human mAbs against infliximab (HCA233) and adalimumab (HCA204) were obtained from Bio-Rad (Hercules, California, USA). A mouse anti-human kappa light chain hybridoma-derived mAb (positive control mAb) was an in-house developed reagent.

Automated Assays for Measurement of ADAs

Two research prototype bridging assays for semiquantitative analysis of ADAs to infliximab and adalimumab were developed based on the ImmunoCAP technology (Thermo Fisher Scientific, Uppsala, Sweden) hereafter referred to as IFX-ADA and ADL-ADA, respectively. Briefly, 14 µL of patient serum is mixed with 266 µL sample diluent containing biotinylated drug (IFX-ADA: 1000 ng/ml and ADL-ADA: 750 ng/ml), this mixture is then incubated for 4 h at room temperature allowing equilibrium binding between serum ADAs and the biotinylated drug. After incubation, the sample mixture containing biotinylated drug-ADA complex is loaded on a Phadia™ 250 automated analyzer (Thermo Fisher Scientific) and 40 µL of the sample mixture is added to the ImmunoCAP solid phase covalently coupled with infliximab or adalimumab (drug coupling concentration of both assays: 25 mcg/ml), to allow for ADA bridging formation between biotinylated drug and solid phase drug (Fig. 1). After incubation for 30 min at 37 °C that ends with extensive washing, enzyme-labeled streptavidin conjugate is added for 24 min. Following another washing step, a developing agent and finally the stop solution are added for detection of the drug-ADA complexes. The generated fluorescence, reported as response units (RU), is regarded as proportional to the concentration of ADAs in the sample.

Fig. 1
figure 1

The ADAs are detected through bridging complex formed by biotin labeled drug, ADAs, and drug immobilized to the ImmunoCAP solid phase. The presence of drug may compete and partition away ADAs, thus leading to a decrease in detected signal intensity. The presence of high RF can non-specifically bind labeled drug with immobilized drug, leading to increased signal intensities

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The IFX-ADA and ADL-ADA assays adopted the same positive controls, a mouse anti-human kappa light chain mAb (positive control mAb) added to rabbit serum at two different levels. A human serum pool from apparently healthy and non-treated subjects is used as negative control.

Comparative Assays for ADAs

The automated ADA assays on the Phadia platform were compared to existing clinical assays for ADAs against infliximab and adalimumab. The existing clinical assays utilize pre-treatment with acetic acid to break any existing complexes between ADAs and their targeted drugs. The clinical infliximab ADA assay is an in-house developed ECL bridging assay using the Meso Scale Discovery platform MSD QuickPlex® SQ120 (Rockville, Maryland, USA) (24, 25). It detects the bridging complex formed by ADAs bound to biotinylated infliximab and infliximab labeled with a SULFO-TAG™. Calibrators containing different concentrations of human anti-infliximab mAb HCA233 are used to establish correlation between signal intensity and ADA concentrations (U/mL). The ADAs in patient samples were measured in signal intensity and reported in concentration units (U/mL). The reportable range is 20–1250 U/mL.

The comparison adalimumab ADA assay was a laboratory developed test utilizing a commercially available microtiter-based ELISA (Immundiagnostik, Bensheim, Germany) that detects bridging complex formed by ADAs, biotinylated adalimumab, and peroxidase-conjugated adalimumab (16, 26). The drug-ADA complexes bind to a streptavidin-coated microtiter plate and are detected via enzymatic conversion of substrate to a colorimetric product. A cutoff calibrator is used to correlate between signal intensity and ADA concentration. The ADAs in patient samples were measured in signal intensity and reported in arbitrary units (AU)/mL. The reportable range of the clinical ELISA is 10–500 AU/mL.

A subset of patient samples was further analyzed using another clinical ADA assay for adalimumab. The assay principle was previously described (16, 27). Briefly, it was a functional assay based on TNF-sensitive reporter cells. The presence of neutralizing ADAs would block the TNF-binding capacity of adalimumab, and the exogenous unbound TNF would trigger a positive response from the cells.

Method Evaluation of the Automated ImmunoCAP ADA assays

Screening cut point

For IFX-ADA assay and ADL-ADA assay respectively, 40 serum samples from the negative control cohort were each measured in two separate runs over at least two days. The preliminary screening cut point for each assay was established based on the 95th percentile of the negative serum samples. The analytical sensitivity of the assays near respective screening cut points was also determined by analyzing decreasing concentrations of the positive control mAb following protocols described by Shankar et al. (11).

Imprecision

Serum pools with varying levels of infliximab and adalimumab ADAs (Table 1) were measured by the IFX-ADA and ADL-ADA assays with twenty replicates in one run (intra-assay) or across five runs (inter-assay). Each pool was made by mixing equal volume of three individual patient samples. The mean and standard deviation (SD) of replicate measurements (measured and reported in RU) were evaluated. For each assay, five individual ADA-positive patient samples near the preliminary screening cut point were evaluated in triplicate (measured and reported in RU) in a single assay run (Table S1). The mean and SD of triplicate measurement were evaluated.

Table 1 Assay Imprecision and Reproducibility at Different Infliximab and Adalimumab ADA Levels
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Interference

Serum pools with different levels of infliximab and adalimumab ADAs (Table S2) were spiked with interference substances and evaluated in triplicate. The following substances were tested: conjugated bilirubin at 40 mg/dL (Millipore Sigma, Burlington, MA), free bilirubin at 40 mg/dL (Millipore Sigma), biotin at 3500 ng/mL (Millipore Sigma), hemoglobin at 1000 mg/dL (prepared in-house), and intralipid at 15 mg/mL (Millipore Sigma). The measured infliximab ADAs or adalimumab ADAs in serum pools spiked with interference substances were compared with measured ADA levels in unspiked pools to discern any interference effects.

Drug Tolerance Profiles of the Automated ADA Assays

To evaluate the drug tolerance profiles of the ImmunoCAP IFX-ADA and ADL-ADA assays that are performed without an acid dissociation step, patient samples were selected containing varying levels of ADAs to each drug. Each patient sample was divided into 5 aliquots and four of them were spiked with serum containing different amounts of infliximab or adalimumab (Millipore Sigma) to obtain increasing concentrations of free drugs. One remaining aliquot was spiked with the same volume of blank serum (zero infliximab or adalimumab) for comparison. All aliquots were incubated overnight before analysis by the IFX-ADA or ADL-ADA assay in duplicate. Serum pools containing anti-infliximab mAb HCA233 at 50 and 350 ng/mL; or anti-adalimumab mAb HCA204 at 10,000 ng/mL were also analyzed. Relative signal reduction was calculated by comparing aliquots spiked with infliximab or adalimumab with the aliquot spiked with blank serum.

Interference from RF

Serum pools containing four different levels of infliximab or adalimumab ADAs were spiked with known high RF-containing patient serum samples (> 500 IU/mL RF IgM; P1 and P2 for IFX-ADA; P3-P6 for ADL-ADA) at a 9:1 ratio. The RF levels in serum samples (P1-P6) were determined by EliA™ RF IgM assay (Thermo Fisher Scientific). The ADAs serum pools spiked with RF were compared to pools spiked with an equal volume of RF-negative serum by analyzing them with the automated ADA assays to discern any interference caused by RF.

Binding Kinetics of Drug Specific mAbs

Binding kinetics analysis was performed on a Biacore™ T100 biosensor (Cytiva, Uppsala, Sweden) based on SPR technology. Briefly, infliximab and adalimumab were immobilized on Sensor chip CM5 in different channels. Five concentrations of HCA233, HCA204, and positive control mAb (obtained from 1:3 serial dilutions starting from 1 × 10–6 M) were sequentially added to the chip at a flow rate of 30 µl/min to allow binding to the corresponding drug. The binding kinetics was calculated using a 1:1 model fitting following the recommended method [Single-Cycle Kinetics (SCK)™] of the manufacturer.

Statistical Analysis

Statistical analyses were performed in Microsoft Excel and Analyse-it®. Non-parametric quantile regression was used to establish the preliminary screening cut-points. Spearman’s rank correlation was used to derive correlation coefficient (rs) for quantitative comparison based on authentic patient samples. Simple linear regression was used to derive linear relationship between IFX-ADA or ADL-ADA assay responses and concentrations of mAbs against infliximab or adalimumab. In qualitative agreement analysis, a Wilson score interval-based 95% confidence interval was calculated for positive and negative agreement. Tukey–Kramer HCD test was used to evaluate statistically significant differences from RF interference based on p-value < 0.05. Figures and graphic illustration were drawn and created in Adobe Illustrator and Biorender.com.

Results

ADA Binding and Assay Design

Assay optimization was performed to identify appropriate drug conjugate and solid phase coupling concentrations to balance assay sensitivity and drug tolerance (data not shown). When testing the serially diluted positive control mAb, both assays exhibited a detection limit below 25 ng/ml and could differentiate varying concentrations of positive control mAb over a 3-log range (Figure S1). Based on the SPR experiment, the positive control mAb binds to infliximab and adalimumab with similar binding affinities (dissociation constant KD approx. 9.0 nM, Fig. S2). In comparison, adalimumab-specific HCA204 exhibited a much slower dissociation rate and higher affinity binding (KD = 0.88 nM, Fig. S2). Compared to the positive control mAb, HCA233 exhibited a much higher binding affinity towards infliximab and a very low dissociation rate, and no reliable affinity determinations could be calculated from our analysis (Fig. S2).

Screening Cut Point

The 40 serum samples from healthy and drug naïve donors were used to define the screening cut points. When analyzed by the IFX-ADA assay over four different days, the average response for the samples was 120.1 RU (95% confidence interval CI: 111.4 to 135.0), with a standard deviation (SD) of 29.4 (Fig. 2a). The screening cut point of the IFX-ADA assay based on 95th percentile of the healthy controls was calculated to be 171 RU.

Fig. 2
figure 2

Infliximab and adalimumab ADAs were measured in healthy volunteers and patients receiving infliximab or adalimumab treatment. Group median and quartiles were represented in the figure and the IFX-ADA and ADL-ADA assay results were plotted on a logarithmic scale. a Infliximab ADA levels measured by IFX-ADA assay in healthy subjects (N = 40, “healthy control”), patients with positive infliximab ADAs exceeding the reportable range based on the comparison clinical ECL assay (N = 7, “high titer”), patients with positive infliximab ADAs within reportable range of the comparison assay (N = 47, “intermediate titer”), patients with negative infliximab ADAs below reportable range of the comparison assay (N = 25, “negative”) and patient with detectable infliximab ADAs but deemed negative based on the assay cutoff (N = 5, “negative titer”). Screening cut point 171 RU was plotted as the dashed horizontal line. b Adalimumab ADA levels measured by ADL-ADA assay in healthy subjects (N = 40, “healthy control”), patients with positive adalimumab ADAs exceeding the reportable range of the comparison clinical ELISA assay (N = 19, “high titer”), patients with positive adalimumab ADAs within reportable range of the comparison assay (N = 47, “intermediate”), and patients with negative adalimumab ADAs based on the comparison assay (N = 22, “negative”). Screening cut point 117 RU was plotted as the dashed horizontal line

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The same set of healthy samples was analyzed by the ADL-ADA assay over two days, yielding an average of 81.9 RU (95% CI: 76.6 to 87.2), SD = 16.6 (Fig. 2B). The screening cut point for the ADL-ADA assay based on the 95th percentile was 117 RU.

Imprecision

The intra- and inter-assay imprecision were evaluated at three different ADA levels in 20 replicates and the coefficient of variation (CV%) were all less than 10% for both the IFX-ADA assay and the ADL-ADA assay (Table 1). Close to the screening cut point of each assay, five patient samples were also evaluated in triplicate. For both assays, the CVs% near respective screening cut point were less than 20% (Table S1).

Matrix Interference

The presence of free hemoglobin, triglyceride, or free or conjugated bilirubin did not impact the measurements in either of the ImmunoCAP ADA assays (Table S2). Similarly, the clinical ECL for infliximab ADAs and ELISA for adalimumab ADAs are also tolerant for hemolysis, lipemia, and icterus in patient serum samples (24, 26). Nevertheless, both IFX-ADA and ADL-ADA assays showed significant negative biases in measured ADA levels due to the presence of biotin at 3500 ng/mL. For IFX-ADA, three positive patient pools exhibited 88% relative reduction when biotin was added. For ADL-ADA, the relative reduction in RU was between 52 and 61%. In both IFX-ADA and ADL-ADA assays, enzyme-labeled streptavidin conjugate was used to detect the formation of the ADA bridging complex. It is postulated that free biotin in the patient sample can bind to the enzyme-labeled streptavidin conjugate. The unbound biotin-conjugate complex would be washed away prior to signal detection, resulting in negative biases in the overall assay measurement. In clinical ECL for infliximab ADAs, signal reduction was observed in serum samples containing more than 12.5 ng/mL biotin. In clinical ELISA for adalimumab ADAs, it is recommended that patient should stop taking biotin/vitamin B7 supplement at least 12 h prior to sample collection.

Method Comparison with Clinical Assays

Patient specimens (n = 84) were analyzed using the automated IFX-ADA assay and the results were compared with the clinical ECL assay. When classified by the screening cut point of the IFX-ADA assay (171 RU) and the clinical cutoff of the ECL assay (50 U/mL), positive agreement was 1.00 and negative agreement was 0.53 (Table 2). All 14 discrepant results were positive on the IFX-ADA assay and negative on the ECL assay (Table 2). In 5 patient samples with discrepant results, there were measurable levels of infliximab ADAs based on the clinical ECL assay (Fig. 2a), but these levels were lower than the assay cutoff of 50 U/mL and considered negative. These 5 patient samples were shown to contain significantly higher levels of infliximab ADAs compared to healthy controls based on the ImmunoCAP IFX-ADA assay. There were 32 patient samples containing infliximab ADAs outside the reportable range of the clinical ECL assay (Fig. 2a). Based on 52 patient samples with quantitative measurement from both the IFX-ADA and the clinical ECL assays, we observed a strong correlation between the two assays (Fig. 3a; rs = 0.673, 95% CI: 0.484–0.802). When analyzing different dilutions of the anti-infliximab mAb HCA233 with the IFX-ADA assay, we observed a strong linear correlation (R2 = 0.9965) between observed RU and the HCA233 concentrations (Fig. 3b). This indicates a linear correlation between the IFX-ADA assay and the clinical ECL assay since HCA233 is also used as calibrator antibody in the latter assay. The discrepant results between the two assays observed in patient samples are largely due to different cutoffs established for the two assays.

Table 2 Qualitative Agreement Between ImmunoCAP IFX-ADA and the Clinical ECL Assay
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Fig. 3
figure 3

Quantitative analysis of ImmunoCAP IFX-ADA and ADL-ADA assays. a Quantitative correlation between IFX-ADA and a clinical ECL assay based on patient samples within the reportable range of the ECL assay (20–1250 U/ml). b Linear responses between IFX-ADA measurements and concentrations of infliximab specific mAb HCA233 (20–1250 ng/ml). c Quantitative correlation between ADL-ADA and a clinical ELISA based on patient samples within the reportable range of the clinical ELISA (10–500 AU/ml). d Responses of ADL-ADA assay at different concentrations of adalimumab specific mAb HCA204 (50–1000 ng/ml)

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Eighty-eight patient samples were evaluated by both ADL-ADA and the clinical ELISA assay. For qualitative agreement, results were classified according to the screening cut point of the ADL-ADA assay (117 RU) and the clinical assay cutoff of the ELISA assay (14 AU/mL). Positive agreement between the two assays was 0.68 and negative agreement was 1.00 (Table 3). All 21 discrepant results were negative by the ADL-ADA assay and positive by the clinical ELISA assay (Table 3). There were 40 samples outside the reportable range for the clinical ELISA assay (Fig. 3b). Based on the remaining 48 patient samples with quantitative values from both the ADL-ADA and clinical ELISA assays, we observed a moderate quantitative correlation between the two assays (Fig. 3c; rs = 0.510, 95% CI = 0.198–0.728). We investigated concentrations of adalimumab in patient samples with qualitatively agreeable and discrepant ADA results between ADL-ADA and clinical ELISA (Table S3). In 21 patient samples with discrepant results (negative by ADL-ADA, positive by clinical ELISA), their ADA results measured by the clinical ELISA ranged from 18.7 AU/mL to 215.2 AU/mL; the adalimumab concentrations varied from less than 0.8 mcg/mL (lower limit of quantification of the adalimumab assay) to 6.1 mcg/mL (median 2.1 mcg/mL). The remaining patient samples contained positive adalimumab ADAs according to both assays and the overall trend of their adalimumab drug concentrations decreased with increasing levels of adalimumab ADAs. In 21 patient samples with matching ADA levels (15.2–255.7 AU/mL based on the clinical ELISA), the adalimumab drug concentrations were from less than 0.8 mcg/mL to 6.9 mcg/mL, with a median 1.7 mcg/mL. The RF IgM in all patient samples were less than 5.0 IU/mL, indicating negative or equivocal results.

Table 3 Qualitative Agreement Between ImmunoCAP ADL-ADA and the Clinical ELISA Assay
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We also evaluated a subset of patient samples (n = 34) using another clinical ADA assay for adalimumab, a cell-based reporter gene activity (RGA) assay sensitive to TNF activity. Generally, the three assays agreed among each other for certain “high positive” and negative samples (Table S4). All 16 negative samples on the ADL-ADA assays were also negative on the RGA assay. Nine of the 18 positive samples on the ADL-ADA assay were negative on the RGA assay, although they were all positive on the clinical ELISA assay.

When using the ADL-ADA assay to evaluate mAb HCA204 spiked at different concentrations in serum, we observed a linear response at mAb concentrations between 0.25 and 50 mcg/mL (R2 = 0.9975, Fig. 3D). The ADL-ADA assay was not able to detect HCA204 at concentrations less than 250 ng/mL. This observation contrasted the high binding affinity observed between HCA204 and adalimumab during binding kinetics characterization (Fig. S2).

Drug Tolerance Profile

Drug tolerance of the IFX-ADA assay was evaluated by studying the inhibition profile of the measured infliximab ADAs in response to added infliximab. The addition of infliximab resulted in decreased RU in all but one patient samples (Fig. 4a). The amplitude of signal reduction varied at different concentrations of infliximab. The measured ADA levels underwent more drastic reduction at 2 and 5 mcg/mL infliximab, followed by a continued albeit lessened reduction at 10 and 15 mcg/mL infliximab. The average relative signal reduction changed from 50.4% (2 mcg/mL) to 69.2% (15 mcg/mL). The inhibition profiles in patient samples were highly dependent on their existing ADA titers. Samples with ADA titers less than 700 RU in the IFX-ADA assay were more likely to drop below the screening cut point with increasing amounts of added infliximab, while samples with ADAs levels corresponding to more than 700 RU remained positive at 15 mcg/mL infliximab (Fig. 4a). The drug tolerance profile of the mAb HCA233 at 50 and 350 ng/mL was also evaluated; the mAb HCA233 displayed a different inhibition profile compared to the patient samples, reaching maximal signal reduction below the assay screening cut point at an infliximab concentration of 2 mcg/mL (Fig. 4A).

Fig. 4
figure 4

Drug tolerance profiles of patient samples containing different levels ADAs. a Inhibition of IFX-ADA results with the addition of infliximab at different concentrations. 6 patient samples were evaluated with added infliximab at 0, 2, 5, 10 mcg/ml. 10 patient samples were evaluated with spiked infliximab at 0, 2, 5, 10, 15 mcg/ml. Human anti-infliximab mAb HCA233 at 50 ng/ml and 350 ng/ml were also evaluated with spiked infliximab at 0, 2, 5, 10, 15 mcg/ml. The measurement results (RU) based on the IFX-ADA assay were plotted on a logarithmic scale with a base of 5. Screening cut point 171 RU was plotted as the dashed horizontal line. b Inhibition of ADL-ADA results with the addition of adalimumab at different concentrations. 15 patient samples were evaluated with added adalimumab at 0, 2, 4, 8, 16 mcg/ml. 5 patient samples were evaluated with spiked adalimumab at 0, 5, 10, 20 mcg/ml. Human anti-adalimumab mAb HCA204 at 10,000 ng/ml was also evaluated with spiked adalimumab at 0, 5, 10, 20 mcg/ml. The measurement results (RU) based on the ADL-ADA assay were plotted on a logarithmic scale with a base of 5. Screening cut point 117 RU was plotted as the dashed horizontal line

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When evaluating the ADL-ADA assay, increasing concentrations of adalimumab were spiked into 20 patient samples containing different levels of adalimumab ADA. While the addition of free adalimumab decreased the measured RU in some patient samples, other samples did not show significant changes (Fig. 4b). In patient specimens with adalimumab ADA levels between 300 and 2000 RU by the ADL-ADA assay, greater magnitudes of signal reduction were observed. In patient samples with ADA titers less than 200 RU, the relative response reduction to added adalimumab was not statistically significant considering the assay imprecision. The average relative signal reduction due to adalimumab addition ranged from 15.8% (2 mcg/mL) to 33.5% (16 mcg/mL). A constant decline in RU was observed when increasing adalimumab concentrations were added to the human anti-adalimumab mAb HCA204 (Fig. 4b). However, the mAb HCA204 (10,000 ng/mL) remained positive by the ADL-ADA assay (i.e. above screening cut point) in the presence of up to 20 mcg/mL adalimumab spiked to the sample.

Interference from Rheumatoid Factors

For the IFX-ADA assay, RF serum P1 appeared to negatively interfere with the ADA measurement when spiked into the mid and high ADA serum pools, whereas RF serum P2 interfered by increasing the ADA result of the low patient serum pool (Table 4). Nevertheless, there were no false positive or false negative infliximab ADA qualitative results observed for the ADA serum pools due to RF interference.

Table 4 Potential RF Interferences Evaluated in IFX-ADA and ADL-ADA Assays Based on Patient Pools Containing Different Levels of ADAs
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For the ADL-ADA assay, all four individual RF serum (P3-P6) interfered in the assay by increasing the ADA results when spiked into the negative, low, and mid ADA sample pools (Table 4). RF serum P5 gave the largest relative increase, and it caused a false positive result for adalimumab ADAs when spiked into the ADA-negative serum pool (final RF IgM concentration: 176 IU/mL) (Table 4). No false positive results in the ADL-ADA assay were observed for any of the other three RF serum used for spiking (RF serum P3, P4 and P6).

Discussion

The development of ADAs is crucial clinical information when evaluating treatment responses and personalizing dose adjustment in patients undergoing TNF-inhibitor therapy (28). Laboratory tests to detect and measure circulating ADAs against infliximab and adalimumab are routinely utilized clinically for patients with various autoimmune disorders during treatment with TNF-antagonists (2, 29). Growing interests in proactive monitoring of ADA development before treatment non-responses occur further require the assays to detect ADAs in the presence of significant levels of antibody drugs. In this study we evaluated two new automated immunoassays developed to measure ADAs to infliximab and adalimumab.

Design Consideration for Automated ADA Assays

We applied high coupling concentrations for both adalimumab and infliximab to maximize the amount of ADA binding targets for each assay. The excess of solid phase-coupled drug is designed to drive drug-bound ADAs to bind to ImmunoCAP in accordance with the law of mass action without acid dissociation of drug-bound ADA complexes (23). In comparison, drug-bound ADAs may more significantly interfere with the bridging formation in conventional microtiter plate-based bridging ELISAs and TNF functional activity assays (15,16,17, 25). Utilizing the sponge-like cellulose solid phase, both ImmunoCAP ADA assays have large antibody-binding capacities, approximately 150 times more than a conventional microtiter well (30). Additional design consideration was to balance between drug tolerance and assay sensitivity by varying serum sample input and the concentration of the biotinylated drug. A higher biotinylated drug concentration can increase the drug tolerance but decrease the analytical sensitivity of the assay.

The durations of the binding incubation steps were also optimized. In the first binding step between patient ADAs and biotinylated drug, the incubation time is set longer so binding between ADAs and biotinylated drugs reaches equilibrium. In the second ImmunoCAP binding step, kinetic dissociation of the drug-ADA complexes precedes the formation of the drug-ADA-ImmunoCAP bridging complex. As a result, the assay can exhibit seemingly low sensitivity when measuring high affinity ADA mAbs if their binding dissociation rates are low, as in the case for HCA204 in the ADL-ADA assay. In comparison, naturally occurring human ADAs in patient samples are largely heterogenous and can exhibit various binding affinities and higher dissociation rates (31). Hence it is important to examine assay sensitivity using authentic patient samples.

Comparability Across Different ADA Assays

Due to the lack of commutable standards and the polyclonal nature of ADAs, results generated by different ADA assays are not always readily comparable (21). In both the IFX-ADA and ADL-ADA assays, we established preliminary screening cut points based on the upper 95th percentile of negative serum samples from healthy volunteers. This approach is commonly adopted in immunogenicity assays during clinical trials when evaluating immune responses among trial participants. Based on this intended use, such an approach generally allows a 5% false positive rate to maximize screening sensitivity (8, 11). However, when adopted clinically to monitor patients receiving TNF-inhibitor treatment, a positive cutoff established based on drug naïve patients with specific autoimmune disorders would be more clinically relevant. For the clinical ECL assay, the cutoff of positive infliximab ADAs was established based on clinical responses of the patients receiving infliximab treatment. Based on human anti-infliximab mAb HCA233, there was a strong linear correlation between quantitative measurements obtained from the IFX-ADA assay and the clinical ECL assay. Furthermore, when comparing infliximab ADA results of autoimmune patients receiving infliximab therapies, there was a positive correlation between the two methods (rs = 0.673), albeit with a rather scattered distribution. Qualitative agreement analysis between the two assays further revealed that the preliminary screening cut point of the IFX-ADA assay classifies more patient samples as positive, although the clinical relevance of infliximab ADAs at such low levels is not known.

For the ADL-ADA evaluation, we observed a moderate quantitative correlation (rs = 0.510) with the clinical ELISA when analyzing authentic patient samples with adalimumab ADAs. The two assays exhibited 100% negative qualitative agreement, but the ADL-ADA assay classified more patients as negative than the clinical ELISA (Fig. 2B). We further investigated the adalimumab concentrations and RF levels in patient serum samples (Table S3 and S4). As expected, adalimumab concentrations tend to be low or not detected when there are high titer levels of ADAs. In patient serum samples with discrepant results between ADL-ADA and clinical ELISA, there were detectable levels of adalimumab in more than half of them (14 out of 21). Nevertheless, in patient samples with agreeable results between the two assays and similar levels of adalimumab ADAs (according to the clinical ELISA), there were comparable levels of adalimumab in majority of the samples (Table S3). Hence, the discrepant results do not appear to be caused by the presence of adalimumab. We did not observe any significantly levels of RF that can cause potential interference. The ADL-ADA assay was able to differentiate high adalimumab ADAs levels in patient samples exceeding the upper limit of the clinical ELISA assay reporting range (> 500 AU/ml).

One potential limitation of the prototype IFX-ADA and ADL-ADA assays is that they measure binding and not neutralizing ADAs. However, early studies showed a strong association between the levels of binding antibodies to infliximab and their neutralizing capacity (32). It has been indicated that most ADAs against therapeutic antibodies are directed against the idiotope in the antigen-binding region (28). Our observation of high agreement between the ADL-ADA and the TNF-activity based RGA further supported this.

Distinctive Inhibition Profiles in Drug Tolerance Studies

ADAs towards biological drugs are typically polyclonal, hence their drug-binding affinities and properties may vary (31). In patients receiving adalimumab or infliximab for various autoimmune disorders, the typical drug trough concentrations are expected to be less than 20 mcg/mL (6, 9, 21). The inhibition profiles of the IFX-ADA and ADL-ADA assays in response to increasing amounts of infliximab or adalimumab further demonstrated the heterogenous nature of ADAs. In the IFX-ADA assay, low concentrations of added infliximab (2 and 5 mcg/mL) caused a larger reduction in measured signals, whereas continued addition of infliximab to 10 and 15 mcg/mL resulted in less signal change (Fig. 4A). The amplitude of signal reduction varied across patient samples, further demonstrating the heterogeneity of human ADAs. Such heterogeneity was further revealed by the different drug inhibition profile observed for mAb HCA233, where the signal reduction maximized at 2 mcg/mL of added infliximab (Fig. 4A). The binding affinity of HCA233 to infliximab is high, hence the incubation times adopted in the IFX-ADA assay was not sufficient for dissociation of HCA233-infliximab bound complexes, resulting in low signals. Although affinity matured mAbs are commonly adopted as calibrators for ADA assays, there are limitations of these high affinity mAbs to be used as materials to evaluate the performance characteristics of the ADA assay (33).

In the ADL-ADA assay, only samples with high ADA levels exhibited significant reduction in measured ADA results after addition of adalimumab. Several patient samples with low or negative ADA levels (< 200 RU) showed no significant reduction in their ADL-ADA assay results. The distinctive inhibition profiles observed in the present drug tolerance studies may indicate different binding properties of ADAs for infliximab and adalimumab. Adalimumab is a fully humanized monoclonal IgG antibody, whereas infliximab is a chimeric antibody containing murine sequence in its heavy and light chain variable domains. Previous immunogenicity studies have also shown a higher prevalence of ADAs development in patients treated with infliximab than with adalimumab (34).

Interference of Biotin and Rheumatoid Factor

Both ImmunoCAP IFX-ADA and ADM-ADA assays are susceptible to the presence of biotin. Biotin interference has been an ongoing issue for numerous clinical assays utilizing the biotin-streptavidin binding as detection mechanism (35). Depending on assay design principles, free biotin in patient sample can either cause false negative or false positive results. As biotin is increasingly utilized as a nutritional supplement, it is important to identify such limitation and notify patients to refrain from biotin supplements at least two days before sample collection.

Potential interferences from RF were observed in the two automated ADA assays. RF in one out of four high RF serum caused a false positive result in the ADL-ADA assay when spiked into an ADA-negative serum pool. RF belongs to a class of autoantibodies targeting the fragment crystallizable (Fc) region of human IgG. Both ImmunoCAP assays use biotinylated drug and immobilized drug on the solid phase to detect bridging ADAs. It is postulated that RF can bind to exposed Fc domain of conjugated therapeutic IgG1 antibodies such as infliximab and adalimumab (36), thus bridge the biotinylated and immobilized drugs and result in falsely high results in ADA bridging assays (Fig. 1). Such RF interference could pose a problem in autoimmune disorders where RF elevations are commonly observed and where the patients may be treated with TNF-blockers such as infliximab and adalimumab (37). Therefore, potential interferences from high levels of RF should be considered when evaluating positive results from bridging assays utilizing intact therapeutic antibodies with a human IgG1 Fc region as ADA-binding reagents. It should be noted that there were no patient samples containing elevated RF among the included patient samples in the present study (Table S4).

Expanded Applications in Immunogenicity Monitoring

Based on its large binding capacity and equilibrium driven binding, the automated ImmunoCAP assays can be applied to detect ADAs towards other therapeutic proteins. Both infliximab and adalimumab are IgG1 kappa antibodies, accordingly the assays utilize intact therapeutic antibodies for the biotin-labeled drug and the solid-phase coupled drug. The assay can be modified to use specific antibody fragments or domains as construct to detect ADAs towards specific functional motifs, such as the TNF-binding VL domain. One potential disadvantage of using antigen-binding fragments is that neo-epitopes in the hinge region may be exposed and lead to false positive ADA results due to pre-existing anti-hinge antibodies (38).

In addition to antibody therapeutics, immunogenicity monitoring and the detection of ADAs play important roles in candidate selection, safety monitoring, and efficacy evaluation of other protein biologics, including peptide-based protein therapeutics, engineered bispecific antibodies (39), and antibody drug conjugates (40). In multiple domain biotherapeutics, ADA assays differentiating epitope specificity of binding antibodies and neutralizing antibodies may be needed to understand the effects of immunogenic responses (41). Immune responses to nucleotide-based therapeutics such as mRNA are normally driven by RNA-specific Toll-like receptors. The safety monitoring of nucleotide-based therapeutics such as the mRNA vaccines were primarily carried out by cytokine responses (42).

Conclusion

In summary, we evaluated two automated immunoassays for ADAs against infliximab and adalimumab. Even without an acid dissociation step, the two research prototype assays were able to detect ADAs in the presence of drugs. The analytical performances of the two assays show reproducible results and robustness. However, results from various ADA assays are not readily comparable to each other, and there are no established reference standards for ADAs to infliximab and adalimumab. The findings from this study can provide valuable insights into the suitability and reliability of automated immunoassays for routine clinical monitoring of ADA levels in patients receiving TNF inhibitor therapies. Ultimately, this study can provide new analytical alternatives for proactive therapeutic monitoring of ADAs against adalimumab and infliximab, and to potentially improve the efficacy of these widely used TNF-blockers in treating autoimmune diseases.