Infliximab (IFX) is a chimeric mouse-human monoclonal anti-tumor necrosis factor (TNF) antibody effective for induction and maintenance of remission in Crohn's disease (CD) and ulcerative colitis (UC). However, 30%–84% of the patents lose response to the medication along the course of the treatment.1–5 Trough levels of IFX significantly correlate with clinical response to IFX in CD6, 7 and UC8 patients.
Anti-infliximab antibodies (ATI) develop in up to 61% of the patients.6 The development of ATI is correlated with increased clearance and lower serum levels of IFX as well as increased risk for infusion reactions in CD patients.6 However, data regarding the relationship between development of ATI and clinical response to IFX in inflammatory bowel disease (IBD) patients has been equivocal. One possible reason for this may be ascribed to the technique of conventional double-antigen (DA) enzyme-linked immunosorbent assays (ELISAs) for ATI measurement, which employs IFX as the capture antigen and labeled IFX as the detection antibody. Consequently, this method is susceptible to various technical limitations, including the inability to detect monovalent IgG4 ATI, which could possibly lead to false-negative results on the one hand, and plastic rheumatoid factor interactions yielding false-positive results7 on the other. Moreover, IFX in serum competes with the detection antibody, thereby precluding the detection of ATI. This situation is commonly reported as inconclusive ATI and is reported in clinical trials and case series. For instance, inconclusive results of ATI levels were reported in 72% of patients included in the SONIC study8 and in 39% of UC patients in a case series from Toronto.9 These limitations of the DA technique hamper the ability to accurately define the role of ATI levels in predicting loss of response to IFX.
As an alternative to the DA technique, a radioimmunoassay technique for measuring ATI levels was described for both rheumatoid arthritis and CD patients.10, 11 Both of these studies demonstrated a significant correlation between ATI levels and loss of clinical response to IFX. However, this method is more cumbersome and is not widely available yet.
Another alternative to the DA ELISA was developed in our laboratory. It comprises an ELISA employing antihuman lambda chain (AHLC) conjugated antibody in the detection phase, taking advantage of the fact that IFX is composed of kappa chains. Notably, the presence of ATI demonstrated by this method correlates with loss of clinical response in CD patients treated with IFX.7 Conceptually, the implementation of AHLC-conjugated antibody in the detection phase may allow detection of IgG4 ATIs and also diminish the magnitude of the interference caused by positive IFX levels, but this has not been hitherto tested. In fact, while the method of testing may bear significant implications for the ability to detect ATI and may thus impact clinical decisions derived from these results, so far there are no studies comparing the different ATI assays with respect to their clinical utility. Moreover, the inability of DA ELISA to detect ATI in the presence of IFX renders it impossible to accurately assess the evolution of antidrug antibody response over time and to investigate the clinical significance of coexistence of ATI with IFX.
Therefore, the aim of this study was to compare the accuracy of ATI detection by each of these two ELISA techniques (DA and AHLC), as defined by its correlation with clinical response or loss of response to treatment with IFX. The secondary objective was to evaluate the clinical characteristics and outcome of IFX-treated CD patients who developed ATI in the presence of detectable serum IFX (double positivity [DP]) as compared with patients who have never developed DP.
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- PATIENTS AND METHODS
- Supporting Information
To the best of our knowledge, the present study is the first to compare two methods of ATI detection and investigate their clinical utility. The results show that the AHLC-based ELISA performs with a similar accuracy as a tested commercial DA-based ELISA for the detection of ATI in patients with undetectable serum IFX. However, AHLC-based ELISA was able to detect ATI in the sera of four patients with detectable serum IFX that were undetectable by the conventional DA-based ELISA. As 3/4 of these IFX+ATI+ were still responding at the time of sampling, the DA technique showed a numeric trend toward better specificity and positive predictive value of ATI detection for clinical LOR.
What are the implications of these findings for predicting clinical outcome and directing medical interventions in IFX-treated patients? There was a significant correlation between detectable ATI (by both methods) and loss of clinical response as reported by the treating physician. This finding is consistent with the previous data published by our group.7 However, the correlation of ATI status with clinical course is not perfect, since nearly half of the patients losing response may not have detectable ATI in serum by any of the methods (Table 2). Conversely, a proportion of patients may still enjoy a sustained response in the face of detectable ATI. A number of factors may explain these observations. Loss of response may occur due to causes other than immunogenicity, antibodies may be nonneutralizing and neutralizing antibodies may have escaped detection due to the “window phenomena.” The latter may be unmasked if patients are tested again at a later timepoint postinfusion. In this respect, the AHLC and DA assays achieved an overall comparable correlation with the clinical outcome in our study, likely because of the relatively small sample size.
In the lack of a gold standard technique for ATI measurement, comparing the technical performance accuracy of ATI assays may be problematic, especially regarding their ability to detect ATI in the presence of IFX. When measured within 10 days from IFX infusion, an IFX+ATI+ pattern was demonstrated in 3/3 patients with known positive ATI and undetectable serum trough IFX level before the index infusion. This observation is consistent with the existing data regarding IFX pharmacokinetics12 and attests to the in vivo validity of this double-positive status. Moreover, double positivity for ATI and IFX was also reproducible in spiking experiments in vitro. Notably, addition of extrinsic IFX in concentrations that are commonly detected in clinical practice resulted in a moderate reduction in measured ATI levels by the AHLC method, presumably due to displacement of polyclonal/heterologous low-affinity ATI. However, ATI levels remained above the cutoff levels of positivity in 3/9 (33%) of the patients at serum concentrations of IFX commonly occurring in clinical practice. In contrast, addition of extrinsic IFX resulted in undetectable ATI in all tested samples by the DA technique. These experiments corroborated that the AHLC method is less affected by the presence of IFX in serum compared to the DA-based assays and is still able to detect “true” ATI in this situation, at least in some patients.
From the clinical perspective, ATI+IFX+ pattern can also be demonstrated in samples obtained 6–8 weeks from IFX infusion. When tested by the DA ELISA, these patients' ATI status would have been reported as “inconclusive.” This ATI status was associated with higher rates of steroid-free remission8 and longer median duration of remission.13 Because all of these patients had detectable serum IFX that is associated with a superior clinical outcome compared to patients with undetectable trough IFX levels,6, 8 this observation is not surprising. However, in the subgroup of patients who develop positive ATI when IFX is still detectable in the serum, this may serve as an indicator of evolving immune response to IFX that would result in future low trough levels and loss of response. This specific patient population has never been previously addressed due to technical inability to detect this double positivity status.
We examined the clinical outcome of these double-positive patients. Thirty-one percent of these patients experienced LOR upon detection of double positivity status, while 10/12 patients (78%) developed LOR within 24 months of an index ATI+IFX+ measurement. Interestingly, development of LOR was associated with abatement of trough serum IFX level in only half of the patients. This observation supports the existence of an alternative pathway for development of LOR, which may be nonimmune or mediated by neutralizing antibodies that do not influence the drug level as measured by available solid-phase ELISA assays. Thus, coexistence of detectable serum IFX and ATI may have a considerable clinical significance. This finding could be an indicator of a present or evolving immune response to IFX and imminent development of LOR. Conceptually, the ability to predict this reaction early may prove useful for introducing preemptive interventions to prevent the occurrence of LOR to IFX. Nonetheless, further studies in a larger cohort of patients are required to clarify the significance of these findings and to elucidate the value of potential preventive strategies once IFX+ATI+ status is detected.
Our study has several limitations. The clinical data for the patients were collected retrospectively, and no objective clinical scoring system was employed. A prospective study employing established clinical and endoscopic scoring system is required for the formal clinical validation of the AHLC-ATI ELISA. However, from the practical point of view, the clinician's decision to discontinue or escalate IFX coupled with subjective patients' clinical deterioration probably reflects the real-life assessment of the severity of the disease and the clinical decision making.14 Another limitation is that the assays examined are solid-phase assays and a fluid phase test such as a radioimmunoassay (RIA) was not tested in the present study. Nonetheless, RIA is more cumbersome and less available in clinical laboratories compared to standard ELISA techniques. Notwithstanding, it would be of value as a next step to perform similar comparative studies of both solid phase and fluid phase methodologies for ATI detection. Finally, the number of patients with ATI+IFX+ was small and does not enable us to draw any statistically definitive conclusions. However, it does point to the existence and significance of this phenomenon as a possible harbinger of a future abatement of serum drug level and development of LOR.
In conclusion, the AHLC antibody-based ELISA method is at least as accurate as the DA-based ELISA for detection of anti-IFX antibodies. In a subgroup of patients this method identifies ATI in the presence of detectable IFX in the serum. This finding may be clinically significant for being a forerunner of LOR to IFX in some patients, and for possibly allowing us to undertake early preventive interventions.