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Immunogenicity Assessment of Tumor Necrosis Factor Antagonists in the Clinical Laboratory.

Due to the central role of tumor necrosis factor (TNF) [2] in inflammation and the pathogenesis of autoimmune and chronic inflammatory diseases, TNF antagonists have revolutionized the treatment of rheumatoid arthritis (RA), ankylosing spondylitis, psoriasis, and inflammatory bowel diseases (IBD), including Crohn disease (CD) and ulcerative colitis (1, 2). The therapeutic benefits have been so dramatic that TNF antagonists are among the best-selling, most-prescribed pharmaceuticals (3). Although most patients respond favorably to these drugs by showing dramatic improvements of the clinical symptoms, many others fail to respond or develop clinical relapse after initial favorable response (4). The mechanisms for the development of response failure are complex; however, immunogenicity of these protein-based TNF antagonists has been recognized as a major factor (5). The emergence of antidrug antibodies (ADAs) contributes to therapeutic failure by multiple mechanisms, such as neutralization of drug activity or drug removal. In this review, we present the currently used laboratory methodologies for the detection of ADA leading to therapeutic failure, focusing on clinically relevant technologies; assays that are used only in research laboratories are not presented here in detail (6-9).

Furthermore, to improve therapeutic efficacy and patient safety, a rational, laboratory-based approach is provided to aid the management of patients on TNF antagonists. A personalized approach in which therapy is tailored to the individual patient's needs and based on immunopharmacological evidence enables better, safer, and more cost-effective therapies.

TNF Antagonists Currently in Clinical Use

Discovery of the causal role of the proinflammatory cytokine TNF in the pathogenesis of RA in the early 1990s (10), followed by clinical trials involving blocking antibodies to TNF, led to the development of the first biological therapies for RA (11, 12) and subsequently for CD (13).

In the past decades, several monoclonal antibody (mAb)-based TNF antagonists have been developed (Fig. 1, Table 1). All of these molecules have been designed by genetic engineering and include nonhuman sequences to various extents. Infliximab is a chimeric mouse/human antibody in which the mouse Fab region is preserved, but the rest of the molecule is replaced by human sequences. Adalimumab and golimumab are human IgG1 antibodies produced by phage display or transgenic-mouse technology, respectively. Despite containing predominantly human sequences, these molecules may show structural features that are different from those of the endogenous proteins, such as glycosylation patterns that are characteristic of the producing cells [Chinese hamster ovary (CHO) cells or mouse myeloma cells] and not necessarily identical to the human glycosylation pattern, which may also contribute to immunogenicity (14). Certolizumab pegol is a humanized antibody Fab fragment, designed by engrafting the mouse complementarity determining region (CDR) into a human IgG4 k Fab framework, and it has a polyethylene glycol (PEG) molecule attached that prolongs the half-life in the circulation; the construct is expressed in bacteria and therefore it lacks glycosylation. Etanercept is a molecule composed of the extracellular part of human TNF type 2 receptor (TNF-R2, p75) fused to dimeric human IgG1 Fc, and is able to neutralize both ligands, TNF and lymphotoxin-[alpha].

In addition to these approved drugs, several others are currently in development. One next-generation TNF-[alpha] blocker currently in clinical trials, ozoralizumab, has been designed using nanobody technology based on proprietary camelid type single-domain antibody fragments (15) and is expected to show low immunogenicity by design; however, clinical studies will be needed to confirm this. In addition, a biological product similar to infliximab, CT-P13, has recently been approved by the US Food and Drug Administration (FDA) and is expected to show immunogenicity similar to that of the reference drug (16).

Treatment Failure to TNF-Antagonist Therapy

Although administration of these drugs lowers disease activity, inducing clinical remission in the majority of patients, therapeutic challenges with TNF antagonists are substantial and may result in inadequate responses in some patients. Some do not respond at all (primary response failure), and others lose response over time (secondary response failure), despite increased doses or more frequent administration of the drug (4, 17). The mechanisms of treatment failure can be attributed to many factors, including drug-related immunogenicity, treatment-related factors including dosing and bioavailability issues, pharmacokinetics and pharmacodynamics of the drug, individual patient-related factors, and disease-specific differences in the pathogenic role of TNF (18).

PRIMARY RESPONSE FAILURE

Primary response failure is clinically defined as a lack of improvement in clinical signs and symptoms during the induction therapy and has been reported to occur in about one-third of patients treated with TNF antagonists, including both RA and CD patients (4). The underlying reason for primary response failure is not always clear, but it may involve rapid clearance of the drug in the absence of immunogenicity or bioavailability issues such as inadequate dosing or patient compliance. Beyond altered pharmacokinetics, other causes of primary treatment failure include pharmacodynamics, such as non-TNF-driven disease mechanisms. Immunogenicity is usually not a contributing factor for primary treatment failure because the development of antibodies takes some time (several weeks to months).

SECONDARY RESPONSE FAILURE

Secondary response failure occurs when patients who had shown improvement of clinical symptoms during the induction therapy with TNF antagonists lose response over time and show relapses, despite increased or more frequent dosing. It has been shown that up to 50% of patients with RA and CD develop secondary nonresponsiveness due to loss of effectiveness of the drug over time (17). The mechanisms of secondary response failure can be diverse, but the most common cause is immunogenicity resulting in the development of antibody response to the TNF inhibitor (5). AD As will neutralize the drug rendering it ineffective or induce its rapid clearance through immune complex formation.

In addition to immunogenicity, other issues may also contribute to secondary nonresponsiveness, such as pharmacokinetic factors including inadequate drug concentrations in the circulation and subsequently in target tissues, which can result from accelerated drug clearance or increased consumption in periods of high disease activity. Pharmacodynamic issues that affect the pathological mechanisms driving inflammation such as infections, which can markedly alter the inflammatory process and cytokine balance, or concomitant treatment with other drugs may also result in secondary response failure and recurrence of the symptoms (14).

IMMUNOGENICITY TO TNF ANTAGONISTS

Repeated injections of exogenous proteins have been shown to trigger the immune response and the development of antibodies against the protein, which is commonly associated with a decrease in the effectiveness of the treatment (19,20). The ability of a therapeutic protein to trigger the immune system is related to its "foreignness"--the more it differs from the endogenous counterpart, the higher the chance that it elicits immunogenicity (20). Fully human antibodies are now produced by phage display technology or by using humanized mouse models; however, immunogenicity is still an issue. Etanercept is less immunogenic than monoclonal antibodies because only the fusion part of the protein may contain foreign T-cell epitopes (21 ). Certolizumab is expected to show reduced immunogenicity because it lacks the Fc fragment, which mediates binding and uptake of TNF/antibody complexes by antigen presenting cells; however, this needs to be confirmed by further clinical studies.

The mechanism of immunogenicity involves the uptake of these drugs by antigen-presenting cells, followed by cleavage into peptides that can be presented by HLA molecules. These peptide-HLA complexes are then recognized by T-cell receptors, thus inducing proliferation and expansion of the T-cell clone specific for the peptide. The efficiency of the antigen-presentation process (i.e., immunogenicity) depends on the peptides available or the immunogenic T-cell epitopes that can be found in the therapeutic protein and their ability to trigger an immune response (22). Immunogenicity is further influenced by the patient's genetic background, primarily the HLA haplotype, and the strength of binding between immunogenic drug-derived peptides and various HLA alleles (23 ). Further modulation of immunogenicity may occur by genetic variations in other immune regulatory genes such as interleukin (IL)-10 (24) and, importantly, the microenvironment in which the antigen-specific CD4+ T cells encounter antigen, including the presence of inflammatory cytokines that may skew T-cell differentiation into certain lineages (25 ). This is of interest given the autoimmune or chronic inflammatory status of the patient population treated with TNF antagonists, which may impact the overall immune response to these drugs. Patients who develop antibodies against one type of TNF antagonist are at higher risk for developing antibodies against another type of TNF inhibitor when switching therapy.

IBD patients with previous antiinfliximab antibodies were significantly more prone to develop antiadalimumab antibodies (33%) when switched to adalimumab compared to patients without previous antiinfliximab antibodies (26). Further investigation of patient-related immunogenicity factors could potentially predict the patients who are at increased risk for developing ADAs before starting the treatment.

Treatment-related factors such as the mode of administration also play an important role in immunogenicity. Use of high doses of a protein drug may reduce its immunogenicity by inducing immunological tolerance (27). Repeated doses over prolonged treatment time increase the risk of ADA development. Intravenous administration is generally thought to be less immunogenic than subcutaneous or intramuscular administration (28). Most TNF inhibitors are administered through repeated subcutaneous injections (with the exception of infliximab given intravenously), which is similar to the delivery of immunogenic vaccines. Of interest, combination therapy using TNF antagonists and immunosuppressive drugs such as methotrexate often decreases the incidence of antibody formation and enhances clinical efficacy (29, 30). However, because of risks commonly associated with long-term immunosuppressive therapy, an individualized approach based on risk--benefit evaluation is preferred (31).

ADA RESPONSE

According to a recent comprehensive review, 25.3% of all patients treated with infliximab developed ADAs (30). Adalimumab, a fully human antibody, was shown to elicit antibody response in 14.1% of patients in a combined cohort of RA, spondylarthritis, and IBD (32).

Neutralizing ADAs have the ability to directly interfere with the biological effect of the drug by decreasing or eliminating its ability to inhibit TNF-mediated signaling through cell surface TNF receptors. These antibodies may directly bind to the idiotype of the drug or to other sites in its proximity, eliciting steric hindrance that, in turn, prevents binding of the drug to TNF.

Nonneutralizing antibodies do not directly interfere with TNF binding but may compromise therapeutic efficacy by contributing to enhanced clearance of the drug from the circulation through immune complex formation and binding to Fc receptors on phagocytic cells (14). In case of subcutaneous administration, immune complexes may form around the injection site, which will prevent absorption of the drug into the circulation, reducing bioavailability in the target tissues (33).

In addition to the neutralizing vs nonneutralizing effect, the isotype of the ADA has additional relevance in influencing the pharmacokinetics and the types of adverse effects. Infliximab-specific antibodies are primarily IgG isotypes, although other isotypes such as IgA, IgM, and rarely IgE have been reported (34, 35). Of the IgG-type antibodies, IgG1 and IgG4 are most common for both infliximab (34) and adalimumab (18,36). The presence of the IgG4 types of antibodies is consistent with previous observations showing that this subtype is induced by repeated antigen exposure (37).

IgG antibodies (with the exception of IgG3) are known to bind to neonatal Fc receptors (FcRn) in the endosomes of endothelial cells following internalization, where they are protected from catabolism and recycled so that their half-lives are significantly longer than the half-lives of other antibodies (38). In addition, some IgG subclasses, and most effectively IgG1, are able to interact and trigger a variety of effector mechanisms such as immune complex formation, activation of complement, and Fc receptor--mediated processes, which may account for the altered pharmacokinetics, therapeutic nonresponsiveness, and side effects observed.

Overall, ADAs are associated with low trough drug concentration, reduced clinical response, or remission of clinical disease and increased incidence of infusion or injection-site reactions (39, 40). Drug bioactivity has been shown to disappear from circulation as soon as ADAs appear (9).

MANAGEMENT OF THERAPEUTIC FAILURE

In clinical practice, therapeutic failure to TNF antagonists is usually managed by increasing the dose of the drug or shortening the dose interval while continuing treatment with the same drug. Although this empirical approach may work well for some patients, there is a risk for worsening of symptoms for others during treatment optimization. If nonresponsiveness persists, the patient is usually switched to another type of TNF antagonist, anticipating the possible emergence of immunogenicity against the first drug. When treatment with the second drug fails, the patient is changed to a different class of biologicals such IL-6 antagonists, because treatment history is suggestive of the involvement of non-TNF-mediated pathways. In this empiric strategy, no early attempt is made to identify the mechanism for loss of response to TNF antagonist.

A rational and mechanistic alternative to this empiric strategy is to employ laboratory testing to measure drug concentration, or bioactivity, and detect ADA to select the best treatment strategy based on the most likely mechanism responsible for loss of response.

Support in favor of the test-guided strategy comes from recent studies showing significantly reduced mean treatment costs per patient, compared to the empirical approach, without differences in clinical efficacy (41-44).

Methods for Measuring Serum Drug Concentrations and ADAs

Several methodologies are currently available for measuring serum TNF antagonist drug concentrations and for detecting ADAs, which show significant variations in both analytical sensitivity and specificity (45-48). Most of these are primarily binding-based assays, including solid-phase ELISA methods (8, 45, 49) and liquid-phase, HPLC-based homogenous mobility shift assay (HMSA) (50); in addition, a functional cell-based reporter gene assay (RGA) has been developed (51 ) and is currently available for clinical use (52).

One of the most important criteria in assay development is the ability of the assay to accurately and reliably measure concentrations of functionally active drugs and ADAs. The difficulty in developing such assays derives from the fact that both the ADA and the most commonly used TNF antagonists are immunoglobulins themselves. In addition, they are measured from serum, which itself has high concentrations of endogenous immunoglobulins and frequently includes interfering factors such as rheumatoid factor and antiallotypic or heterophilic antibodies (53).

One shortcoming of all binding assays is that they do not distinguish between functional neutralizing vs non-neutralizing ADAs, although this information is essential for a more precise understanding of the mechanism of treatment failure. Functional assays, on the other hand, allow for the detection of biologically active, drug-neutralizing antibodies, which interfere with the drug-mediated blocking of the TNF-signaling pathway, leading to therapeutic nonresponsiveness. It is important to note that regulatory authorities such as the FDA recommend that functional cell-based assays be used to measure antibodies to therapeutic drugs, because bioassays are inherently more reflective of the in vivo situation (54).

Furthermore, detection of ADAs poses an additional challenge that affects nearly all methodologies, and that is drug interference caused by the presence of high concentrations of TNF antagonists in the sera during therapy. Most assays predominantly detect free ADAs in circulation, when they are present at concentrations that stoichiometrically exceed drug concentrations (Fig. 2). During continuous treatment, drug interference may lead to lower analytical sensitivity for antibody detection, which could result in underestimation of the total amount of ADA present or a false-negative result. The ability of detecting ADAs varies between assays; some are more prone to interference than others (Fig. 2). Although there are some newer methods developed for detecting ADA in the presence of a drug (6), these methods are not yet widely in use. Immunogenicity data should always be interpreted in the context of the assay used and considering factors such as timing of blood samples (using trough concentrations when possible) and assay strategy and characteristics. It is important to realize that even when these factors are considered, there can be discrepancies between ADAs produced in patients and the ADA concentrations detected by various different assays in different laboratories.

DETECTION OF ADAS BY BINDING ASSAYS

ELISA. The most commonly used assays for the detection of ADAs are ELISA methods, including traditional sandwich ELISA and bridging ELISA. Sandwich ELISAs use plastic-immobilized Fab or [F(ab').sub.2] fragments of the TNF antagonists for capture. Although this assay is affordable and easy to use, it can give false-positive or aberrant results due to nonspecific binding of cross-reacting antibodies to the immobilized drug, or due to aggregation of drug molecules on the plastic surface, which can either create new epitopes or hide the existing epitopes (14).

The bridging ELISA is a modified ELISA method (Fig. 3A), which takes advantage of the bivalency or multivalency of the main antibody isotypes. It uses plastic-immobilized drugs as solid phase, which will bind and pull down the ADAs from the patient's serum. Instead of using a detection antibody, enzyme-tagged drug is used for detection, with the ADA forming a bridge between the immobilized vs enzyme-tagged drug molecules due to the bivalency of IgG. This method is able to detect most types of IgGs and is easy to use, but there are several drawbacks that need to be recognized. False-positive results (Fig. 3A, left panel) can occur due to the presence of interfering antibodies that can cross-link the drug molecules added in the assay, such as rheumatoid factor that notoriously binds to the Fc part of IgG nonspecifically or the presence of antiidiotype antibodies. False-negative results (Fig. 3A, right panel) can occur due to drug interference, when the drug is present in high concentrations in the serum and forms complexes with the ADA, which will prevent its detection in the bridging ELISA (55). Furthermore, this method is unable to detect ADAs that are IgG4 isotype because IgG4 is usually monovalent and bispecific with half the molecule being exchanged after synthesis, which makes it invisible in bridging ELISA (36).

HMSA. This method uses size exclusion HPLC for the measurement of both drug and ADAs (Fig. 3B), and it is available for clinical use in the US (50). ADA in patient serum is detected by adding a fluorescently labeled drug, which will associate with the antibody present (fluid-phase binding). This is followed by size exclusion HPLC-based separation of ADA-bound vs free labeled drug.

Because the antigen--antibody binding takes place in homogenous liquid phase, the HMSA method is expected to show increased analytical specificity by overcoming potential artifacts related to solid-phase ELISAs, such as aggregation and nonspecific binding. Another advantage of the HMSA method compared to bridging ELISA is the ability to detect antibodies of all isotypes and all subclasses, including IgG4. Due to the incorporation of an acid-dissociation step during the ADA detection, the tolerance of HMSA for drug interference is dramatically improved and allows for the detection of ADAs in the presence of up to 60 [micro]g/mL serum infliximab (50).

Because of the need for fluorescent labeling, this method is expensive, which may be a limitation for routine clinical use. Another drawback of this test is its inability to distinguish between neutralizing and nonneutralizing antibodies. Method comparison studies have shown that the majority (68%) of HMSA-reported antibodies in infliximab-treated patients were not functionally active when tested in parallel with a functional test (47). These antibodies may lack a drug-neutralizing effect or may be blocked from detection by functional assays due to being complexed with a circulating drug in the serum or they may just be false-positive results indicating the presence of nonfunctional antibodies, which may not result in treatment failure.

FUNCTIONAL ASSAYS FOR THE DETECTION OF NEUTRALIZING ADAS

Initial cell-based assays were based on the ability of TNF to kill susceptible tumor cell lines (56); however, these assays did not translate into clinical use since they were too cumbersome and difficult to standardize.

Recently, a cell-based RGA has become available (51), which is currently the only method that allows for direct detection of functionally active drug and neutralizing antibodies and is available for clinical use (52). This method is based on the use of reporter cells carrying a TNF-inducible, NF-[kappa]B (nuclear factor-[kappa]B)-regulated firefly luciferase reporter-gene construct. The reporter gene turns on when TNF is added to the cells and binds to the TNF receptor, inducing firefly luciferase expression (Fig. 4). To control for serum matrix effects, affecting viability and cell numbers, this signal is normalized to the constitutively expressed Renilla luciferase carried within the same reporter cell. To measure drug activity, the patient serum is mixed with a fixed amount of TNF and then added to the cells. If the drug is present, it will inhibit the activity of TNF and will prevent the expression of the reporter gene. Thus, the amount of infliximab present in the serum will inversely correlate to the amount of luminescence produced by the cells (Fig. 4A).

For the detection of neutralizing ADA, the serum is preincubated first with a known concentration of the drug, and the assay is performed as described above. If the serum contains neutralizing antibodies, these antibodies will prevent the drug from interfering with the TNF-induced induction of the reporter gene, resulting in a luminescent signal (Fig. 4B). The amount of neutralizing antibody in the serum directly correlates with the amount of luminescence produced by the cells. The antibody is currently quantified by testing serial dilutions of the serum, and by identifying the highest dilution at which blocking of infliximab activity is no longer observed. The availability of ADA standards to infliximab (57) and adalimumab (58) will allow for reporting of absolute antibody concentrations once these standards are adopted and appropriately validated and correlated against the existing methods.

In contrast to binding assays, RGA measures TNF bioactivity, providing a direct functional assessment of the biologically active drug and the neutralizing activity of ADA. Neutralizing ADAs block the idiotype of the drug molecule directly or by steric hindrance, with sufficient strength to interfere with, or eliminate, the activity of the drug. Nonneutralizing ADAs that do not directly interfere with the activity of the drug (e.g., bind to regions other than the idiotype, such as the Fc region) will not be detected. This explains why the RGA may be less sensitive than binding assays (HMSA) or RIAs (47) because it only detects antibodies that truly neutralize and interfere with the biological activity, such as TNF signaling at the cellular level and likely in vivo. Direct detection of functionally active, neutralizing antibodies provides a more direct understanding of why therapies fail in some individuals and not in others. For example, binding but nonneutralizing antibodies detected by binding-based methods only, such as HMSA, may be transient or have no impact on treatment response (59) because they do not directly interfere with the biological activity of the drug in vivo. Nonneutralizing antibodies may, however, still alter the pharmacokinetics of drugs by other mechanisms, such as formation of immune complexes and subsequent removal of the drug by the mononuclear phagocyte system (17). The individual contributions of these different types of antibodies to therapeutic nonresponse are not well characterized, partly because the specific assays have not been widely available. Combined use of binding assays along with functional tests provides the necessary tools to clarify the role of both neutralizing and non-neutralizing antibodies in therapeutic response failure.

Due to the nature of the assay, that it is measuring TNF activity, the presence of any TNF-a inhibitor in the serum would cause interference with drug-activity measurement. As with the other assays, detection of ADA by RGA is sensitive to drug interference; increasing amounts of drug in the serum resulted in decreasing titers of ADAs detected (52). Therefore, it is important to follow the recommended approach to test for ADA when drug concentrations are lowest (trough).

Another cell-based method for measuring TNF inhibitors has been described for research use, which is based on measuring IL-6 released from a fibrosarcoma cell line upon TNF stimulation (58). However, the longer turnaround time of this test will likely prevent it from being used in clinical laboratories.

APPLYING LC-MS/MS FOR MEASURING TNF ANTAGONISTS

In addition to the binding and functional assays used to measure both drug and ADA, additional novel methodologies have recently been established for quantification of drug concentrations only, such as LC-MS/MS, which is available for clinical use (60). The method is based on using clonotypic peptides obtained by trypsin digestion from the heavy- and light-chain variable regions of primarily chimeric immunoglobulins, which are then quantified using LCMS/MS. The challenge of this method lies in being able to distinguish the tryptic peptides from the vast repertoire of endogenous immunoglobulins present in human serum, which is easier for chimeric antibodies such as infliximab but can be more challenging for fully human antibodies such as adalimumab. Another disadvantage of the method is the higher limit of detection compared to reported ELISA methods; nonetheless, it is analytically sensitive enough to differentiate clinically significant concentrations of infliximab in the serum and is comparable to the HMSA method (50).

Although most assays for TNF antagonists are thought to measure predominantly free drugs (not complexed with ADAs), the trypsin digestion step used in this method allows for the measurement of total infliximab, regardless whether it is free or bound in immune complex (50).

Test-Guided Strategy in TNF Antagonist Treatment

The use of a test-guided strategy in the setting of TNF antagonist treatment failure significantly reduces the cost of therapy per patient, as compared to empirical drug dose escalation (43, 44, 61). Testing-based strategies are not only cost-effective, but allow therapies to be tailored to the individual needs of patients, providing a personalized, rather than a universal, approach and reducing delays in effective treatment (45).

Patients are currently evaluated by monitoring disease activity using clinical criteria; monitoring TNF antagonists is not part of the routine clinical care. However, given the high frequency of loss of response to these drugs and their recognized immunogenic potential, laboratory methods to measure drug concentrations and detect ADA are increasingly useful in patient management.

The workup for therapeutic response failure includes measuring trough serum drug concentrations right before the patient is about to receive the next dose. Practices vary between laboratories; some laboratories measure both drug and ADA at the same time while others offer them as reflex tests, in which, if the drug is undetectable or very low, then the patient is reflexed to an ADA test. Depending on the results of the 2 tests, and in the clinical context of therapeutic failure, 4 different scenarios may occur, which require different management strategies (Fig. 5). This algorithm has been proposed by several investigators (4, 14, 41, 45, 47, 62) and has been supported by clinical trials involving CD patients treated with infliximab (43).

One scenario based on the test outcome is that both drug and ADA are below the level of detection at trough, despite the patient being compliant with therapy. In this group of patients, the response failure is likely nonantibody mediated, involving bioavailability or pharmacokinetic issues; therefore, the recommendation is to intensify treatment using the same TNF antagonist by increasing the dose, or shortening the dose interval, followed by reevaluation of clinical response.

The second scenario is when the drug is not detectable but ADAs are present, ideally confirmed by serial measurements, providing laboratory evidence for the emergence of ADAs, followed by declining drug concentrations. In this setting of persistent treatment failure, the recommendation is that these patients be switched to another TNF antagonist because antibodies are usually drug specific.

The third scenario is when the patient has detectable drug concentrations in the therapeutic range or higher and undetectable ADAs, but is still not responding to treatment. In this group of patients, if treatment failure persists, the recommendation is to reassess the clinical condition and to rule out other noninflammatory causes for treatment failure. Switching to another class of biologicals, targeting a different inflammatory mediator, for example, IL-6, may be useful.

Finally, the fourth and rarest scenario is when both drug and ADAs are detected in the serum. In this case, the recommendation is to repeat testing to rule out false-positive results. This scenario is expected to occur pre dominantly when the ADA concentrations are tested using binding-based assays, which detect all antibodies, including those that are nonfunctional. Factors that may bias test results include the occurrence of low avidity and/or transient antibodies, neutralization of excess antibodies by the drug (window phenomenon), variations due to serum albumin concentrations, and FcRn salvage activity (47). This group of patients would benefit from functional testing for the presence of neutralizing antibodies. If the results of the repeat test are the same, and the response failure persists, switching to another type of therapy, as in the previous scenario, would be beneficial.

Currently, measuring TNF antagonists and ADA is primarily indicated for the management of patients developing treatment failure and not for drug monitoring. Prospective randomized studies are necessary to establish appropriate testing intervals and clinical cutoff levels for therapeutic drug monitoring using these assays.

Summary and Future Directions

TNF antagonists constitute an important class of biologics that are effective and used worldwide for the treatment of chronic inflammatory diseases, including RA, IBD, and others. Immunogenicity of these antibody-based drugs constitutes an important issue that can lead to therapeutic nonresponsiveness in a number of patients. ADAs are associated with reduced serum drug concentrations, impaired treatment efficacy, and adverse effects such as hypersensitivity reactions and immune complex disease. Over 40% of patients treated with infliximab or adalimumab become sensitized and develop antibodies (63). Screening for ADAs is required for marketing of all novel biological therapeutics by regulatory agencies, including the FDA and European Medicines Agency (64-66), which further supports the importance of monitoring individual patients for drug immunogenicity. Given the increasing role of biopharmaceuticals, it is expected that clinical testing for ADA will be extended to other protein-based therapeutic drugs as well.

Currently, testing for TNF antagonist concentrations and ADAs is recommended in the context of therapeutic failure. Understanding the concepts and limitations of the available clinical tests is important for correct interpretation of test results; assays that report biological activities of both the TNF antagonist and the ADA are more meaningful because they mimic more closely the in vivo situation. Binding assays may over-report the presence of ADAs because they detect both neutralizing and nonneutralizing antibodies, which may or may not lead to therapeutic failure in vivo.

With the availability of clinical laboratory tests for the major types of TNF antagonists and ADAs, monitoring individual patients for circulating active drug concentrations and antibodies will likely become part of routine clinical practice.

Increasing evidence suggests that the use of a test-guided strategy significantly reduces the cost of therapy per patient compared to empirical drug dose escalation and also reduces delays in effective treatment. Implementing a personalized approach in which therapy is tailored to the individual needs of the patient, rather than using a universal approach with the costly and widely used TNF antagonists, is essential for safer, cheaper, and more effective treatment strategies.

Author Contributions: AH authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Acknowledgments: The authors acknowledge the expert help of Mary Paul in preparing the graphics for the figures.

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Eszter Lazar-Molnar [1] and Julio C. Delgado [1] *

[1] Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT.

* Address correspondence to this author at: Department of Pathology, University of Utah School of Medicine, 500 Chipeta Way MS115, Salt Lake City, UT, 84108. Fax 801-5845207; e-mailjulio.delgado@aruplab.com.

Received March 29, 2016; accepted May 23, 2016.

Previously published online at DOI: 10.1373/clinchem.2015.242875

[2] Nonstandard abbreviations: TNF, tumor necrosis factor; RA, rheumatoid arthritis; IBD, inflammatory bowel disease; CD, Crohn disease; ADA, antidrug antibody; mAb, monoclonal antibody; CHO, Chinese hamster ovary; CDR, complementarity determining region; PEG, polyethylene glycol; TNF-R2, TNF type 2 receptor; FDA, US Food and Drug Administration; FcRn, neonatal Fc receptor; HMSA, homogenous mobility shift assay; RGA, reporter gene assay.

Caption: Fig. 1. TNF antagonists approved for clinical use.

Infliximab is a chimeric antibody having mouse Fab and human IgG1 constant region. Adalimumab and Golimumab are fully human IgG1 antibodies. Certolizumab is a PEGylated humanized antibody Fab fragment with mouse CDR regions. Etanercept is a chimeric fusion protein of TNFR2 and IgG1 Fc. The binding targets of the drugs are indicated. LT-[alpha], lymphotoxin-[alpha].

Caption: Fig. 3. Binding-based assays used for detecting antibodies to TNF antagonists.

(A), The solid-phase bridging ELISA is based on bivalency of IgG, which can bridge plate-bound unlabeled and soluble enzymatically labeled drug molecules. Nonspecific antibodies that can crosslink drug molecules will cause false-positive results. Excess drug or the presence of IgG4 type ADAs will cause false-negative results. (B), The HMSA detects ADAs by complex formation with fluorescently labeled drug molecules added to the serum, followed by chromatographic (size-exclusion HPLC) separation of ADA-bound versus free labeled drug. HMSA detects any antibody that binds to the drug, including functional (neutralizing) and nonfunctional ADA.

Caption: Fig. 4. Functional cell-based RGA for detection of antibodies to TNF antagonists.

RGA uses cells carrying a TNF-inducible luciferase reporter-gene construct, which is turned on by TNF, generating luminescence. (A), For drug activity assay, serum is mixed with TNF and incubated with the cells. Presence of drug in the serum blocks TNF activity, decreasing luminescence. (B), For measuring neutralizing antibodies, the same assay is used but the serum samples are pre-incubated with the drug. In the absence of neutralizing antibodies, the drug blocks TNF activity, and there is no luminescence. When neutralizing ADA is present, the inhibition of TNF activity by the exogenous drug is released, the reporter gene is turned on and luminescence is generated.

Caption: Fig. 5. Proposed algorithm for interpretation of laboratory test results in patients showing therapeutic nonresponse to TNF antagonists.

Four scenarios are encountered for patients becoming nonresponsive to TNF antagonists. Serum trough concentrations for drug and ADA are measured using clinically validated test with predetermined cutoff levels for both drug and ADA.
Table 1. TNF antagonists currently in clinical use and their
structural and pharmacological properties.

TNF inhibitors          Infliximab         Certolizumab pegol

Molecule           Chimeric TNF antibody   Humanized antibody
                                           Fab, PEGylated

Expression         hIgG 1 [kappa],         hlgG4 Fab, mouse
construct          (a) murine Fab          CDR

Cell line used     Sp2/0 hybridoma         Escherichia coli
for expression

Sequence origin    [congruent to] 75%      [congruent to] 80%
                   human                   human

Method of          IV infusion every 6-8   SC every 2 weeks
administration     weeks

Half-life (days)           8-10                    14

Year of first              1998                   2008
FDA approval

TNF inhibitors            Etanercept                 Adalimumab

Molecule           Human TNF receptor fusion   Human TNF antibody
                   protein

Expression         DimerichlgGI Fc, fused to   hlgG1[kappa], selected
construct          hTNF-R2 (p75)               by phage display

Cell line used     CHO cells                   CHO cells
for expression

Sequence origin    Human                       Human

Method of          SC once or twice weekly     SC every 2 weeks
administration

Half-life (days)              3-5                      10-20

Year of first                1998                       2002
FDA approval

TNF inhibitors             Golimumab

Molecule           Human TNF antibody

Expression         hlgG1[kappa], produced by
construct          transgenic mouse
                   technology

Cell line used     SP2/0 hybridoma
for expression

Sequence origin    Human

Method of          SC once a month
administration

Half-life (days)              14

Year of first                2009
FDA approval

(a) hlg, human Ig; IV, intravenous; SC, subcutaneous injection.

Fig. 2. Detection of ADAs in the presence of TNF antagonists.

Detection of ADAs varies depending on the assay used and the amount of
drug in the serum, which may form immune complexes, decreasing the
availability of free ADA detected by most assays. The HMSA uses acid
treatment to dissociate immune complexes, which makes it less
sensitive to drug interference.

1 ADA assay
ELISA          -         -            -           +
HMSA           -        +/-           +           +
RGA            -         -           +/-          +
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