Antibodies Toward Vedolizumab Appear from the First Infusion Onward and Disappear Over Time

Background:

Vedolizumab (VDZ) has been approved for the treatment of patients with moderate-to-severe Crohn's disease and ulcerative colitis. The GEMINI trials reported a low prevalence of anti-VDZ antibodies (AVA). However, the AVA assays used in GEMINI were drug sensitive, resulting in a possible underestimation of the rate of AVA formation. This study aimed to monitor immunogenicity of VDZ in a real-life cohort using a drug-resistant AVA assay.

Methods:

Using a combination of VDZ/AVA complex precipitation and acid dissociation, a drug-resistant assay for AVA detection in the presence of high VDZ concentrations has been developed and analytically validated. The assay was applied on serum samples of 179 VDZ-treated patients with inflammatory bowel disease to evaluate the prevalence and time course of AVA.

Results:

A dose–response curve ranging from 25 to 1600 ng/mL using 1/125 diluted serum was obtained, allowing the detection of AVA concentrations up to 200 μg/mL of MA-VDZ19C11 equivalents, a calibrator antibody to VDZ. This assay was highly AVA specific and drug resistant. Four of 179 VDZ-treated patients (2.2%) were AVA positive and AVA were detected already after the first VDZ infusion. AVA were all transient in these patients without need for any dosage optimization. There was no correlation between VDZ and AVA concentrations in the AVA-positive samples.

Conclusions:

AVA appear from the first VDZ infusion onward and disappear over time. The low prevalence of AVA suggests that immunogenicity does not influence response to treatment.

vedolizumab, immunogenicity, antidrug antibody, drug interference

Vedolizumab (VDZ) is a recombinant humanized IgG1 monoclonal antibody that exclusively targets α₄β₇ integrin on gut-homing lymphocytes and interacts with mucosal addressin cell-adhesion molecules-1 (MAdCAM-1), thereby preventing the adhesion of lymphocytes to the vascular endothelium and reducing intestinal inflammation.¹ VDZ has been approved for the treatment of patients with moderate-to-severe Crohn's disease (CD) and ulcerative colitis (UC) refractory or intolerant to conventional therapy.^2–4

Immunogenicity remains an important issue to biological agents as it triggers the development of antidrug antibodies, which may neutralize the active drug and increase drug clearance.⁵ Immunogenicity can negatively influence the treatment outcome, the effective treatment duration, and can induce a secondary loss of response (LOR). This can occur at any point during the treatment after an initial response at induction. Although this is not the only cause of LOR, it is by far the most studied.^6,7 Clinically, immunogenic-mediated LOR is managed by shortening dose intervals, increasing the dosage, adding an immunomodulator,⁸ or by switching the patients to another biological within or out of class.

Currently, the proportion of inflammatory bowel disease (IBD)-related health care costs on anti–tumour necrosis factor (anti-TNF) therapy is rising while proportions for hospitalization and surgical interventions are declining.^9,10 Treatment algorithms based on therapeutic drug monitoring (TDM) and antidrug antibody monitoring have the potential to guide to more effective dosing in individual patients receiving anti-TNF therapy. A study in Denmark further concluded such monitoring-guided decision making is more cost-effective than the empirical approach in patients with CD with secondary infliximab failure, which can potentially save up to 56% in cost with no significant difference in response rates.¹¹ Such treatment algorithms may also have a role in optimizing the efficacy and cost efficiency in the use of VDZ therapy.

In GEMINI trials, the prevalence of anti-VDZ antibodies (AVA) was reported in 3.7% and 4.1% of patients with UC and CD, respectively.^2,3 Available immunogenicity assays for antidrug antibody detection, however, are either drug sensitive or drug tolerant, resulting in no or limited drug tolerance. The presence of drug in serum samples could possibly lead to an underestimation of antidrug antibody concentrations.^12,13 VDZ trough concentrations are typically >25 μg/mL during induction and >10 μg/mL during maintenance therapy according to GEMINI trials, making it challenging for immunogenicity assays to detect AVA in patient samples. Potential interference from serum components could also be a concern for an immunogenicity assay. Serum proteins such as rheumatoid factor and residual antidrug antibodies from previous exposure to an anti-TNF biological may cause false-positive signals on AVA detection.^12,14

Recent studies constructed and validated novel drug-resistant assays for the detection of antidrug antibodies in the presence of high drug concentrations, achieved through a combination of drug/antidrug antibody complex precipitation and acid dissociation.^15–17 This study aimed to validate the drug-resistant assay for AVA detection in the presence of high VDZ concentrations and to investigate the prevalence and the time course of AVA in a cohort of 179 VDZ-treated patients with IBD.

Materials and Methods

Polyethylene glycol (molar weight 8000) and glycine were purchased from Sigma-Aldrich (Steinheim, Germany) and Acros Organics (Geel, Belgium), respectively. Streptavidin poly-horse radish peroxide (Poly-HRP) and Sulfo-NHS-LC-Biotin were purchased from IBL International GmbH (Hamburg, Germany) and Thermo Fisher Scientific Inc. (Waltham, MA), respectively. Both 96-well clear V-Bottom Polypropylene Not Treated Microplate No. 3363, Nonsterile and 96-well microtiter plates No. 3590 were purchased from Corning Incorporated (New York, NY). Healthy control sera were purchased from Valley Biomedical (Winchester, VA). Vedolizumab (Entyvio) was purchased from Takeda Pharmaceuticals (Deerfield, IL). Liquichek Rheumatoid Factor Control (level 3) was purchased from Bio-Rad Laboratories N.V. (Temse, Belgium).

Three mouse monoclonal antibodies toward VDZ (MA-VDZ6F3, MA-VDZ6E6, and MA-VDZ19C11) were in-house developed, produced and purified in a similar way as previously described.^18,19

Assays for VDZ and Circulating AVA Detection

An in-house developed sandwich-type enzyme-linked immunosorbent assay was used to quantify VDZ trough concentrations in patient sera.²⁰ The assay uses MA-VDZ6F3 as capture antibody and biotinylated MA-VDZ6E6 as detection antibody. A nonlinear dose–response curve ranging from 0.2 to 10 ng/mL of VDZ in 1:10,000 diluted serum was obtained, allowing the detection of VDZ concentrations up to 100 μg/mL in patient serum samples.

A drug-sensitive bridging assay was developed for detection of circulating AVA, using VDZ for both capture and detection and MA-VDZ19C11, a monoclonal antibody toward VDZ, as calibrator. A nonlinear dose–response curve ranging from 0.2 to 10 ng/mL of MA-VDZ19C11 was obtained and serum samples were diluted in 1:20, 1:40, 1:80, and 1:160. The assay was able to detect AVA concentrations up to 1600 ng/mL of MA-VDZ19C11 equivalents in patient sera.

A Drug-resistant Assay for Circulating and Complexed AVA Detection

A drug-resistant assay protocol based on precipitation and acidification of drug/antidrug antibody complex described by Bian et al¹⁷ was modified for AVA detection. Briefly, 40 μL of 100 μg/mL of VDZ was added into 10 μL of 1:5 diluted serum sample. After 1 hour of incubation, 50 μL of 16% polyethylene glycol was applied to precipitate the formed VDZ/AVA complexes during overnight incubation. After centrifugation, the pellets were washed; 250 μL of glycine–HCl (0.1 M, pH 2.5) was applied to reconstitute the pellets and separated AVA was immediately coated onto a fresh plate and subject to blocking and specific detection using biotinylated VDZ and streptavidin–HRP. The assay lower limit of quantification (LLOQ) was determined using a panel of VDZ-naive serum samples and calculated based on the mean optical density of the naive serum samples plus 10 SDs.

Assay Specificity

The impact of anti-TNF biological agents (infliximab and adalimumab), antibodies toward anti-TNF biologicals, and rheumatoid factor on AVA determination in the drug-resistant assay was evaluated. Simply, 10 μg/mL of infliximab, adalimumab, monoclonal antibody against adalimumab (MA-ADM6A10),¹⁸ and monoclonal antibody against infliximab (MA-IFX6B7)¹⁹ were separately spiked into 1:5 diluted healthy control serum followed by 1 hour of incubation at 24°C in an Eppendorf Thermomixer R. The response signal of these mixtures was measured after the whole assay protocol. Four VDZ-naive patient sera spiked with 10 μg/mL of MA-VDZ19C11 and individual patient samples with known positive concentrations of anti-infliximab or anti-adalimumab antibody were evaluated to detect any interference. One control serum containing high rheumatoid factor concentration (150 U/mL) was also used to test for unspecific response.

Evaluation of Assay Drug Tolerance

Evaluation of drug interference by VDZ on AVA determination in the drug-resistant assay used a series of internal control samples. MA-VDZ19C11 was spiked at 10, 40, and 200 μg/mL in 1:5 diluted healthy serum with increasing VDZ concentrations (0, 1, 10, and 100 μg/mL), followed by 1 hour of incubation at 24°C in an Eppendorf Thermomixer R. Residual binding of MA-VDZ19C11 mixture was determined.

Evaluation of AVA Status in a Cohort of 179 VDZ-treated Patients

To evaluate the prevalence and the time course of AVA, serum trough samples of 179 VDZ-treated patients with IBD (113 CD, 66 UC)²⁰ were analyzed using the drug-resistant assay. These patients had received an induction schedule with VDZ 300 mg at weeks 0, 2, 6 (and 10 for CD) and a maintenance schedule with VDZ every 8 weeks from week 14 onward. Samples at weeks 6 and 22 (or at the last available time point) were first measured for VDZ and AVA positivity. If AVA were positive, all consecutive trough samples of that patient were further analyzed. AVA concentrations were expressed as μg/mL of MA-VDZ19C11 equivalents and concentrations below the LLOQ at any time point were considered as negative and treated as zero.

Evaluation of Clinical, Biological, and Endoscopic Response

Clinical response to VDZ was defined as a marked decrease or disappearance of symptoms (physicians' global assessment). Biological response was evaluated in the subgroup of patients with CD with an elevated C-reactive protein (CRP) (>5 mg/L) at week 0 and was defined as a decrease from baseline CRP of at least 50% or a normalization of CRP (≤5 mg/L). Mucosal healing was defined in UC based on sigmoidoscopy at week 14 (Mayo endoscopic subscore of 0 or 1) and in CD based on colonoscopy at week 22 (complete absence of ulcerations).

Ethical Considerations

Written informed consent was provided by all patients in the framework of the Institutional Review Board–approved Flemish Inheritance study for Crohn's and colitis (VLECC; B322201213950/S53684).

Results

Assay Characteristics

MA-VDZ19C11, an IgG2b monoclonal antibody toward VDZ, was used as a calibrator to generate a nonlinear dose–response curve ranging from 25 to 1600 ng/mL in 1:125 diluted serum (Fig. 1), allowing detection of AVA concentrations up to 200 μg/mL of MA-VDZ19C11 equivalents. Using 32 VDZ-naive patient samples (week 0), the assay LLOQ was determined to be 3.8 μg/mL equivalents.

Figure 1.

A general nonlinear dose–response curve of MA-VDZ19C11 ranging from 25 to 1600 ng/mL produced from the average of n = 18 repetitions with the SD expressed as error bars.

Assay Specificity

The assay specificity was evaluated using serum samples containing anti-TNF biologicals, antibodies toward anti-TNF biologicals, and rheumatoid factor (Table 1). Full recovery (88%–105%) was obtained when spiking 100 μg/mL of MA-VDZ19C11 into either the assay buffer or the week 0 VDZ-naive patient samples. Control sera spiked with infliximab, adalimumab, or rheumatoid factor (150 U/mL) did not reveal a signal above LLOQ. Additionally, no AVA concentration was detected in patient samples at week 0 and in patient sera that had positive antibodies toward infliximab and adalimumab.

Table 1.

Serum Samples with MA-VDZ19C11, Infliximab, Adalimumab, Anti-infliximab, Anti-adalimumab Antibody, and Rheumatoid Factor for Specificity Testing

Evaluation of Assay Drug Tolerance

Complete recoveries (82%–109%) of increasing concentrations of MA-VDZ19C11 were obtained, regardless of the drug amount present in the samples, as shown in Figure 2, indicating that the assay is drug resistant up to 100 μg/mL VDZ (n ≥ 3).

Figure 2.

Bar chart showing the recovery of 3 different concentrations of MA-VDZ19C11 (10, 40, and 200 μg/mL) at increasing concentrations of VDZ up to 100 μg/mL in the assay (mean ± SD, n ≥ 3).

Prevalence and Time Course of AVA in a Cohort of 179 VDZ-treated Patients

Using the drug-sensitive assay, AVA were detected in 1 of 179 patients (0.6%) at week 6 (patient 1, Fig. 3). This sample revealed undetectable VDZ. VDZ was also undetectable in the week 6 sample of 2 other patients and in the week 22 sample of 1 other patient. In none of these 3 samples AVA were detected using the drug-sensitive assay (nor using the drug-resistant assay).

Figure 3.

Time course of AVA formation in the 4 AVA-positive patients at consecutive time points during VDZ treatment. VDZ trough concentrations (dashed line, left y-axis) are reported in μg/mL and AVA concentrations (solid line, right y-axis) are measured using the drug-resistant AVA assay and expressed in μg/mL of MA-VDZ19C11 equivalents. Week 6 sample of patient 1 with undetectable VDZ concentration is also positive to AVA in a drug-sensitive assay.

The drug-resistant assay identified 4 AVA-positive patients (2.2%) and 3 (1.7%) were AVA positive at 2 or more time points (Fig. 3). In 2 of the 4 patients, AVA appeared already upon the first VDZ infusion (i.e., just before the week 2 VDZ infusion). All AVA disappeared over time, indicating their transient character.

No correlation between VDZ and AVA concentrations was observed in the AVA-positive samples (Pearson's r = 0.10, P = 0.76, n = 12). Two of 4 AVA-positive patients experienced mucosal healing and 2 of 4 AVA-positive patients revealed clinical response (Table 2). All 4 patients continued standard VDZ therapy at least up to 1 year (Table 2). Patient 1 experienced arthralgia during the VDZ therapy, but no other adverse events were reported. Two of 4 patients were previously treated with anti-TNF biologicals and stopped because of loss of response, but no antibodies toward the anti-TNF biologicals have been observed using drug-sensitive assays.

Table 2.

Clinical Characteristics of the 4 Patients with Anti-VDZ Antibodies

Discussion

To accurately evaluate the VDZ immunogenicity, a drug-resistant AVA assay has been validated in this study using a combination of VDZ/AVA complex precipitation and acid dissociation. The assay is highly specific for AVA detection as no interference from any anti-TNF biological, antibodies toward anti-TNF biologicals, and rheumatoid factor was observed, allowing the quantification of AVA concentrations in samples from patients previously exposed to anti-TNF biologicals. In the drug tolerance test, the drug-resistant AVA assay remains accurate in the presence of VDZ concentrations up to 100 μg/mL, supporting its drug-resistant property.

Immunogenicity is one of the key contributors to LOR to biological agents; however, the prevalence differs between agents.^21–24 LOR or inadequate response does not only affect physical health but build social and economic burden. Uncontrolled symptoms of IBD can be stressful and damaging to one's career. A study in Germany found in total cost of CD, 64% were due to indirect costs such as early retirement and sick leave while 32% were from direct medical costs, 54% and 41%, respectively, for UC.²⁵ As the prevalence of IBD is expected to rise in the next decade, the need for biological agents will only grow, and with it, the incidence of immunogenicity.²⁶ Antidrug antibody monitoring combined with TDM has proven to be both effective and cost-effective in anti-TNF biologicals.⁹ However, detection of AVA is restricted by the currently available immunogenicity assays because of the high VDZ concentrations. An assay that provides accurate and reliable detection of AVA can aid in understanding of VDZ efficacy.

Applying the drug-resistant assay into a cohort of 179 VDZ-treated patients revealed that 4 patients (2.2%) were AVA positive at any time point, which is lower than the reported 3.7% and 4.1% in the GEMINI I and II trials, as displayed in Table 3. Of note, a 1.7% rate of AVA formation on ≥2 time points reported here is higher than the percentages reported in all available studies.^2–4,27,28 Regarding this study, the drug-resistant assay demonstrated its major advantage in resistance to drug concentration over the drug-sensitive assay, where only 1 (0.6%) patient was defined as AVA positive. VDZ showed an overall low immunogenicity (0.0%–4.1%) by different type AVA assays in comparison with what has been observed in anti-TNF biologicals. A large literature review conducted by Vermeire et al²⁹ revealed up to 65% infliximab-treated patients and 38% adalimumab-treated patients were positive for antidrug antibody upon exposure.

Table 3.

Percentage of AVA-Positive Patients Reported in Literature

Although the mechanism to the low rate of AVA formation is unclear, 2 reasons might explain the observed low immunogenicity. First, the reformulated Chinese hamster ovary cell–derived VDZ has demonstrated less immunogenicity than the earlier nonsecreting murine myeloma NS0-derived version without affecting idiotype.^30,31 Second, high dosing and a prolonged half-life result in high drug concentrations. Chaigne and Watier³² also proposed that adjusting drug concentrations to a targeted high concentration could suppress the immunization and achieve high-zone immune tolerance. The limitations of the study are that only samples at week 6 and week 22 (or at the last available time point) were screened for AVA positivity and that due to the extensive procedure including multiple washing steps and a final 1:125 dilution, AVA with low affinity or low titers might not be detected. Additionally, all samples analyzed here were of patients either during treatment or immediately after stop of treatment of one single cohort. Validation in an external cohort is essential to generalize its use.

The low prevalence of AVA makes it difficult to study the relation between AVA and the response in this study. Patients 2 and 3 achieved mucosal healing, whereas patients 3 and 4 revealed clinical response (Table 2). Interestingly, the only patient with a lack of clinical response and mucosal healing was also positive using a drug-sensitive AVA assay.

Similar to anti-TNF biologicals, TDM may also guide VDZ dosing in clinical practice. Clinical trials have revealed correlations between higher VDZ concentrations and clinical response, although optimal trough concentrations have not been identified.^2–4,33 With VDZ immunogenicity viewed as a minor hindrance and the favorable effect of high drug levels brought to light, the practicality of an immunogenicity assay as a routine procedure becomes questionable. Routine monitoring of VDZ concentrations alone or in combination with other predictors like α₄β₇ saturation may be adequate to correlate exposure with response to therapy.³⁴ The AVA assay may have a niche as a secondary measure instead, used in cases that require conclusive evidence for the cause of treatment failure, but this needs confirmation using larger and/or external cohorts.

Conclusions

In conclusion, validation of a drug-resistant AVA assay verified its competency in the monitoring of AVA concentrations in patient sera containing high VDZ concentrations. AVA are detected in 2.2% of 179 VDZ-treated patients at any time point. Antibodies appear already upon the first VDZ infusion and are all transient over time. The low prevalence of AVA suggests that immunogenicity does not influence response to treatment. Taken together, the immunogenicity assay for AVA detection is suggested to be used as a second-line monitoring tool to investigate the cause of treatment failure when needed instead of as a routine measure.

Acknowledgments

S. Bian acknowledges the China Scholarship Council (CSC) for the PhD scholarship. Supported in part by TBM Grant T003716N of the Research Foundation—Flanders (FWO), Belgium. G. Van Assche, M. Ferrante, and S. Vermeire are Senior Clinical Investigators of the Research Foundation—Flanders (FWO), Belgium. G. Van Assche received financial support for research from Abbott and Ferring Pharmaceuticals, lecture fees from Janssen, MSD, and Abbott, and consultancy fees from PDL BioPharma, UCB Pharma, Sanofi-Aventis, Abbott, Abbvie, Ferring, Novartis, Biogen Idec, Janssen Biologics, NovoNordisk, Zealand Pharma A/S, Millenium/Takeda, Shire, Novartis, and Bristol Mayer Squibb. M. Ferrante received financial support for research from Takeda, lecture fees from MSD, Janssen, Abbvie, Boehringer-Ingelheim, Ferring, Chiesi, Tillotts, Zeria, and Mitsubishi Tanabe, and consultancy fees from MSD, Janssen, Abbvie, Boehringer-Ingelheim, and Ferring. S. Vermeire received grant support from MSD, Abbvie, and Takeda, lecture fees from Abbvie, MSD, Ferring Pharmaceuticals, Takeda, and Hospira, and consultancy fees from Abbvie, Takeda, Pfizer, Ferring Pharmaceuticals, Shire Pharmaceuticals Group, MSD, Hospira, Mundipharma, Celgene, Galapagos, and Genentech/Roche. A. Gils has served as a speaker for MSD, Janssen Biologicals, Pfizer, Takeda, and Abbvie, as consultant for UCB, and has received Investigator Initiated Research Grants from Pfizer. KU Leuven licensed infliximab and adalimumab enzyme-linked immunosorbent assay to apDia. The remaining authors have no conflict of interest to disclose.

Author notes

Author disclosures are available in the Acknowledgments.