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Detection of anti-cytokine antibodies and their clinical relevance Expert Rev. Clin. Immunol. Early online, 1–19 (2014)
Anthony Meager*1 and Meenu Wadhwa2 1 Regaem Consultants, 62 Whitchurch Gardens, Edgware, Middlesex, HA8 6PD, UK 2 Biotherapeutics Group, The National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK *Author for correspondence: Tel.: +44 208 952 4834 [email protected]
Cytokines regulate many aspects of cell growth and differentiation and play pivotal roles in the orchestration of immune defence against invading pathogens. Though ‘self’ proteins, they are potentially immunogenic and can give rise to anti-cytokine autoantibodies (aCA). The main foci of the article are a critical summary of the various methodologies applied for detecting and measuring aCA and a broad review of studies of the occurrence, characterization and clinical relevance of aCA in normal healthy individuals, patients with autoimmune diseases or microbial infections and aCA in patients whose disease is treated with recombinant cytokine products. The need for technical and methodological improvement of assays, including validation and standardization, together with approaches to harmonize calculation and reporting of results is also discussed. KEYWORDS: anti-cytokine autoantibodies • bioassays • clinical relevance • immunoassays• neutralizing antibodies
Cytokines & anticytokine antibodies Cytokines, a diverse class of inducible, secreted proteins, exert biological activity via specific cell surface membrane receptors coupled to signal transduction pathways. Produced in response to external and endogenous stimuli, they act on cells bearing the cognate receptors to modulate proliferation, differentiation and immune regulatory activity. Since cytokines are protein in nature, they bear antigenic determinants (epitopes) that may be recognized by immune systems to trigger antibody development. This occurs in situations where immune tolerance is broken, as for example in certain autoimmune diseases, or following administration of therapeutic cytokine products. Human anticytokine antibodies (aCAs) are polyclonal autoantibodies mainly of the IgG class and are either nonneutralizing or neutralizing or both [1]. Neutralizing aCAs inhibit cytokine binding to its cognate cell surface receptors and thus severely reduce or abrogate cytokine activities. At high, persistent titers in vivo, such aCAs effectively reduce bioavailability of cytokines, negatively impacting on their pharmacology and roles in normal physiological regulatory processes. Understanding the clinical impact of aCA on disease progression, management and outcomes is of
informahealthcare.com
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prime medical importance. Therefore, development and implementation of analytical tests (assays) to detect and quantify aCA have a crucial role in scientific and clinical studies to investigate the properties of such antibodies and their clinical relevance. Cytokine source for aCA assays
Before describing assays to detect and measure aCA, it is appropriate to consider the sources and characteristics of cytokines used in their construction, which are critical to their performance. Currently, most human cytokines are manufactured in bulk by rDNA processes from bacteria, for example, Escherichia coli or yeasts, for example, Pichia pastorus, Hansuela polymorpha. While their primary amino acid sequences are normally precisely replicated, the recombinant cytokines produced from such sources will often differ structurally from cytokines produced by human cells in vivo due to differences in post-translational modifications, for example, glycosylation, and/or the ‘harsh’ denaturing procedures required for extraction and purification. For example, recombinant proteins derived from E. coli lack the posttranslational modifications, for example, glycosylation of proteins produced by eukaryotic, mammalian cells and are denatured during
2014 Informa UK Ltd
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Review
Meager & Wadhwa
extraction and subsequently renatured. Since some antibodies recognize the structural differences between recombinant and ‘naturally produced’ cytokines, such differences may have significant implications for detection of aCA and may also generate false results [2]. Deformed (purposely), modified (e.g., Histag labeled proteins), denatured or aggregated recombinant cytokines may also negatively impact on the performance and reliability of assays to detect aCA. Therefore, assays should be carried out with more than one preparation of recombinant cytokine, that is, perform as many confirmatory assays as practical. All types of binding immunoassays should be properly validated; however, regulatory criteria are much stricter for assays to detect and measure aCA developed following treatment with therapeutic cytokine than for those used for detection of aCA in autoimmune patients [2–4]. Solid-phase immunoassays
The ELISA is the laboratory standard solid-phase method for quantifying cytokines and is readily adapted for aCA detection and measurement [2,5]. Here, cytokine protein is immobilized by binding to the plastic surface of microtiter plates, aCA in added test samples binds to cytokine, and can be detected by the addition of specific anti-HuIgG antibodies. The latter is either conjugated to an enzyme, for example, alkaline phosphatase, horseradish peroxidase, or a small molecule, for example, biotin, which acts to amplify output signal following binding of an enzyme conjugate, for example, streptavidin–alkaline phosphatase (streptavidin binds with very high affinity to biotin). The performance of ELISA requires both binding and washing steps and the addition of enzyme substrate. The final color is measured spectrophotometrically and is directly proportional to aCA concentration in test samples. For precise measurement of aCA concentration, ELISA requires calibration with a strongly positive aCA control preparation and must display high specificity, sensitivity and robustness including inter-/ intra-assay reproducibility. Further, specificity of aCA binding should be demonstrated by addition of cytokine, which acts as a specific competitive inhibitor, into the test sample mix. ELISAs have the advantage that relatively few reagents and equipment are required and can be performed in standard laboratory conditions, but may not provide satisfactory data when applied to clinical samples such as sera or plasma. They are subject to variable matrix effects and interference due to immune complexes, rheumatoid factor, complement, and so on. ELISAs are often poorly tolerant to high levels of therapeutic cytokine that in some instances coexist with aCA (TABLE 1) [2,5,6]. A frequent modification of ELISA, a ‘bridging’ ELISA, uses cytokine both for capture of aCA and its detection; the latter is appropriately conjugated or tagged in order such that a colorimetric (or other, see below) signal is developed. A typical example is: streptavidin-coated plates to capture biotin-tagged cytokine; aCA test sample; digoxigenin-tagged cytokine to complete trimolecular ‘bridge’; enzyme-labeled antidigoxigenin antibodies to generate colorimetric readout. Advantageously, bridging ELISAs do not require a specific human aCA-positive doi: 10.1586/1744666X.2014.918848
control (a corresponding animal aCA can be substituted in the ‘bridge’) and enzyme-conjugated antihuman IgG for detection. Although they have low backgrounds, they are often poorly tolerant to circulating cytokine and subject to similar matrix effects to conventional ELISA, and may not be able to detect low-affinity antibodies, for example, IgM, or functionally monovalent antibodies such as IgG4 (TABLE 1) [2,5,6]. Replacement of enzyme conjugate with a fluorescent conjugate, such as europium–streptavidin, provides the means to develop immunofluorometric assays. These are read with a fluorimeter and may exhibit greater specificity than ‘traditional’ ELISA [7]. Other adaptations and readout methods, for example, dissociation-enhanced lanthanide fluorescent immunoassay, time-resolved fluorescence resonance energy transfer, are also available. Alternatively, the enzyme conjugate may be replaced with a ruthenium-conjugated protein. An oxidation–reduction reaction of ruthenium ions in the presence of tripropylamine generates electrochemiluminescence (ECL) under appropriate voltage stimulation. As ruthenium ions are recycled, the ECL signal is amplified to yield increased sensitivity. Such ECLbased assays may be developed in either conventional or bridging assay protocols [8,9]. Since they offer greater specific dynamic range, higher sensitivity and are less prone to matrix effects than ELISA, they are becoming more widely used for antibody detection (TABLE 1). BiaCore assays
Virtually all ELISA and related immunoassay methods involve multiple washing steps, which could remove rapidly dissociating low-affinity aCA. In some cases, the latter may constitute a significant proportion of total aCA, for example, in early immune responses, and require other detection methods. Surface plasmon resonance technology, which offers real-time detection of aCA with rapid off-rates, has been developed in BiaCore systems (GE Healthcare). Binding of aCA is measured by a dedicated ‘sensorgram’ as they flow over a cytokine-coated sensor chip. This generates an increase in signal that reaches equilibrium according to the Ka (complex formation rate). Addition of an appropriate buffer releases aCA with a corresponding decrease in signal according to the dissociation rate, Kd. Biacore evaluation software calculates Ka and Kd by fitting data to interaction models [2,10]. Generally, surface plasmon resonance BiaCore systems are less sensitive for high-affinity aCA and ‘low throughput’ as opposed to ELISA, immunofluorometric, dissociation-enhanced lanthanide fluorescent immunoassay and ECL assays. However, they are more likely to detect lowaffinity aCA. Since they can be used for isotyping and kinetics, they are more suited for confirmatory testing and characterization of aCA (TABLE 1). Fluid-phase assays
Increasing criticism of solid-phase assays that they lack specificity and sensitivity (not always the case) and are prone to yield false positives (unacceptable for clinical analyses) has generated interest in the development and use of fluid-phase assays. The Expert Rev. Clin. Immunol.
Detection of aCA & their clinical relevance
Review
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Table 1. Advantages and disadvantages of assay methods for detection and measurement of anticytokine autoantibodies. Method
Advantages
Disadvantages
ELISA
Readily available reagents and equipment; Easy and inexpensive to perform; high throughput
Variable sensitivity and dynamic range for measurement; Low-affinity antibodies, e.g., IgM, may be lost in washing steps; immobilized ‘capture’ cytokine may be deformed so that conformational epitopes no longer recognized; poor tolerance to circulating cytokine, especially in the ‘bridging’ format; cannot detect monovalent IgG4 antibodies; subject to variable matrix effects
IFMA DELFIA ECLA
Higher sensitivity and greater specific dynamic range than ELISA; better tolerance to circulating cytokine than ELISA; high throughput
More expensive reagents and equipment required than for ELISA; rapidly dissociating antibodies may not be detected; immobilized ‘capture’ cytokine may be deformed so that conformational epitopes are no longer recognized; may not detect monovalent IgG4 antibodies; subject to variable matrix effects
SPR
Capable of detecting lowaffinity IgM and monovalent IgG4 antibodies; characterization of antibody capabilities, e.g., kinetics, isotyping
Expensive equipment; slow to perform, especially with multiple samples; less sensitive than other methods; immobilized ‘capture’ cytokine may be deformed so that conformational epitopes are no longer recognized; subject to variable matrix effects
Multiplex assays
Capable of detecting several different aCA within a single assay
Expensive, nontransferable equipment; difficult to establish specific dynamic range for each aCA; immobilized ‘capture’ cytokine may be deformed so that conformational epitopes are no longer recognized; cannot detect monovalent IgG4 antibodies; subject to variable matrix effects
RIA RLBA LIPS
Cytokine in solution exhibits conformational epitopes; good specificity, sensitivity and specific dynamic range; less likely to produce ‘false’ results than solid-phase assays
Use of radioisotopes for RIA and RLBA; coupling of cytokine to radioisotope or fusion with luminescing protein may induce changes in cytokine conformation; prone to high backgrounds; subject to variable matrix effects
Bioassays
Capable of detecting and quantifying neutralizing aCA; can be very sensitive
Not able to measure nonneutralizing aCA, which may constitute a significant proportion of total aCA––these may have clinical relevance for cytokine clearance; require cell culture facilities and expertise and thus relatively expensive to perform; inherently variable and difficult to standardize; difficult to validate; difficulties in calculating and reporting neutralizing titers; subject to variable matrix effects
aCA: Anticytokine autoantibodies; DELFIA: Dissociation-enhanced lanthanide fluorescent immunoassay; IFMA: Immunofluorometric assays; LIPS: Luciferase immunoprecipitation systems; RLBA: Radioligand-binding assays; SPR: Surface plasmon resonance.
obvious benefits are that as antigens are in solution, their 3D structures are intact, their epitopes are not masked on immobilization to solid supports and interaction with aCA is therefore more specific and encompassing. However, cytokines still need to be labeled in some way, either radiolabeled or coupled to a (nonradioactive) tag or protein: these procedures may affect structure. The most familiar methods are radioimmunoassays where the cytokine is first radiolabeled [5]. This may be done directly with radio-iodine isotopes (125I) or indirectly using in vitro eukaryotic (mammalian) transcription and translation systems. Cytokines, for example, may be transcribed and informahealthcare.com
translated from cDNAs in the presence of 35S-methionine. 125 I- or 35S-labelled cytokines are incubated with aCA test samples in fluid phase before immune complexes are isolated by either immunoprecipitation with polyethylene glycol or bound to protein A/G-coated resins or beads [11]. Such assays have been dubbed ‘radioligand binding assays’. Alternatively, the in vitro transcription/translation platform can be used to generate (nonradioactive) fusion proteins of Renilla luciferase and cytokines. In what are known ‘luciferase immunoprecipitation systems (LIPSs)’, Renilla luciferase-tagged cytokines, as cell extracts, are incubated with test aCA samples in solution; doi: 10.1586/1744666X.2014.918848
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complexes are precipitated with protein A/G beads and luminescence measured [12]. Despite the advantages that these assays offer, they are prone to interference by serum/plasma matrix and circulating cytokine levels, have high backgrounds, may not detect low-affinity IgM aCA and are not easy to automate (TABLE 1).
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Bioassays
Although immunoassays can, if properly validated, determine the immunochemical concentrations of aCA, they do not reveal the proportion of neutralizing antibodies (NAbs), if present in a sample, contains. This requires a functional assay in which the inhibitory action of neutralizing aCA on cytokine activity is quantified. Functional assays for cytokines are based on the measurement of biological activity, triggered via cell surface receptors, in in vitro cell-based systems. These activities include stimulation or inhibition of cell proliferation, immune modulatory and antiviral actions, differentiation and cytotoxicity (apoptosis or necrosis) and are cytokine’ concentration dependent, thus permitting generation of dose–response data and thus potency determinations. Assays can be processed at both ‘early’ and ‘late’ stages of cellular responses. It is however beyond the scope of this article to provide detailed information of the plethora of cytokine bioassays, but these have been extensively reviewed [13,14]. The main advantages of such assays are that they are mostly very sensitive (though not always equaling the high sensitivity of immunoassays), endpoints are readily defined and they can be calibrated with WHO international biological standards of cytokines with assigned potencies. The drawbacks are that they are inherently variable, labor intensive to perform, often lack specificity and are time dependent, frequently requiring more than 1 day for reaching endpoints. Nevertheless, they may be adapted to measure the inhibitory activity of cytokine antagonists including neutralizing aCA. These act by preventing cytokine binding to cognate cell surface receptors, thus inhibiting cytokine activity. This ‘blocking’ activity can be measured in so-called ‘neutralization assays’ where activity reduction of a fixed concentration of cytokine by serially diluted aCA is evaluated [2,14,15]. The neutralizing capacity or strength of aCA is dependent on several factors including ability of antibodies to bind, strength of their binding (intrinsic and functional affinities), immunoglobulin class and type and epitope location on the surface of the cytokine molecule. Thus, a wide diversity of NAbs to cytokine molecules exists [15]. Currently, very few reference preparations of human neutralizing aCA, for example, anti-IFN-a and anti-IFN-b [16], anti-GM-CSF [17], are available; they are intended only for monitoring the sensitivity of cell-based assays. They cannot be used in the same way as the WHO IS for interferon (IFN) ligands to calibrate assays for potency determinations. Thus, neutralizing activity cannot be expressed in international (reference) units. This lack of means for uniformly expressing neutralizing activity has led to diverse analytical methods for assessing and calculating it and has already led to wide variations in the reporting of neutralizing doi: 10.1586/1744666X.2014.918848
aCA titers or concentrations. Nevertheless, certain methodological elements are common to most approaches for measuring aCA. First, the majority of assays are based on a ‘constant cytokine method’ where dilutions of test sample are mixed with a fixed concentration of cytokine and incubated together for 1– 2 h to allow neutralization to take place. The mixtures are then applied to the cytokine-sensitive cells and the assays are processed as if cytokine per se were being tested. Neutralizing activity is measured as the aCA dilution that effectively reduces cytokine potency by a significant, quantifiable degree, which is generally a fixed percentage, for example, 50, 80 and 90%. Despite theoretical difficulties inherent in antibody: cytokinebinding kinetics – largely unresolved for polyclonal IgG – the common practice is therefore to express ‘potency’ or neutralizing activity as the dilution, for example, 1/1000, 1/1280, 1/ 5000, of test sample that results in the chosen ‘endpoint’. For reporting purposes, the reciprocals of these dilutions, for example, 1000, 1280, 5000, are often used. In the IFN field, an analytical approach developed by Kawade and colleagues has led to the development of a formula that calculates the neutralizing titer as ‘10-fold reducing units’ from antiviral IFN neutralization assays (AVINA) in which IFN activity is reduced from 10 to 1 laboratory units [16]. While this approach has often found favorable for determining and reporting anti-IFN neutralizing titers, its applicability to other neutralizing aCA has not been established. Thus, in some instances, the simple practice of estimating the volumetric equivalent of the titer is used, for example, 1 ml equivalent to 1/1000 of the starting test material [18]. As with immunoassays, appropriate validation (qualification) is crucial for generating reliable data. Guidance papers, focusing on design, optimization and validation of cell-based assays, have been published [19–21]. Overall, these summarize the practical and analytical considerations needed for the development of bioassays to accurately measure the neutralizing activity of aCA. While it is beyond the present article to cover the extensive details of these guidance reviews, it is pertinent to point out that several of the technical issues that affect the performance and outcome of immunoassays, including variable effects of matrix, circulating cytokines and immune complexes, rheumatoid factor and complement, excipients, are also common to bioassays. Over and above these issues are a host of variables including cell passage levels, cell growth culture medium and serum levels, temperature, pH, microtiter plating effects, impacting on assay performance and sensitivity. Thus, each bioassay will have its own construction and performance criteria, and validation should therefore be carried out on a case-by-case basis. Endogenous (or naturally occurring) aCA in health & disease
Central and peripheral immune ‘tolerance’ prevent the emergence of autoantibodies to self-proteins (antigens). Their breakdown, resulting in autoantibodies, is currently hypothesized to arise due to subpotent thymic expression of target self-antigens Expert Rev. Clin. Immunol.
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Detection of aCA & their clinical relevance
(TSAg), leading to enhanced escape of autoreactive T cells and/or repeated inappropriate stimulation by TSAg in the periphery [22–24]. There are, however, as yet unexplained biases in autoantibody development, which have suggested other regulatory mechanisms impinge on autoimmunization, for example, the actions of Treg. Connections to epidemiological and demographical differences, provoking microbial agents and drugs, tumors, inflammation, genetic associations and old age have all been implicated as potential causative and sustaining factors. Alternatively, it has been hypothesized that any external or intracellular stimulus that creates a ‘dangerous’ environment increases the risk of autoimmunization [25]. aCAs are among diverse autoantibodies that arise sporadically during the lifetime of healthy individuals, or with varying, usually increased, frequency in specific diseases. Using both immunoassay and bioassay approaches, the prevalence and characteristics of these aCA have been investigated [2–4,21]. These typically are present as circulating IgG autoantibodies, usually at low to undetectable prevalence and titers in the blood of normal healthy individuals, but with increased prevalence and persistent high titers in certain autoimmune diseases, where sometimes they correlate with immunodeficiency and pathogenesis [26]. Interestingly, disease-associated aCAs appear to be limited to only about a third of all known cytokines. Most targeted cytokines are type I, II and III IFNs, IL-1a, IL-6, IL-8, IL-12, IL-17A, IL-17F, IL-22, G-CSF, GM-CSF and TNF-a. In health
Regarding aCA in healthy individuals, the expectations are that either they would be completely absent, or occur sporadically in the healthy population at large, or are more widespread, but remain undetected either due to the insufficient sensitivity of assay methods or being ‘hidden’ by association with circulating cytokine or other substances. It is perhaps not surprising that examples of all three of these scenarios have been reported in studies where sera or plasma from healthy individuals have been analyzed for the presence of specific aCA (TABLE 2). Given the significant pitfalls in determining the presence of aCA without a true negative control group of sera or assay standardization, these results often require further authentication and remain controversial in some instances. For example, autoantibodies against IFN-a/w were first reported in 1990, but bone fide neutralization of IFN activity was difficult to demonstrate. Later studies have indicated that they could be present in up to 2% of healthy individuals (TABLE 2) [27,28]. Against IFN-b, another type I IFN or IFN-l (type III IFN), prevalence is likely to be even lower. Although early studies suggested the widespread occurrence of anti-IFN-g (type II IFN) [29], this has not subsequently been confirmed [12,28]. Regarding other cytokines, including many interleukins, G-CSF, GM-CSF and TNF-a, Watanabe and coauthors [30,31] have reported that aCAs against them are ubiquitous in healthy individuals, but would go undetected by most assay protocols as they are bound to circulating cytokine in immune complexes. While these informahealthcare.com
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results, in particular for anti-GM-CSF, are disputed due to technical problems with the assays used [32], the potential presence of cytokine: aCA IgG complexes for several cytokines cannot be disregarded. Indeed, such immune complexes probably do exist for anti-IL-1a [33] and anti-IL-8 (TABLE 1) [30,34]. Currently, literature reports indicate a 5–25% prevalence of antiIL-1a, increasing slightly with age [27,35], 11–13, 8, 0.4 and 0.1% for anti-G-CSF [30,36], anti-GM-CSF [28,30], anti-IL-10 [37] and anti-IL-6 [38], respectively, and absence (if potential immune complexes are discounted) of anti-IL-2, anti-IL-4, anti-IL-12, anti-IL-17A, anti-IL-17F, anti-IL-17F, antiIL-22 and anti-TNF-a (TABLE 2) [9,12,28,39,40]. While considerable uncertainties remain concerning the real prevalence and circulating levels of aCA from data derived from assays using sera or plasma, a clearer picture for certain specific aCA has emerged from the study of pharmaceutical IgG preparations, which include both intravenous (IVIG) and intramuscular (IMIG) immunoglobulin products. While immunoassays, especially ELISA, are not suitable for measuring aCA in IVIG/IMIG products on account of the very high total IgG concentration, sensitive bioassays are capable of quantifying neutralizing aCA. For instance, when tested by AVINA, most IVIG and IMIG products moderately neutralized IFN-a subtypes and also IFN-w (60% sequence homology to IFN-a), but more rarely and not independently of IFN-a, IFN-b (30% sequence homology to IFN-a) and the distantly related IFN-l (TABLE 2) [18,41]. Since neutralizing activity was wholly absent in some IVIG batches, it is believed that the NAbs against IFNs arise from plasma(s) of one or a few individuals with high titers, which are subsequently diluted in the plasma pool derived from thousands of blood donations. Such individuals may have appeared outwardly healthy, but may have had subclinical infection or disease at the time of blood donation. For example, the chickenpox herpes virus, Varicella zoster, has been associated with the development of autoantibodies to IFN-a [42]: interestingly, highest titers of anti-IFN-a NAbs were found in batches of Varicella zoster-specific IMIG [18]. In contrast, despite a report of anti-IFN-g (type II IFN) in sera and IVIG that selectively inhibited IFN-g-mediated HLA-DR and interfered in IFN-g-stimulated mixed lymphocyte reactions [29], no IVIG/IMIG batches neutralized IFN-g-mediated antiviral activity in AVINA [18,29]. Such discrepant results appear odd given that all IFN-g activities are mediated via the same cell surface receptor. Besides IFNs, large numbers of IVIG/IMIG batches strongly neutralize IL-1a and GM-CSF in their respective bioassays, probably reflecting the significant prevalence of these aCA in the sera/plasma of healthy individuals (TABLE 2) [18]. The antiGM-CSF Abs neutralized recombinant GM-CSF derived from E. coli, yeast and CHO cells and bound to these in immunoblots, thus confirming their specificity for GM-CSF [18]. However, no neutralization of IL-2, IL-4, IL-6, IL-10, IL-12, GCSF and TNF-a was detected in multiple IVIG/IMIG batches (TABLE 2). The neutralizing aCAs against IFNs, IL-1a and GMCSF appear to have no overt effects on health, though doi: 10.1586/1744666X.2014.918848
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Table 2. Anticytokine autoantibodies in healthy individuals. Cytokine
Cytokine properties
Test material
Prevalence % positive
Neutralizing or not (+/-)
Comments
Ref.
IFN-a/w
Mixture of 12 related a-helical, monomeric IFN-a subtypes and IFN-w; antiviral, antiproliferative, immune modulatory.
S/P IVIG IMIG
1–2 74-IVIG 40-IMIG
+/+ +
Occasional positives. Significantly neutralize IFN-a2. Most other IFN-a subtypes and IFN-w neutralized to varying degrees
[18,27, 28,41]
IFN-b
Single a-helical, monomeric, glycosylated IFN; Similar activities to IFN-a.
S/P IVIG IMIG
Review
Detection of anti-cytokine antibodies and their clinical relevance Expert Rev. Clin. Immunol. Early online, 1–19 (2014)
Anthony Meager*1 and Meenu Wadhwa2 1 Regaem Consultants, 62 Whitchurch Gardens, Edgware, Middlesex, HA8 6PD, UK 2 Biotherapeutics Group, The National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK *Author for correspondence: Tel.: +44 208 952 4834 [email protected]
Cytokines regulate many aspects of cell growth and differentiation and play pivotal roles in the orchestration of immune defence against invading pathogens. Though ‘self’ proteins, they are potentially immunogenic and can give rise to anti-cytokine autoantibodies (aCA). The main foci of the article are a critical summary of the various methodologies applied for detecting and measuring aCA and a broad review of studies of the occurrence, characterization and clinical relevance of aCA in normal healthy individuals, patients with autoimmune diseases or microbial infections and aCA in patients whose disease is treated with recombinant cytokine products. The need for technical and methodological improvement of assays, including validation and standardization, together with approaches to harmonize calculation and reporting of results is also discussed. KEYWORDS: anti-cytokine autoantibodies • bioassays • clinical relevance • immunoassays• neutralizing antibodies
Cytokines & anticytokine antibodies Cytokines, a diverse class of inducible, secreted proteins, exert biological activity via specific cell surface membrane receptors coupled to signal transduction pathways. Produced in response to external and endogenous stimuli, they act on cells bearing the cognate receptors to modulate proliferation, differentiation and immune regulatory activity. Since cytokines are protein in nature, they bear antigenic determinants (epitopes) that may be recognized by immune systems to trigger antibody development. This occurs in situations where immune tolerance is broken, as for example in certain autoimmune diseases, or following administration of therapeutic cytokine products. Human anticytokine antibodies (aCAs) are polyclonal autoantibodies mainly of the IgG class and are either nonneutralizing or neutralizing or both [1]. Neutralizing aCAs inhibit cytokine binding to its cognate cell surface receptors and thus severely reduce or abrogate cytokine activities. At high, persistent titers in vivo, such aCAs effectively reduce bioavailability of cytokines, negatively impacting on their pharmacology and roles in normal physiological regulatory processes. Understanding the clinical impact of aCA on disease progression, management and outcomes is of
informahealthcare.com
10.1586/1744666X.2014.918848
prime medical importance. Therefore, development and implementation of analytical tests (assays) to detect and quantify aCA have a crucial role in scientific and clinical studies to investigate the properties of such antibodies and their clinical relevance. Cytokine source for aCA assays
Before describing assays to detect and measure aCA, it is appropriate to consider the sources and characteristics of cytokines used in their construction, which are critical to their performance. Currently, most human cytokines are manufactured in bulk by rDNA processes from bacteria, for example, Escherichia coli or yeasts, for example, Pichia pastorus, Hansuela polymorpha. While their primary amino acid sequences are normally precisely replicated, the recombinant cytokines produced from such sources will often differ structurally from cytokines produced by human cells in vivo due to differences in post-translational modifications, for example, glycosylation, and/or the ‘harsh’ denaturing procedures required for extraction and purification. For example, recombinant proteins derived from E. coli lack the posttranslational modifications, for example, glycosylation of proteins produced by eukaryotic, mammalian cells and are denatured during
2014 Informa UK Ltd
ISSN 1744-666X
1
Expert Review of Clinical Immunology Downloaded from informahealthcare.com by Washington University Library on 06/22/14 For personal use only.
Review
Meager & Wadhwa
extraction and subsequently renatured. Since some antibodies recognize the structural differences between recombinant and ‘naturally produced’ cytokines, such differences may have significant implications for detection of aCA and may also generate false results [2]. Deformed (purposely), modified (e.g., Histag labeled proteins), denatured or aggregated recombinant cytokines may also negatively impact on the performance and reliability of assays to detect aCA. Therefore, assays should be carried out with more than one preparation of recombinant cytokine, that is, perform as many confirmatory assays as practical. All types of binding immunoassays should be properly validated; however, regulatory criteria are much stricter for assays to detect and measure aCA developed following treatment with therapeutic cytokine than for those used for detection of aCA in autoimmune patients [2–4]. Solid-phase immunoassays
The ELISA is the laboratory standard solid-phase method for quantifying cytokines and is readily adapted for aCA detection and measurement [2,5]. Here, cytokine protein is immobilized by binding to the plastic surface of microtiter plates, aCA in added test samples binds to cytokine, and can be detected by the addition of specific anti-HuIgG antibodies. The latter is either conjugated to an enzyme, for example, alkaline phosphatase, horseradish peroxidase, or a small molecule, for example, biotin, which acts to amplify output signal following binding of an enzyme conjugate, for example, streptavidin–alkaline phosphatase (streptavidin binds with very high affinity to biotin). The performance of ELISA requires both binding and washing steps and the addition of enzyme substrate. The final color is measured spectrophotometrically and is directly proportional to aCA concentration in test samples. For precise measurement of aCA concentration, ELISA requires calibration with a strongly positive aCA control preparation and must display high specificity, sensitivity and robustness including inter-/ intra-assay reproducibility. Further, specificity of aCA binding should be demonstrated by addition of cytokine, which acts as a specific competitive inhibitor, into the test sample mix. ELISAs have the advantage that relatively few reagents and equipment are required and can be performed in standard laboratory conditions, but may not provide satisfactory data when applied to clinical samples such as sera or plasma. They are subject to variable matrix effects and interference due to immune complexes, rheumatoid factor, complement, and so on. ELISAs are often poorly tolerant to high levels of therapeutic cytokine that in some instances coexist with aCA (TABLE 1) [2,5,6]. A frequent modification of ELISA, a ‘bridging’ ELISA, uses cytokine both for capture of aCA and its detection; the latter is appropriately conjugated or tagged in order such that a colorimetric (or other, see below) signal is developed. A typical example is: streptavidin-coated plates to capture biotin-tagged cytokine; aCA test sample; digoxigenin-tagged cytokine to complete trimolecular ‘bridge’; enzyme-labeled antidigoxigenin antibodies to generate colorimetric readout. Advantageously, bridging ELISAs do not require a specific human aCA-positive doi: 10.1586/1744666X.2014.918848
control (a corresponding animal aCA can be substituted in the ‘bridge’) and enzyme-conjugated antihuman IgG for detection. Although they have low backgrounds, they are often poorly tolerant to circulating cytokine and subject to similar matrix effects to conventional ELISA, and may not be able to detect low-affinity antibodies, for example, IgM, or functionally monovalent antibodies such as IgG4 (TABLE 1) [2,5,6]. Replacement of enzyme conjugate with a fluorescent conjugate, such as europium–streptavidin, provides the means to develop immunofluorometric assays. These are read with a fluorimeter and may exhibit greater specificity than ‘traditional’ ELISA [7]. Other adaptations and readout methods, for example, dissociation-enhanced lanthanide fluorescent immunoassay, time-resolved fluorescence resonance energy transfer, are also available. Alternatively, the enzyme conjugate may be replaced with a ruthenium-conjugated protein. An oxidation–reduction reaction of ruthenium ions in the presence of tripropylamine generates electrochemiluminescence (ECL) under appropriate voltage stimulation. As ruthenium ions are recycled, the ECL signal is amplified to yield increased sensitivity. Such ECLbased assays may be developed in either conventional or bridging assay protocols [8,9]. Since they offer greater specific dynamic range, higher sensitivity and are less prone to matrix effects than ELISA, they are becoming more widely used for antibody detection (TABLE 1). BiaCore assays
Virtually all ELISA and related immunoassay methods involve multiple washing steps, which could remove rapidly dissociating low-affinity aCA. In some cases, the latter may constitute a significant proportion of total aCA, for example, in early immune responses, and require other detection methods. Surface plasmon resonance technology, which offers real-time detection of aCA with rapid off-rates, has been developed in BiaCore systems (GE Healthcare). Binding of aCA is measured by a dedicated ‘sensorgram’ as they flow over a cytokine-coated sensor chip. This generates an increase in signal that reaches equilibrium according to the Ka (complex formation rate). Addition of an appropriate buffer releases aCA with a corresponding decrease in signal according to the dissociation rate, Kd. Biacore evaluation software calculates Ka and Kd by fitting data to interaction models [2,10]. Generally, surface plasmon resonance BiaCore systems are less sensitive for high-affinity aCA and ‘low throughput’ as opposed to ELISA, immunofluorometric, dissociation-enhanced lanthanide fluorescent immunoassay and ECL assays. However, they are more likely to detect lowaffinity aCA. Since they can be used for isotyping and kinetics, they are more suited for confirmatory testing and characterization of aCA (TABLE 1). Fluid-phase assays
Increasing criticism of solid-phase assays that they lack specificity and sensitivity (not always the case) and are prone to yield false positives (unacceptable for clinical analyses) has generated interest in the development and use of fluid-phase assays. The Expert Rev. Clin. Immunol.
Detection of aCA & their clinical relevance
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Table 1. Advantages and disadvantages of assay methods for detection and measurement of anticytokine autoantibodies. Method
Advantages
Disadvantages
ELISA
Readily available reagents and equipment; Easy and inexpensive to perform; high throughput
Variable sensitivity and dynamic range for measurement; Low-affinity antibodies, e.g., IgM, may be lost in washing steps; immobilized ‘capture’ cytokine may be deformed so that conformational epitopes no longer recognized; poor tolerance to circulating cytokine, especially in the ‘bridging’ format; cannot detect monovalent IgG4 antibodies; subject to variable matrix effects
IFMA DELFIA ECLA
Higher sensitivity and greater specific dynamic range than ELISA; better tolerance to circulating cytokine than ELISA; high throughput
More expensive reagents and equipment required than for ELISA; rapidly dissociating antibodies may not be detected; immobilized ‘capture’ cytokine may be deformed so that conformational epitopes are no longer recognized; may not detect monovalent IgG4 antibodies; subject to variable matrix effects
SPR
Capable of detecting lowaffinity IgM and monovalent IgG4 antibodies; characterization of antibody capabilities, e.g., kinetics, isotyping
Expensive equipment; slow to perform, especially with multiple samples; less sensitive than other methods; immobilized ‘capture’ cytokine may be deformed so that conformational epitopes are no longer recognized; subject to variable matrix effects
Multiplex assays
Capable of detecting several different aCA within a single assay
Expensive, nontransferable equipment; difficult to establish specific dynamic range for each aCA; immobilized ‘capture’ cytokine may be deformed so that conformational epitopes are no longer recognized; cannot detect monovalent IgG4 antibodies; subject to variable matrix effects
RIA RLBA LIPS
Cytokine in solution exhibits conformational epitopes; good specificity, sensitivity and specific dynamic range; less likely to produce ‘false’ results than solid-phase assays
Use of radioisotopes for RIA and RLBA; coupling of cytokine to radioisotope or fusion with luminescing protein may induce changes in cytokine conformation; prone to high backgrounds; subject to variable matrix effects
Bioassays
Capable of detecting and quantifying neutralizing aCA; can be very sensitive
Not able to measure nonneutralizing aCA, which may constitute a significant proportion of total aCA––these may have clinical relevance for cytokine clearance; require cell culture facilities and expertise and thus relatively expensive to perform; inherently variable and difficult to standardize; difficult to validate; difficulties in calculating and reporting neutralizing titers; subject to variable matrix effects
aCA: Anticytokine autoantibodies; DELFIA: Dissociation-enhanced lanthanide fluorescent immunoassay; IFMA: Immunofluorometric assays; LIPS: Luciferase immunoprecipitation systems; RLBA: Radioligand-binding assays; SPR: Surface plasmon resonance.
obvious benefits are that as antigens are in solution, their 3D structures are intact, their epitopes are not masked on immobilization to solid supports and interaction with aCA is therefore more specific and encompassing. However, cytokines still need to be labeled in some way, either radiolabeled or coupled to a (nonradioactive) tag or protein: these procedures may affect structure. The most familiar methods are radioimmunoassays where the cytokine is first radiolabeled [5]. This may be done directly with radio-iodine isotopes (125I) or indirectly using in vitro eukaryotic (mammalian) transcription and translation systems. Cytokines, for example, may be transcribed and informahealthcare.com
translated from cDNAs in the presence of 35S-methionine. 125 I- or 35S-labelled cytokines are incubated with aCA test samples in fluid phase before immune complexes are isolated by either immunoprecipitation with polyethylene glycol or bound to protein A/G-coated resins or beads [11]. Such assays have been dubbed ‘radioligand binding assays’. Alternatively, the in vitro transcription/translation platform can be used to generate (nonradioactive) fusion proteins of Renilla luciferase and cytokines. In what are known ‘luciferase immunoprecipitation systems (LIPSs)’, Renilla luciferase-tagged cytokines, as cell extracts, are incubated with test aCA samples in solution; doi: 10.1586/1744666X.2014.918848
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complexes are precipitated with protein A/G beads and luminescence measured [12]. Despite the advantages that these assays offer, they are prone to interference by serum/plasma matrix and circulating cytokine levels, have high backgrounds, may not detect low-affinity IgM aCA and are not easy to automate (TABLE 1).
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Bioassays
Although immunoassays can, if properly validated, determine the immunochemical concentrations of aCA, they do not reveal the proportion of neutralizing antibodies (NAbs), if present in a sample, contains. This requires a functional assay in which the inhibitory action of neutralizing aCA on cytokine activity is quantified. Functional assays for cytokines are based on the measurement of biological activity, triggered via cell surface receptors, in in vitro cell-based systems. These activities include stimulation or inhibition of cell proliferation, immune modulatory and antiviral actions, differentiation and cytotoxicity (apoptosis or necrosis) and are cytokine’ concentration dependent, thus permitting generation of dose–response data and thus potency determinations. Assays can be processed at both ‘early’ and ‘late’ stages of cellular responses. It is however beyond the scope of this article to provide detailed information of the plethora of cytokine bioassays, but these have been extensively reviewed [13,14]. The main advantages of such assays are that they are mostly very sensitive (though not always equaling the high sensitivity of immunoassays), endpoints are readily defined and they can be calibrated with WHO international biological standards of cytokines with assigned potencies. The drawbacks are that they are inherently variable, labor intensive to perform, often lack specificity and are time dependent, frequently requiring more than 1 day for reaching endpoints. Nevertheless, they may be adapted to measure the inhibitory activity of cytokine antagonists including neutralizing aCA. These act by preventing cytokine binding to cognate cell surface receptors, thus inhibiting cytokine activity. This ‘blocking’ activity can be measured in so-called ‘neutralization assays’ where activity reduction of a fixed concentration of cytokine by serially diluted aCA is evaluated [2,14,15]. The neutralizing capacity or strength of aCA is dependent on several factors including ability of antibodies to bind, strength of their binding (intrinsic and functional affinities), immunoglobulin class and type and epitope location on the surface of the cytokine molecule. Thus, a wide diversity of NAbs to cytokine molecules exists [15]. Currently, very few reference preparations of human neutralizing aCA, for example, anti-IFN-a and anti-IFN-b [16], anti-GM-CSF [17], are available; they are intended only for monitoring the sensitivity of cell-based assays. They cannot be used in the same way as the WHO IS for interferon (IFN) ligands to calibrate assays for potency determinations. Thus, neutralizing activity cannot be expressed in international (reference) units. This lack of means for uniformly expressing neutralizing activity has led to diverse analytical methods for assessing and calculating it and has already led to wide variations in the reporting of neutralizing doi: 10.1586/1744666X.2014.918848
aCA titers or concentrations. Nevertheless, certain methodological elements are common to most approaches for measuring aCA. First, the majority of assays are based on a ‘constant cytokine method’ where dilutions of test sample are mixed with a fixed concentration of cytokine and incubated together for 1– 2 h to allow neutralization to take place. The mixtures are then applied to the cytokine-sensitive cells and the assays are processed as if cytokine per se were being tested. Neutralizing activity is measured as the aCA dilution that effectively reduces cytokine potency by a significant, quantifiable degree, which is generally a fixed percentage, for example, 50, 80 and 90%. Despite theoretical difficulties inherent in antibody: cytokinebinding kinetics – largely unresolved for polyclonal IgG – the common practice is therefore to express ‘potency’ or neutralizing activity as the dilution, for example, 1/1000, 1/1280, 1/ 5000, of test sample that results in the chosen ‘endpoint’. For reporting purposes, the reciprocals of these dilutions, for example, 1000, 1280, 5000, are often used. In the IFN field, an analytical approach developed by Kawade and colleagues has led to the development of a formula that calculates the neutralizing titer as ‘10-fold reducing units’ from antiviral IFN neutralization assays (AVINA) in which IFN activity is reduced from 10 to 1 laboratory units [16]. While this approach has often found favorable for determining and reporting anti-IFN neutralizing titers, its applicability to other neutralizing aCA has not been established. Thus, in some instances, the simple practice of estimating the volumetric equivalent of the titer is used, for example, 1 ml equivalent to 1/1000 of the starting test material [18]. As with immunoassays, appropriate validation (qualification) is crucial for generating reliable data. Guidance papers, focusing on design, optimization and validation of cell-based assays, have been published [19–21]. Overall, these summarize the practical and analytical considerations needed for the development of bioassays to accurately measure the neutralizing activity of aCA. While it is beyond the present article to cover the extensive details of these guidance reviews, it is pertinent to point out that several of the technical issues that affect the performance and outcome of immunoassays, including variable effects of matrix, circulating cytokines and immune complexes, rheumatoid factor and complement, excipients, are also common to bioassays. Over and above these issues are a host of variables including cell passage levels, cell growth culture medium and serum levels, temperature, pH, microtiter plating effects, impacting on assay performance and sensitivity. Thus, each bioassay will have its own construction and performance criteria, and validation should therefore be carried out on a case-by-case basis. Endogenous (or naturally occurring) aCA in health & disease
Central and peripheral immune ‘tolerance’ prevent the emergence of autoantibodies to self-proteins (antigens). Their breakdown, resulting in autoantibodies, is currently hypothesized to arise due to subpotent thymic expression of target self-antigens Expert Rev. Clin. Immunol.
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Detection of aCA & their clinical relevance
(TSAg), leading to enhanced escape of autoreactive T cells and/or repeated inappropriate stimulation by TSAg in the periphery [22–24]. There are, however, as yet unexplained biases in autoantibody development, which have suggested other regulatory mechanisms impinge on autoimmunization, for example, the actions of Treg. Connections to epidemiological and demographical differences, provoking microbial agents and drugs, tumors, inflammation, genetic associations and old age have all been implicated as potential causative and sustaining factors. Alternatively, it has been hypothesized that any external or intracellular stimulus that creates a ‘dangerous’ environment increases the risk of autoimmunization [25]. aCAs are among diverse autoantibodies that arise sporadically during the lifetime of healthy individuals, or with varying, usually increased, frequency in specific diseases. Using both immunoassay and bioassay approaches, the prevalence and characteristics of these aCA have been investigated [2–4,21]. These typically are present as circulating IgG autoantibodies, usually at low to undetectable prevalence and titers in the blood of normal healthy individuals, but with increased prevalence and persistent high titers in certain autoimmune diseases, where sometimes they correlate with immunodeficiency and pathogenesis [26]. Interestingly, disease-associated aCAs appear to be limited to only about a third of all known cytokines. Most targeted cytokines are type I, II and III IFNs, IL-1a, IL-6, IL-8, IL-12, IL-17A, IL-17F, IL-22, G-CSF, GM-CSF and TNF-a. In health
Regarding aCA in healthy individuals, the expectations are that either they would be completely absent, or occur sporadically in the healthy population at large, or are more widespread, but remain undetected either due to the insufficient sensitivity of assay methods or being ‘hidden’ by association with circulating cytokine or other substances. It is perhaps not surprising that examples of all three of these scenarios have been reported in studies where sera or plasma from healthy individuals have been analyzed for the presence of specific aCA (TABLE 2). Given the significant pitfalls in determining the presence of aCA without a true negative control group of sera or assay standardization, these results often require further authentication and remain controversial in some instances. For example, autoantibodies against IFN-a/w were first reported in 1990, but bone fide neutralization of IFN activity was difficult to demonstrate. Later studies have indicated that they could be present in up to 2% of healthy individuals (TABLE 2) [27,28]. Against IFN-b, another type I IFN or IFN-l (type III IFN), prevalence is likely to be even lower. Although early studies suggested the widespread occurrence of anti-IFN-g (type II IFN) [29], this has not subsequently been confirmed [12,28]. Regarding other cytokines, including many interleukins, G-CSF, GM-CSF and TNF-a, Watanabe and coauthors [30,31] have reported that aCAs against them are ubiquitous in healthy individuals, but would go undetected by most assay protocols as they are bound to circulating cytokine in immune complexes. While these informahealthcare.com
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results, in particular for anti-GM-CSF, are disputed due to technical problems with the assays used [32], the potential presence of cytokine: aCA IgG complexes for several cytokines cannot be disregarded. Indeed, such immune complexes probably do exist for anti-IL-1a [33] and anti-IL-8 (TABLE 1) [30,34]. Currently, literature reports indicate a 5–25% prevalence of antiIL-1a, increasing slightly with age [27,35], 11–13, 8, 0.4 and 0.1% for anti-G-CSF [30,36], anti-GM-CSF [28,30], anti-IL-10 [37] and anti-IL-6 [38], respectively, and absence (if potential immune complexes are discounted) of anti-IL-2, anti-IL-4, anti-IL-12, anti-IL-17A, anti-IL-17F, anti-IL-17F, antiIL-22 and anti-TNF-a (TABLE 2) [9,12,28,39,40]. While considerable uncertainties remain concerning the real prevalence and circulating levels of aCA from data derived from assays using sera or plasma, a clearer picture for certain specific aCA has emerged from the study of pharmaceutical IgG preparations, which include both intravenous (IVIG) and intramuscular (IMIG) immunoglobulin products. While immunoassays, especially ELISA, are not suitable for measuring aCA in IVIG/IMIG products on account of the very high total IgG concentration, sensitive bioassays are capable of quantifying neutralizing aCA. For instance, when tested by AVINA, most IVIG and IMIG products moderately neutralized IFN-a subtypes and also IFN-w (60% sequence homology to IFN-a), but more rarely and not independently of IFN-a, IFN-b (30% sequence homology to IFN-a) and the distantly related IFN-l (TABLE 2) [18,41]. Since neutralizing activity was wholly absent in some IVIG batches, it is believed that the NAbs against IFNs arise from plasma(s) of one or a few individuals with high titers, which are subsequently diluted in the plasma pool derived from thousands of blood donations. Such individuals may have appeared outwardly healthy, but may have had subclinical infection or disease at the time of blood donation. For example, the chickenpox herpes virus, Varicella zoster, has been associated with the development of autoantibodies to IFN-a [42]: interestingly, highest titers of anti-IFN-a NAbs were found in batches of Varicella zoster-specific IMIG [18]. In contrast, despite a report of anti-IFN-g (type II IFN) in sera and IVIG that selectively inhibited IFN-g-mediated HLA-DR and interfered in IFN-g-stimulated mixed lymphocyte reactions [29], no IVIG/IMIG batches neutralized IFN-g-mediated antiviral activity in AVINA [18,29]. Such discrepant results appear odd given that all IFN-g activities are mediated via the same cell surface receptor. Besides IFNs, large numbers of IVIG/IMIG batches strongly neutralize IL-1a and GM-CSF in their respective bioassays, probably reflecting the significant prevalence of these aCA in the sera/plasma of healthy individuals (TABLE 2) [18]. The antiGM-CSF Abs neutralized recombinant GM-CSF derived from E. coli, yeast and CHO cells and bound to these in immunoblots, thus confirming their specificity for GM-CSF [18]. However, no neutralization of IL-2, IL-4, IL-6, IL-10, IL-12, GCSF and TNF-a was detected in multiple IVIG/IMIG batches (TABLE 2). The neutralizing aCAs against IFNs, IL-1a and GMCSF appear to have no overt effects on health, though doi: 10.1586/1744666X.2014.918848
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Table 2. Anticytokine autoantibodies in healthy individuals. Cytokine
Cytokine properties
Test material
Prevalence % positive
Neutralizing or not (+/-)
Comments
Ref.
IFN-a/w
Mixture of 12 related a-helical, monomeric IFN-a subtypes and IFN-w; antiviral, antiproliferative, immune modulatory.
S/P IVIG IMIG
1–2 74-IVIG 40-IMIG
+/+ +
Occasional positives. Significantly neutralize IFN-a2. Most other IFN-a subtypes and IFN-w neutralized to varying degrees
[18,27, 28,41]
IFN-b
Single a-helical, monomeric, glycosylated IFN; Similar activities to IFN-a.
S/P IVIG IMIG
