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Detection and characterization of antibodies against recombinant human erythropoietin by RIPA, ELISA and neutralization assay in patients with renal anemia
Journal of Immunological Methods 336 (2008) 152–158
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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
Detection and characterization of antibodies against recombinant human erythropoietin by RIPA, ELISA and neutralization assay in patients with renal anemia Johann Gross a,⁎, Renate Moller a, Steffen Bischoff c, Sima Canaan-Kühl b, Manfred Fromme d, Wolfgang Henke a a b c d
Molecular Biology Research Laboratory, Department of Otorhinolaryngology, University Medicine Charité, Berlin, Germany Department of Nephrology and Medical Intensive Care, University Medicine Charité, Berlin, Germany Dialysis Centre, Gutenbergstrasse 5, 01307 Dresden, Germany Nephrological Center Stuttgart, Wolframstr. 60-62, 70191 Stuttgart, Germany
a r t i c l e
i n f o
Article history: Received 22 November 2007 Received in revised form 9 April 2008 Accepted 10 April 2008 Available online 5 May 2008 Keywords: Affinity Antibodies Erythropoietin Neutralization
a b s t r a c t The aim of the present study was to analyze the relations between the serum antierythropoietin antibody (AEAb) levels and the antibodies' neutralizing activity in 20 patients with renal anemia and rhEPO-induced antibodies. AEAb levels were determined by the enzyme-linked immunosorbent assay (ELISA, double antigen-bridging) and by radioimmunoprecipitation assay (RIPA). The bone marrow neutralization test was used to determine the neutralizing activity of the antibodies. RIPA and ELISA data resulted in closely correlated measurements. The relations between AEAb levels and the neutralizing activity of the antibodies are variable as shown by follow-up and cross-sectional evaluations of the data. Serum samples with a high antibody level (N 1000 ng/ml) are associated with 100% neutralizing activity, whereas serum samples with lower AEAb levels show partial neutralizing activities or have no effect. Determining the neutralizing activity might be helpful when it comes to deciding of whether or not rhEPO therapy should be continued, specifically in patients who have low antibody levels. The apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC50) did not change in the course of the disease, nor did it correlate to the AEAb levels or the neutralizing activities. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Erythropoietin (EPO) is the primary regulator of erythropoiesis. EPO binds and activates the EPO receptor expressed on the surface of cells. Activation of the EPO receptor by EPO binding induces a cascade of signaling events that support both erythroid proliferation and differentiation (Fisher, 1997). Recombinant human erythropoietin (rhEPO) is a sialoglycoprotein hormone whose action is biologically equivalent to that of endogenous EPO. It is used pharmaceutically for the ⁎ Corresponding author. Molecular Biology Research Laboratory, Department of Otorhinolaryngology, University Medicine Charité, Charitéplatz1, D-10117 Berlin, Germany. Tel.: +49 30 450 555 311; fax: +49 30 450 555 908. E-mail address: [email protected] (J. Gross). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.04.009
stimulation of red blood cell development in anemia patients who have suffered kidney failure or are undergoing tumor therapies. A rare side effect associated with rhEPO therapy is the development of antibody-mediated pure red cell aplasia (PRCA) (Bennett et al., 2004; Casadevall et al., 2002). Between January 1998 and April 2004, 175 cases of epoetin-associated PRCA were reported for Eprex, 11 cases for NeoRecormon, and 5 cases for Epogen (Bennett et al., 2004). Since December 2002 there has been a significant decrease in newly reported cases of rhEPO induced PRCA. As of 30 November 2005, a total of 215 antibody positive PRCA cases had been reported [http: www.jnjpharmarnd.com/company/n-casereports.html]. Recommendations for hematological criteria for use in the diagnosis of epoetin-induced pure red cell aplasia have been
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published (Casadevall et al., 2004). The criteria for diagnosing rhEPO-induced PRCA include mainly changes in hemoglobin levels, reticulocyte numbers or bone-marrow morphology and the detection of serum anti-erythropoietin antibodies (AEAb). The identification of AEAb in the serum is the key feature for diagnosing rhEPO-induced PRCA. Several tests are available for the diagnosis of AEAb, these being mainly radioimmunoprecipitation assays (RIPA), enzyme-linked immunosorbent assays (ELISA) and BIAcore biosensor immunoassays (Hoesel et al., 2004; Swanson et al., 2004; Tacey et al., 2003; Thorpe and Swanson, 2005b). The advantages and disadvantages of these methods were reviewed by Wadhwa et al. (2003) and Thorpe and Swanson (2005a). Whereas tests for determining AEAb measure the binding capacity between EPO and the antibody, tests for neutralizing antibodies (NAb) assess the ability of such antibodies to inhibit biological activity, i.e., the proliferation of erythroid precursor cells (Gupta et al., 2007; Kawade et al., 2003; Schellekens, 2007). The results of bioassays of different laboratories are even more difficult to compare than the results of antibody quantification, because the methods use different cells and activity units (Thorpe and Swanson, 2005a). In order to determine neutralizing antibodies, different tumor cell lines have been used such as the IL-3-dependent murine 32D cell line transfected with human EPO receptors (Wei et al., 2004) or human UT-7/EPO cells, an immortalized cell line (Kelley et al., 2005; Wu et al., 2004). Casadevall et al. (2002) used normal bone marrow cells to characterize the neutralizing activity of anti-erythropoietin antibodies. The mechanisms leading to the development of antibodies to rhEPO are based on breaking the immune tolerance that normally exists to human homologue proteins. It is assumed that the appearance of AEAb and consequently the inhibition of the proliferation of erythroid precursor cells seem to be
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associated with the formation of aggregates during rhEPO storage or with the way rhEPO is administered. The clinical consequences of the presence of antibodies are, in general, a function of the antibodies' binding specificities, their affinity and neutralizing activity (Schellekens, 2007). The aim of the present study was to analyze the relations between the serum anti-erythropoietin antibody levels and their neutralizing activity in 20 patients suffering from renal anemia. The AEAb levels were determined using the double antigen-bridging ELISA (Hoesel et al., 2004), and the neutralizing activity was determined by the bone marrow neutralization test as described by Casadevall et al. (2002). Because the AEAb levels of some of the patients were determined by both RIPA and ELISA, we correlated the results obtained from the two methods. In addition, we determined the apparent affinity of the antibodies (IC50). Analyzing the relations between these criteria may contribute to understanding the characteristics of the rhEPOinduced antibodies and may allow conclusions to be drawn about the necessity of determining the neutralizing activity in the diagnostics of patients developing anti-rhEPO antibodies. 2. Patients and methods 2.1. Patients Serum samples from 20 patients who were suspected of having anti-erythropoietin antibodies were obtained from nephrologists and dialysis centers from Germany and abroad. Details concerning the patients were obtained from the adverse event reports for epoetin beta from its manufacturer Roche, Basel, Switzerland, and from clinicians treating such patients. For follow-up, two to six samples were sequentially collected from nine patients to determine their AEAb levels. The samples submitted to the laboratory for diagnosis
Table 1 Basic data of patients (age, gender, diagnosis, treatment, level of anti-EPO antibodies and neutralizing activity) Patient (age, sex)
Main diagnosis, treatment
1
a
Goodpaster's syndrome; Chronic renal failure; Epoetin beta; 2PRCA (1361) Chronic renal failure; diabetes mellitus; Erypo for 244 d, then NeoRecormon for 59 d; PRCA (291) Chronic renal insufficiency; Erypo, then NeoRecormon (duration unknown); PRCA (257) Chronic renal failure; NeoRecormon; PRCA (449) Diabetic renal insufficiency; NeoRecormon; PRCA (297) End stage renal disease; Epopetin alpha for 37d, then NeoRecormon for 238d; PRCA (273) Chronic renal failure; NeoRecormon; PRCA (449) End stage renal failure, peritoneal dialysis, renal transplant; Epoetin beta; PRCA (734) Chronic glomerulonephritis; Epoetin alpha for 365 d, then Epoetin beta for 31d and Darbepoetin alpha for 7 d; PRCA (396) Chronic renal failure; Eprex for 2559 d, then NeoRecormon for 364 d; PRCA (2920) Diabetic nephropathy; Epoetin beta; PRCA suspected (191) Chronic pyelonephritis; Epoetin alpha for 523 d, then Epoetin beta for 170 d; PRCA (559) Chronic renal failure; Epoetin alpha for 214 d, then Epoetin beta for 1025 d; PRCA (1022) End stage renal failure; NeoRecormon; PRCA (771) Chronic tubulointestinal nephritis; Eprex for 129 d and then NeoRecormon for 227 d; PRCA (368) Chronic renal insufficiency; Epoetin beta; PRCA (458) End stage renal insufficiency, kidney transplantation; Epoetin beta; PRCA (1109) Terminal renal insufficiency, dialysis; Epoetin beta; PRCA (1076) Congenital kidney dysplasia; NeoRecormon; bone marrow normal (233 and 309) Chronic renal failure; NeoRecormon; bone marrow normal (196); absent iron incorporation into bone marrow cells (243)
6000 (1377); 100% (1399) 8365 (338); 83% (772) 7547 (234); 100% (347) 2047 (468); 100% (519) 30030 (328); 100% (398) 6426 (287); 100% (287) 37450 (469); 100% (477) 2500 (759); 100% (759) 3333 (396). 15% (552)
P1 (52,f) P2 (70,m) P3 (62,f) P4 (65,f) P5 (75,m) P6 (80, m) P7 (79,m) b P8 (51,m) c P9 (48, m) P10 (67, m) P11 (87, m) P12 (76, m) P13 (57, m) P14 (65, m) P15 (83, m) P16 (65, f) P17 (24, m) d P18 (68, m) P19 (21, m) P20 (49, f)
AEAb and NAb
1188 (3024); 0% (2920) 926 (232); NAb — nd 522 (690); NAb — nd 4200 (1050); NAb — nd 24 (784); NAb — nd 4500 (427); NAb – nd 18538 (501); NAb — nd 1195 (1120); 25% (1248) 189 (1172); 42% (1172) 184 (272); 26% (232); 0% (260) 248 (320); 0% (243)
a Patient 2 (Tolman et al., 2004); bPatient 4 (Tolman et al., 2004); c(Weber et al., 2002); d(Kruger et al., 2003); 1AEAb levels in ng/ml and NAb activity in % inhibition. In brackets: time (days) between the start of the rhEPO treatment and the blood sampling for AEAb and NAb determination. 2PRCA diagnosis. In brackets: the time (days) between the start of rhEPO treatment and the diagnosis of PRCA. d — days. nd — not determined.
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originated from patients treated between 2000 and 2006. Basic data of the patients analyzed in this study are summarized in Table 1. 2.2. ELISA and RIPA In order to compare the results obtained by RIPA with those of ELISA, samples were analyzed for AEAb levels in both Prof. Casadevall's laboratory using RIPA (Tacey et al., 2003) and our own laboratory using ELISA (Hoesel et al., 2004). In the RIPA, antibodies are determined by the amount of 125J-EPO bound in an immune complex which can be isolated by binding to Protein G Sepharose. RIPA results are expressed in mU of erythropoietin bound by one ml serum as previously described (Casadevall et al., 2002). In the ELISA, biotinylated rhEPO was incubated in streptavidin-coated microtiter plates at a final volume of 125 µl/well. Human serum or reference antibody preparation were mixed with an equal volume of digoxin labeled rhEPO and added to the wells. After incubation and washing, antidigoxin Fab fragment HRP conjugate was added to each well and incubated. ABTS solution was used as a substrate for HRP. Absorbance was measured at 405 nm and 492 nm. The antibody concentration was calculated by using a fourparameter regression curve. A polyclonal anti-EPO IgG fraction from rabbits, purified on an EPO-Sepharose column, was used as calibration material in the ELISA. The results are given in ng/ml serum. Quality control was carried out as described previously (Hoesel et al., 2004). Briefly, upon routine analysis of sera, three samples of reference material covering the assay concentration range were included in the assays in each case, and the recoveries observed were all within 20% of the expected value. The quality control samples yield the same results in both the previous assays and the assessment of the samples studied here. The cut-off in the ELISA methodology was determined as described (Hoesel et al., 2004). Briefly, serum samples (n = 159) randomly chosen from different hospitals and blood banks, and not considered to contain anti-EPO Abs, were analyzed by the standard assay as a representative collection of negative samples. The 90th percentile of these samples was determined at 5 ng/ml and the 99th percentile at 10 ng/ml. 2.3. Bone marrow colony forming assay The inhibition of the growth of erythroid progenitors by patients' serum was tested in Paris as described, using bone marrow from otherwise normal patients who underwent hipreplacement surgery and gave their written informed consent for the collection of bone marrow cells (Casadevall et al., 2002). Briefly, erythroid cells in normal bone marrow were stimulated with 1 U of epoetin per ml in the presence of 20% pooled control or 20% patients serum. Erythroid colonies were scored after seven days in culture. The neutralizing activity was expressed as a percentage inhibition of colonies formed in the presence of patients serum compared to control serum. 2.4. Apparent affinity (IC50) The concentration of unlabeled rhEPO that produces a 50% inhibition of antibody binding (IC50) was determined by in-
cubating antibodies with increasing concentrations of unlabelled rhEPO in the range of 0.5–30 ng/ml using the ELISA method. The unlabelled rhEPO was added to the well together with DIG-labeled rhEPO. The IC50 of rhEPO was calculated by nonlinear regression analysis of the experimental data using the following equation: antiEPOAbbound ¼
antiEPOAbmax IC50 rhEPO þ IC50
where AntiEPOAbmax denotes the bound antiEPO antibody in the absence of rhEPO. IC50 denotes the rhEPO concentration required to trigger 50% inhibition of the antigen-antibody binding in ELISA. 3. Results 3.1. Patients Table 1 indicates the basic data of 20 patients from eight different countries analyzed for anti-erythropoietin antibody levels and bone-marrow morphology. All of these patients had been treated with rhEPO for renal anemia, with twelve patients having received treatment with epoetin beta and eight patients with epoetin alpha and beta. The bone-marrow analysis resulted in PRCA diagnosed in 18 patients and the exclusion of PRCA in two patients with a normal morphology (P19, P20). The time between the start of rhEPO treatment and the diagnoses of PRCA varied between 191 and 2920 days (median 454 days). Generally, the detection of AEAb formation coincided with that of PRCA. The AEAb levels measured for all these patients comprised a range of 3–37000 ng/ml with a median of about 3000 ng/ml. Analyses of neutralizing antibodies were carried out in the serum samples of 14 patients. The serum samples used for determining neutralizing antibodies were collected about three months after the analyses of the bone marrow morphology (median 80 days, range 0–481 days). The antibodies' neutralizing activity varied between 0% and 100% inhibition. The two patients without PRCA (P19, P20) did not show any neutralizing activity. Among the PRCA patients analyzed for NAb (n = 12), eight patients showed complete (80–100% inhibition) and three patients partial inhibition (15–42%), whereas one patient (P10) had non-neutralizing antibodies. For technical reasons, the activity of neutralizing antibodies could not be determined in six patients. 3.2. Relations between antibody levels measured with RIPA and ELISA During this study, 35 serum samples were analyzed in parallel by both RIPA and ELISA to compare whether the two different methods were a) detecting antibodies and b) assess amounts of antibodies were comparable. The statistical analysis of the results showed the two measurements to be closely correlated with a correlation coefficient of R2 = 0.92 (Fig. 1). The samples included in this correlation comprise samples from Casadevall`s laboratory (n = 15), from the patients of this study (n = 19) and one sample from a patient from a different laboratory (see legend of Fig. 1). Samples from
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a given patient were collected at different time points during the course of disease and were blindly analyzed in the corresponding laboratory. From the equation found to be describing the correlation between RIPA and ELISA, it can be calculated that 5 ng/ml (cut-off value for ELISA) correspond to a RIPA value of 47 mU/ml, a number very close to the cut-off value reported for RIPA (40 mU/ml, personal communication). Parallel analyses of AEAb levels of follow-up samples from patient P17 using ELISA and RIPA showed a similar pattern in the course of the disease, in both the increasing and the decreasing phases of the disease (Fig. 2). Thus, the results obtained by RIPA and ELISA are comparable in quantitative terms. 3.3. Relations between the levels of anti-erythropoietin antibodies and their neutralizing activities In order to get an insight into the relations between the levels of anti-erythropoietin antibodies and their neutralizing activity we compared follow-up values of AEAb and NAb levels during a period of about four years in eight patients. To normalize the course of these curves, the time at which the highest AEAb value occurred was used as the reference point. This point corresponds roughly to the time of the diagnosis of the PRCA and the withdrawal of rhEPO. For all patients we observed a continuous decrease in AEAb levels (Figs. 3 and 4). This decrease followed an exponential function, with a steep decrease during the first year of the observational period and a slower one in subsequent years. A slower decrease rate of the AEAb levels was observed in two patients (P3, P8), indicating heterogeneity in the decrease rate of AEAb levels. The NAb levels of five patients (Fig. 3) showed 100% inhibition of erythroid colonies at the reference point and decreased continuously, roughly in parallel to the log 10 of the AEAb levels (Fig. 3). In three patients, the neutralizing activity
Fig. 1. Correlation of anti-erythropoietin antibody levels as determined by RIPA and ELISA. Data are given in logarithmic terms because of the high range of pathological values. The data follow the equation y = 0.107 x; R2 = 0.92, n = 30. Five samples showed negative values and therefore were not included in the logarithmic correlation. The samples included in this correlation comprise one sample from each patient of Casadevall`s laboratory (n = 15), nine samples of patient P17, five samples from patient P8, three samples from patient P2, two samples from patient P3 (see Table 1) and one sample from a patient from a different laboratory. No significant differences were observed in the regression and correlation coefficients of the groups with n ≥ 5.
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Fig. 2. Anti-erythropoietin antibody levels of patient P17 determined in parallel by RIPA and by ELISA. Abscissa: Time in days after start of rhEPO treatment. For details of patient P17 see Table 1. The first five samples of this patient were originally collected for other diagnostic purposes and were stored at −20 °C.
followed a different course. At the reference point, a partial neutralizing activity was observed, which remained unchanged in two patients for six months irrespective of the fact that the AEA levels decreased continuously (Fig. 4). In one patient, we observed the neutralizing activity to rise by up to 100% inhibition within a period of 300 days. A cross-section correlation analysis of all available parallel AEAb and the NAb determinations (Fig. 5) shows complete neutralizing activities
Fig. 3. Decrease of anti-erythropoietin antibody levels and neutralizing activity during the disease in five patients. Top: Anti-erythropoietin antibody levels. Bottom: Neutralizing activity. For details of the patients see Table 1. The AEAb levels are shown as a log scale because of the high range of pathological values. For better comparison, the curves have been normalized (time at which the highest AEAb level was observed =day 0).
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Fig. 6. Inhibition of anti-erythropoietin antibody binding by unlabelled rhEPO. Samples were preincubated with different concentrations of unlabeled rhEPO added to the microtiter plate. Samples originate from different phases of the disease of patient P2 (see Table 1). Samples were collected on day 411 (full circles; 943 ng/ml), day 690 (full squares; 31 ng/ml) and on day 851 (open triangles; 12 ng/ml) after the start of treatment. The corresponding IC50 values are 5.8, 15.0 und 3.7 ng/ml, respectively.
Fig. 4. Decrease of anti-erythropoietin antibody levels and different changes of neutralizing activity during the disease in three patients. Top: Antierythropoietin antibody levels. Bottom: Neutralizing activity. For details see Table 1 and legend of Fig. 3.
at high AEAb levels above about 1000 ng/ml. Below this level, neutralizing activities between zero and 60% were observed. 3.4. Apparent affinity of anti-erythropoietin antibodies To further characterize the AEAb, we wanted to know whether the neutralizing activity relates to the apparent affinity of the antibodies. Samples of patient P2 (parallel decrease of AEAb and NAb levels, Fig. 3) were used to determine the half-
Fig. 5. Relation between anti-erythropoietin antibody levels and neutralizing activity. AEAb levels and neutralization activity were determined from aliquots of the same sample. Data originate from 10 patients. The AEAb levels are given in logarithmic terms because of the high range of pathological values.
maximal inhibition concentration (IC50) of unlabeled rhEPO as an indicator of the antibodies' apparent affinity. Fig. 6 illustrates that the addition of rhEPO (0.25–30 ng/ml) displaces 70–90% of the labeled rhEPO from the AEAb binding, independent of the AEAb level (12–943 ng/ml) and the disease phase. The IC50 values for rhEPO ranged from 3.7–15.0 ng/ml (200–800 pM) in this patient. Determination of IC50 values of samples collected from five patients showing different AEAb levels confirmed that the IC50 values are independent of the disease phase and the AEAb levels. In the mean, an antibody apparent affinity of 7.2 ±2.0 ng/ml (n =11) was observed. To exclude possible effects of the AEAb level on the IC50 value we determined the IC50 values in a sample by diluting a positive serum containing antibodies of 673 ng/ml. No significant changes in the apparent affinity were observed between undiluted samples and those diluted at rates of 1/10 and 1/100 (data not shown). 4. Discussion Several major findings have been made in this paper. (1) We observed a close correlation between the antibody levels determined by RIPA and those determined by the double antigen-bridging ELISA using biotinylated rhEPO. (2) Serum samples with a high antibody level (N1000 ng/ml) are in general associated with a complete neutralizing activity. In samples with lower levels the antibodies induce partial neutralization or have no effect. (3) The apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC50) did not change in the course of the disease, nor did it correlate to the AEAb levels or the neutralizing activity. Controversy exists over the correlation between AEAb levels determined by RIPA and those determined by ELISA (Thorpe and Swanson, 2005b). In the present study, we found a close correlation to exist between serum levels determined by RIPA and those determined by the double antigen-bridging ELISA (Hoesel et al., 2004). In a previous study, we had shown that the format of the ELISA tests dramatically affects the result (Gross et al., 2006). The present data confirm our previous findings that the double antigen-bridging ELISA using
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biotinylated rhEPO and digoxin labeled rhEPO results in a sensitive, specific and accurate determination of AEAb levels. The relationship between AEAb level and the antibodies' neutralizing activity in patients with rhEPO-induced antibodies are highly variable as shown by follow-up and crosssectional evaluations of the data. In the course of the disease, some patients showed a decreasing neutralizing activity in parallel to decreasing AEAb levels, whereas in other patients the neutralizing activity remained unchanged or even increased despite falling AEAb levels. Cross-sectional analysis has shown that serum samples with a high antibody level (N1000 ng/ml) are associated with a complete neutralizing activity whereas serum samples with lower AEAb levels have differing levels of neutralizing antibodies, with their activity ranging from partially neutralizing to non-neutralizing. On the one hand, these findings might support the coating theory of neutralization assuming that high AEAb levels prevent the binding of the rhEPO molecule to target cell receptors due to steric hindrance (Wilson and McElwain, 2004). On the other hand, the patient-specific time course of the neutralizing activity suggests that antibodies with differing neutralizing activity occur. Most probably, there exists a preferential synthesis of specific neutralizing antibodies with more or less pronounced inhibitory capacities. This assumption is supported by experimental findings. For example, Feldman et al. (1992) produced a series of monoclonal antibodies to amino acid region 99–129 of human erythropoietin that display unique structural features within the putative receptor binding domain of the hormone. Three of the four monoclonal antibodies neutralized the biological activity of EPO in a concentration-dependent fashion in vitro. Similarly, Fibi et al. (1993) produced and characterized recombinant human erythropoietin specific mouse monoclonal antibodies, which were specifically reactive with recombinant human erythropoietin in the enzyme-linked immunosorbent assay. Epitope exclusion studies showed the three distinct epitope regions A, B and C recognized by neutralizing monoclonal antibodies. Non-neutralizing monoclonal antibodies recognized an additional epitope region D. Antibodies defining an epitope region competed with one another for binding sites, but did not compete with antibodies defining a different epitope region. The finding that the apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC50) did not change in the course of the disease and did not correlate to the neutralizing activity of the antibodies confirms previous observations (Casadevall et al., 2002). 5. Conclusion The heterogeneity of the relations between the AEAb levels and the antibodies' neutralizing activity illustrates that the antibodies of the individual patients have an inherent property of being neutralizing or non-neutralizing. This feature might be important in determining the efficiency of therapy or the clinical sequelae (Gupta et al., 2007). Therefore, it is important to decide when and how neutralizing antibodies should be measured in patients treated with rhEPO. A negative result following an ELISA or RIPA test eliminates the need for neutralizing-activity testing, because the binding assay binds all antibodies, both neutralizing and non-neutralizing (Deisenhammer et al., 2004). The present
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study indicates that anti-erythropoietin antibodies above about 1000 ng/ml are most likely to show a complete neutralizing activity, which, in general, makes it unnecessary to determine the antibodies' neutralizing activity. However, in patients with a lower antibody level, where the clinician suspects a loss of clinical efficiency of rhEPO treatment, the determination of neutralizing activities may be helpful when it comes to deciding of whether or not rhEPO therapy should be continued. Therapy with erythropoietin should be discontinued, when neutralizing anti-erythropoietin antibodies are diagnosed. Where non-neutralizing antibodies are diagnosed, continuation of the rhEPO therapy or its restart seem possible as long as the AEA level and the neutralizing activity are monitored, because more rhEPO treatment may induce neutralizing antibodies. Measuring the neutralizing activity of antibodies is no trivial matter (Kawade et al., 2003). There are several methods available for measuring the neutralizing activity of antierythropoietin antibodies (Casadevall et al., 2002; Kelley et al., 2005; Wei et al., 2004). The present study shows that a useful neutralizing test has to be sensitive enough to identify anti-erythropoietin antibody levels between about 10 ng/ml and 1000 ng/ml. Theoretically, human bone marrow cells are best-suited for such tests, but using human bone marrow from healthy donors is neither easily manageable nor ethical. Acknowledgments This work was supported by grants from the University Medicine Charité Berlin (J.G.) and from Roche Diagnostics GmbH (J.G. and R.M.). We are grateful to T. Haselbeck and W. Hoesel (Roche) and to several physicians and nurses for their support and for collecting the serum samples for this study. We thank Prof. Casadevall for performing the RIPA and the neutralization assays and for her cooperation. We wish to thank K.-U. Eckardt for criticism and support. Responsibilities for interpretation of the data and conclusions lies with the authors only. The protocol and the patient information sheet were reviewed and approved by the regulatory authorities of Roche. References Bennett, C.L., Luminari, S., Nissenson, A.R., Tallman, M.S., Klinge, S.A., McWilliams, N., McKoy, J.M., Kim, B., Lyons, E.A., Trifilio, S.M., Raisch, D.W., Evens, A.M., Kuzel, T.M., Schumock, G.T., Belknap, S.M., Locatelli, F., Rossert, J., Casadevall, N., 2004. Pure red-cell aplasia and epoetin therapy. N. Engl. J. Med. 351, 1403. Casadevall, N., Nataf, J., Viron, B., Kolta, A., Kiladjian, J.J., Martin-Dupont, P., Michaud, P., Papo, T., Ugo, V., Teyssandier, I., Varet, B., Mayeux, P., 2002. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N. Engl. J. Med. 346, 469. Casadevall, N., Cournoyer, D., Marsh, J., Messner, H., Pallister, C., ParkerWilliams, J., Rossert, J., 2004. Recommendations on haematological criteria for the diagnosis of epoetin-induced pure red cell aplasia. Eur. J. Haematol. 73, 389. Deisenhammer, F., Schellekens, H., Bertolotto, A., 2004. Measurement of neutralizing antibodies to interferon beta in patients with multiple sclerosis. J. Neurol. 251 (Suppl 2), II31. Feldman, L., Heinzerling, R., Hillam, R.P., Chern, Y.E., Frazier, J.G., Davis, K.L., Sytkowski, A.J., 1992. Four unique monoclonal antibodies to the putative receptor binding domain of erythropoietin inhibit the biological function of the hormone. Exp. Hematol. 20, 64. Fibi, M.R., Aslan, M., Hintz-Obertreis, P., Pauly, J.U., Gerken, M., Luben, G., Lauffer, L., Siebold, B., Stuber, W., Nau, G., 1993. Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization. Blood 81, 670.
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Tacey, R., Greway, A., Smiell, J., Power, D., Kromminga, A., Daha, M., Casadevall, N., Kelley, M., 2003. The detection of anti-erythropoietin antibodies in human serum and plasma. Part I. Validation of the protocol for a radioimmunoprecipitation assay. J. Immunol. Methods 283, 317. Thorpe, R., Swanson, S.J., 2005a. Assays for detecting and diagnosing antibody-mediated pure red cell aplasia (PRCA): an assessment of available procedures. Nephrol. Dial. Transplant. 20 (Suppl 4), iv16. Thorpe, R., Swanson, S.J., 2005b. Current methods for detecting antibodies against erythropoietin and other recombinant proteins. Clin. Diagn. Lab. Immunol. 12, 28. Tolman, C., Duja, S., Richardson, D., Rashid, R., McVerry, A., Mooney, A., Baker, R., Will, E., 2004. Four cases of pure red cell aplasia secondary to epoetin beta, with strong temporal relationships. Nephrol. Dial. Transplant. 19, 2133. Wadhwa, M., Bird, C., Dilger, P., Gaines-Das, R., Thorpe, R., 2003. Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals. J. Immunol. Methods 278, 1. Weber, G., Gross, J., Kromminga, A., Loew, H.H., Eckardt, K.U., 2002. Allergic skin and systemic reactions in a patient with pure red cell aplasia and anti-erythropoietin antibodies challenged with different epoetins. J. Am. Soc. Nephrol. 13, 2381. Wei, X., Swanson, S.J., Gupta, S., 2004. Development and validation of a cellbased bioassay for the detection of neutralizing antibodies against recombinant human erythropoietin in clinical studies. J. Immunol. Methods 293, 115. Wilson, D.P., McElwain, D.L., 2004. A model of neutralization of Chlamydia trachomatis based on antibody and host cell aggregation on the elementary body surface. J. Theor. Biol. 226, 321. Wu, G., Wadgymar, A., Wong, G., Ting, R., Nathoo, B., Mendelssohn, D., Pandeya, S., Sapir, D., Tam, P., 2004. A cross-sectional immunosurveillance study of anti-EPO antibody levels in CRF patients receiving epoetin alfa in 5 Ontario Renal Centers. Am. J. Kidney Dis. 44, 264.
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
Detection and characterization of antibodies against recombinant human erythropoietin by RIPA, ELISA and neutralization assay in patients with renal anemia Johann Gross a,⁎, Renate Moller a, Steffen Bischoff c, Sima Canaan-Kühl b, Manfred Fromme d, Wolfgang Henke a a b c d
Molecular Biology Research Laboratory, Department of Otorhinolaryngology, University Medicine Charité, Berlin, Germany Department of Nephrology and Medical Intensive Care, University Medicine Charité, Berlin, Germany Dialysis Centre, Gutenbergstrasse 5, 01307 Dresden, Germany Nephrological Center Stuttgart, Wolframstr. 60-62, 70191 Stuttgart, Germany
a r t i c l e
i n f o
Article history: Received 22 November 2007 Received in revised form 9 April 2008 Accepted 10 April 2008 Available online 5 May 2008 Keywords: Affinity Antibodies Erythropoietin Neutralization
a b s t r a c t The aim of the present study was to analyze the relations between the serum antierythropoietin antibody (AEAb) levels and the antibodies' neutralizing activity in 20 patients with renal anemia and rhEPO-induced antibodies. AEAb levels were determined by the enzyme-linked immunosorbent assay (ELISA, double antigen-bridging) and by radioimmunoprecipitation assay (RIPA). The bone marrow neutralization test was used to determine the neutralizing activity of the antibodies. RIPA and ELISA data resulted in closely correlated measurements. The relations between AEAb levels and the neutralizing activity of the antibodies are variable as shown by follow-up and cross-sectional evaluations of the data. Serum samples with a high antibody level (N 1000 ng/ml) are associated with 100% neutralizing activity, whereas serum samples with lower AEAb levels show partial neutralizing activities or have no effect. Determining the neutralizing activity might be helpful when it comes to deciding of whether or not rhEPO therapy should be continued, specifically in patients who have low antibody levels. The apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC50) did not change in the course of the disease, nor did it correlate to the AEAb levels or the neutralizing activities. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Erythropoietin (EPO) is the primary regulator of erythropoiesis. EPO binds and activates the EPO receptor expressed on the surface of cells. Activation of the EPO receptor by EPO binding induces a cascade of signaling events that support both erythroid proliferation and differentiation (Fisher, 1997). Recombinant human erythropoietin (rhEPO) is a sialoglycoprotein hormone whose action is biologically equivalent to that of endogenous EPO. It is used pharmaceutically for the ⁎ Corresponding author. Molecular Biology Research Laboratory, Department of Otorhinolaryngology, University Medicine Charité, Charitéplatz1, D-10117 Berlin, Germany. Tel.: +49 30 450 555 311; fax: +49 30 450 555 908. E-mail address: [email protected] (J. Gross). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.04.009
stimulation of red blood cell development in anemia patients who have suffered kidney failure or are undergoing tumor therapies. A rare side effect associated with rhEPO therapy is the development of antibody-mediated pure red cell aplasia (PRCA) (Bennett et al., 2004; Casadevall et al., 2002). Between January 1998 and April 2004, 175 cases of epoetin-associated PRCA were reported for Eprex, 11 cases for NeoRecormon, and 5 cases for Epogen (Bennett et al., 2004). Since December 2002 there has been a significant decrease in newly reported cases of rhEPO induced PRCA. As of 30 November 2005, a total of 215 antibody positive PRCA cases had been reported [http: www.jnjpharmarnd.com/company/n-casereports.html]. Recommendations for hematological criteria for use in the diagnosis of epoetin-induced pure red cell aplasia have been
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published (Casadevall et al., 2004). The criteria for diagnosing rhEPO-induced PRCA include mainly changes in hemoglobin levels, reticulocyte numbers or bone-marrow morphology and the detection of serum anti-erythropoietin antibodies (AEAb). The identification of AEAb in the serum is the key feature for diagnosing rhEPO-induced PRCA. Several tests are available for the diagnosis of AEAb, these being mainly radioimmunoprecipitation assays (RIPA), enzyme-linked immunosorbent assays (ELISA) and BIAcore biosensor immunoassays (Hoesel et al., 2004; Swanson et al., 2004; Tacey et al., 2003; Thorpe and Swanson, 2005b). The advantages and disadvantages of these methods were reviewed by Wadhwa et al. (2003) and Thorpe and Swanson (2005a). Whereas tests for determining AEAb measure the binding capacity between EPO and the antibody, tests for neutralizing antibodies (NAb) assess the ability of such antibodies to inhibit biological activity, i.e., the proliferation of erythroid precursor cells (Gupta et al., 2007; Kawade et al., 2003; Schellekens, 2007). The results of bioassays of different laboratories are even more difficult to compare than the results of antibody quantification, because the methods use different cells and activity units (Thorpe and Swanson, 2005a). In order to determine neutralizing antibodies, different tumor cell lines have been used such as the IL-3-dependent murine 32D cell line transfected with human EPO receptors (Wei et al., 2004) or human UT-7/EPO cells, an immortalized cell line (Kelley et al., 2005; Wu et al., 2004). Casadevall et al. (2002) used normal bone marrow cells to characterize the neutralizing activity of anti-erythropoietin antibodies. The mechanisms leading to the development of antibodies to rhEPO are based on breaking the immune tolerance that normally exists to human homologue proteins. It is assumed that the appearance of AEAb and consequently the inhibition of the proliferation of erythroid precursor cells seem to be
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associated with the formation of aggregates during rhEPO storage or with the way rhEPO is administered. The clinical consequences of the presence of antibodies are, in general, a function of the antibodies' binding specificities, their affinity and neutralizing activity (Schellekens, 2007). The aim of the present study was to analyze the relations between the serum anti-erythropoietin antibody levels and their neutralizing activity in 20 patients suffering from renal anemia. The AEAb levels were determined using the double antigen-bridging ELISA (Hoesel et al., 2004), and the neutralizing activity was determined by the bone marrow neutralization test as described by Casadevall et al. (2002). Because the AEAb levels of some of the patients were determined by both RIPA and ELISA, we correlated the results obtained from the two methods. In addition, we determined the apparent affinity of the antibodies (IC50). Analyzing the relations between these criteria may contribute to understanding the characteristics of the rhEPOinduced antibodies and may allow conclusions to be drawn about the necessity of determining the neutralizing activity in the diagnostics of patients developing anti-rhEPO antibodies. 2. Patients and methods 2.1. Patients Serum samples from 20 patients who were suspected of having anti-erythropoietin antibodies were obtained from nephrologists and dialysis centers from Germany and abroad. Details concerning the patients were obtained from the adverse event reports for epoetin beta from its manufacturer Roche, Basel, Switzerland, and from clinicians treating such patients. For follow-up, two to six samples were sequentially collected from nine patients to determine their AEAb levels. The samples submitted to the laboratory for diagnosis
Table 1 Basic data of patients (age, gender, diagnosis, treatment, level of anti-EPO antibodies and neutralizing activity) Patient (age, sex)
Main diagnosis, treatment
1
a
Goodpaster's syndrome; Chronic renal failure; Epoetin beta; 2PRCA (1361) Chronic renal failure; diabetes mellitus; Erypo for 244 d, then NeoRecormon for 59 d; PRCA (291) Chronic renal insufficiency; Erypo, then NeoRecormon (duration unknown); PRCA (257) Chronic renal failure; NeoRecormon; PRCA (449) Diabetic renal insufficiency; NeoRecormon; PRCA (297) End stage renal disease; Epopetin alpha for 37d, then NeoRecormon for 238d; PRCA (273) Chronic renal failure; NeoRecormon; PRCA (449) End stage renal failure, peritoneal dialysis, renal transplant; Epoetin beta; PRCA (734) Chronic glomerulonephritis; Epoetin alpha for 365 d, then Epoetin beta for 31d and Darbepoetin alpha for 7 d; PRCA (396) Chronic renal failure; Eprex for 2559 d, then NeoRecormon for 364 d; PRCA (2920) Diabetic nephropathy; Epoetin beta; PRCA suspected (191) Chronic pyelonephritis; Epoetin alpha for 523 d, then Epoetin beta for 170 d; PRCA (559) Chronic renal failure; Epoetin alpha for 214 d, then Epoetin beta for 1025 d; PRCA (1022) End stage renal failure; NeoRecormon; PRCA (771) Chronic tubulointestinal nephritis; Eprex for 129 d and then NeoRecormon for 227 d; PRCA (368) Chronic renal insufficiency; Epoetin beta; PRCA (458) End stage renal insufficiency, kidney transplantation; Epoetin beta; PRCA (1109) Terminal renal insufficiency, dialysis; Epoetin beta; PRCA (1076) Congenital kidney dysplasia; NeoRecormon; bone marrow normal (233 and 309) Chronic renal failure; NeoRecormon; bone marrow normal (196); absent iron incorporation into bone marrow cells (243)
6000 (1377); 100% (1399) 8365 (338); 83% (772) 7547 (234); 100% (347) 2047 (468); 100% (519) 30030 (328); 100% (398) 6426 (287); 100% (287) 37450 (469); 100% (477) 2500 (759); 100% (759) 3333 (396). 15% (552)
P1 (52,f) P2 (70,m) P3 (62,f) P4 (65,f) P5 (75,m) P6 (80, m) P7 (79,m) b P8 (51,m) c P9 (48, m) P10 (67, m) P11 (87, m) P12 (76, m) P13 (57, m) P14 (65, m) P15 (83, m) P16 (65, f) P17 (24, m) d P18 (68, m) P19 (21, m) P20 (49, f)
AEAb and NAb
1188 (3024); 0% (2920) 926 (232); NAb — nd 522 (690); NAb — nd 4200 (1050); NAb — nd 24 (784); NAb — nd 4500 (427); NAb – nd 18538 (501); NAb — nd 1195 (1120); 25% (1248) 189 (1172); 42% (1172) 184 (272); 26% (232); 0% (260) 248 (320); 0% (243)
a Patient 2 (Tolman et al., 2004); bPatient 4 (Tolman et al., 2004); c(Weber et al., 2002); d(Kruger et al., 2003); 1AEAb levels in ng/ml and NAb activity in % inhibition. In brackets: time (days) between the start of the rhEPO treatment and the blood sampling for AEAb and NAb determination. 2PRCA diagnosis. In brackets: the time (days) between the start of rhEPO treatment and the diagnosis of PRCA. d — days. nd — not determined.
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originated from patients treated between 2000 and 2006. Basic data of the patients analyzed in this study are summarized in Table 1. 2.2. ELISA and RIPA In order to compare the results obtained by RIPA with those of ELISA, samples were analyzed for AEAb levels in both Prof. Casadevall's laboratory using RIPA (Tacey et al., 2003) and our own laboratory using ELISA (Hoesel et al., 2004). In the RIPA, antibodies are determined by the amount of 125J-EPO bound in an immune complex which can be isolated by binding to Protein G Sepharose. RIPA results are expressed in mU of erythropoietin bound by one ml serum as previously described (Casadevall et al., 2002). In the ELISA, biotinylated rhEPO was incubated in streptavidin-coated microtiter plates at a final volume of 125 µl/well. Human serum or reference antibody preparation were mixed with an equal volume of digoxin labeled rhEPO and added to the wells. After incubation and washing, antidigoxin Fab fragment HRP conjugate was added to each well and incubated. ABTS solution was used as a substrate for HRP. Absorbance was measured at 405 nm and 492 nm. The antibody concentration was calculated by using a fourparameter regression curve. A polyclonal anti-EPO IgG fraction from rabbits, purified on an EPO-Sepharose column, was used as calibration material in the ELISA. The results are given in ng/ml serum. Quality control was carried out as described previously (Hoesel et al., 2004). Briefly, upon routine analysis of sera, three samples of reference material covering the assay concentration range were included in the assays in each case, and the recoveries observed were all within 20% of the expected value. The quality control samples yield the same results in both the previous assays and the assessment of the samples studied here. The cut-off in the ELISA methodology was determined as described (Hoesel et al., 2004). Briefly, serum samples (n = 159) randomly chosen from different hospitals and blood banks, and not considered to contain anti-EPO Abs, were analyzed by the standard assay as a representative collection of negative samples. The 90th percentile of these samples was determined at 5 ng/ml and the 99th percentile at 10 ng/ml. 2.3. Bone marrow colony forming assay The inhibition of the growth of erythroid progenitors by patients' serum was tested in Paris as described, using bone marrow from otherwise normal patients who underwent hipreplacement surgery and gave their written informed consent for the collection of bone marrow cells (Casadevall et al., 2002). Briefly, erythroid cells in normal bone marrow were stimulated with 1 U of epoetin per ml in the presence of 20% pooled control or 20% patients serum. Erythroid colonies were scored after seven days in culture. The neutralizing activity was expressed as a percentage inhibition of colonies formed in the presence of patients serum compared to control serum. 2.4. Apparent affinity (IC50) The concentration of unlabeled rhEPO that produces a 50% inhibition of antibody binding (IC50) was determined by in-
cubating antibodies with increasing concentrations of unlabelled rhEPO in the range of 0.5–30 ng/ml using the ELISA method. The unlabelled rhEPO was added to the well together with DIG-labeled rhEPO. The IC50 of rhEPO was calculated by nonlinear regression analysis of the experimental data using the following equation: antiEPOAbbound ¼
antiEPOAbmax IC50 rhEPO þ IC50
where AntiEPOAbmax denotes the bound antiEPO antibody in the absence of rhEPO. IC50 denotes the rhEPO concentration required to trigger 50% inhibition of the antigen-antibody binding in ELISA. 3. Results 3.1. Patients Table 1 indicates the basic data of 20 patients from eight different countries analyzed for anti-erythropoietin antibody levels and bone-marrow morphology. All of these patients had been treated with rhEPO for renal anemia, with twelve patients having received treatment with epoetin beta and eight patients with epoetin alpha and beta. The bone-marrow analysis resulted in PRCA diagnosed in 18 patients and the exclusion of PRCA in two patients with a normal morphology (P19, P20). The time between the start of rhEPO treatment and the diagnoses of PRCA varied between 191 and 2920 days (median 454 days). Generally, the detection of AEAb formation coincided with that of PRCA. The AEAb levels measured for all these patients comprised a range of 3–37000 ng/ml with a median of about 3000 ng/ml. Analyses of neutralizing antibodies were carried out in the serum samples of 14 patients. The serum samples used for determining neutralizing antibodies were collected about three months after the analyses of the bone marrow morphology (median 80 days, range 0–481 days). The antibodies' neutralizing activity varied between 0% and 100% inhibition. The two patients without PRCA (P19, P20) did not show any neutralizing activity. Among the PRCA patients analyzed for NAb (n = 12), eight patients showed complete (80–100% inhibition) and three patients partial inhibition (15–42%), whereas one patient (P10) had non-neutralizing antibodies. For technical reasons, the activity of neutralizing antibodies could not be determined in six patients. 3.2. Relations between antibody levels measured with RIPA and ELISA During this study, 35 serum samples were analyzed in parallel by both RIPA and ELISA to compare whether the two different methods were a) detecting antibodies and b) assess amounts of antibodies were comparable. The statistical analysis of the results showed the two measurements to be closely correlated with a correlation coefficient of R2 = 0.92 (Fig. 1). The samples included in this correlation comprise samples from Casadevall`s laboratory (n = 15), from the patients of this study (n = 19) and one sample from a patient from a different laboratory (see legend of Fig. 1). Samples from
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a given patient were collected at different time points during the course of disease and were blindly analyzed in the corresponding laboratory. From the equation found to be describing the correlation between RIPA and ELISA, it can be calculated that 5 ng/ml (cut-off value for ELISA) correspond to a RIPA value of 47 mU/ml, a number very close to the cut-off value reported for RIPA (40 mU/ml, personal communication). Parallel analyses of AEAb levels of follow-up samples from patient P17 using ELISA and RIPA showed a similar pattern in the course of the disease, in both the increasing and the decreasing phases of the disease (Fig. 2). Thus, the results obtained by RIPA and ELISA are comparable in quantitative terms. 3.3. Relations between the levels of anti-erythropoietin antibodies and their neutralizing activities In order to get an insight into the relations between the levels of anti-erythropoietin antibodies and their neutralizing activity we compared follow-up values of AEAb and NAb levels during a period of about four years in eight patients. To normalize the course of these curves, the time at which the highest AEAb value occurred was used as the reference point. This point corresponds roughly to the time of the diagnosis of the PRCA and the withdrawal of rhEPO. For all patients we observed a continuous decrease in AEAb levels (Figs. 3 and 4). This decrease followed an exponential function, with a steep decrease during the first year of the observational period and a slower one in subsequent years. A slower decrease rate of the AEAb levels was observed in two patients (P3, P8), indicating heterogeneity in the decrease rate of AEAb levels. The NAb levels of five patients (Fig. 3) showed 100% inhibition of erythroid colonies at the reference point and decreased continuously, roughly in parallel to the log 10 of the AEAb levels (Fig. 3). In three patients, the neutralizing activity
Fig. 1. Correlation of anti-erythropoietin antibody levels as determined by RIPA and ELISA. Data are given in logarithmic terms because of the high range of pathological values. The data follow the equation y = 0.107 x; R2 = 0.92, n = 30. Five samples showed negative values and therefore were not included in the logarithmic correlation. The samples included in this correlation comprise one sample from each patient of Casadevall`s laboratory (n = 15), nine samples of patient P17, five samples from patient P8, three samples from patient P2, two samples from patient P3 (see Table 1) and one sample from a patient from a different laboratory. No significant differences were observed in the regression and correlation coefficients of the groups with n ≥ 5.
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Fig. 2. Anti-erythropoietin antibody levels of patient P17 determined in parallel by RIPA and by ELISA. Abscissa: Time in days after start of rhEPO treatment. For details of patient P17 see Table 1. The first five samples of this patient were originally collected for other diagnostic purposes and were stored at −20 °C.
followed a different course. At the reference point, a partial neutralizing activity was observed, which remained unchanged in two patients for six months irrespective of the fact that the AEA levels decreased continuously (Fig. 4). In one patient, we observed the neutralizing activity to rise by up to 100% inhibition within a period of 300 days. A cross-section correlation analysis of all available parallel AEAb and the NAb determinations (Fig. 5) shows complete neutralizing activities
Fig. 3. Decrease of anti-erythropoietin antibody levels and neutralizing activity during the disease in five patients. Top: Anti-erythropoietin antibody levels. Bottom: Neutralizing activity. For details of the patients see Table 1. The AEAb levels are shown as a log scale because of the high range of pathological values. For better comparison, the curves have been normalized (time at which the highest AEAb level was observed =day 0).
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Fig. 6. Inhibition of anti-erythropoietin antibody binding by unlabelled rhEPO. Samples were preincubated with different concentrations of unlabeled rhEPO added to the microtiter plate. Samples originate from different phases of the disease of patient P2 (see Table 1). Samples were collected on day 411 (full circles; 943 ng/ml), day 690 (full squares; 31 ng/ml) and on day 851 (open triangles; 12 ng/ml) after the start of treatment. The corresponding IC50 values are 5.8, 15.0 und 3.7 ng/ml, respectively.
Fig. 4. Decrease of anti-erythropoietin antibody levels and different changes of neutralizing activity during the disease in three patients. Top: Antierythropoietin antibody levels. Bottom: Neutralizing activity. For details see Table 1 and legend of Fig. 3.
at high AEAb levels above about 1000 ng/ml. Below this level, neutralizing activities between zero and 60% were observed. 3.4. Apparent affinity of anti-erythropoietin antibodies To further characterize the AEAb, we wanted to know whether the neutralizing activity relates to the apparent affinity of the antibodies. Samples of patient P2 (parallel decrease of AEAb and NAb levels, Fig. 3) were used to determine the half-
Fig. 5. Relation between anti-erythropoietin antibody levels and neutralizing activity. AEAb levels and neutralization activity were determined from aliquots of the same sample. Data originate from 10 patients. The AEAb levels are given in logarithmic terms because of the high range of pathological values.
maximal inhibition concentration (IC50) of unlabeled rhEPO as an indicator of the antibodies' apparent affinity. Fig. 6 illustrates that the addition of rhEPO (0.25–30 ng/ml) displaces 70–90% of the labeled rhEPO from the AEAb binding, independent of the AEAb level (12–943 ng/ml) and the disease phase. The IC50 values for rhEPO ranged from 3.7–15.0 ng/ml (200–800 pM) in this patient. Determination of IC50 values of samples collected from five patients showing different AEAb levels confirmed that the IC50 values are independent of the disease phase and the AEAb levels. In the mean, an antibody apparent affinity of 7.2 ±2.0 ng/ml (n =11) was observed. To exclude possible effects of the AEAb level on the IC50 value we determined the IC50 values in a sample by diluting a positive serum containing antibodies of 673 ng/ml. No significant changes in the apparent affinity were observed between undiluted samples and those diluted at rates of 1/10 and 1/100 (data not shown). 4. Discussion Several major findings have been made in this paper. (1) We observed a close correlation between the antibody levels determined by RIPA and those determined by the double antigen-bridging ELISA using biotinylated rhEPO. (2) Serum samples with a high antibody level (N1000 ng/ml) are in general associated with a complete neutralizing activity. In samples with lower levels the antibodies induce partial neutralization or have no effect. (3) The apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC50) did not change in the course of the disease, nor did it correlate to the AEAb levels or the neutralizing activity. Controversy exists over the correlation between AEAb levels determined by RIPA and those determined by ELISA (Thorpe and Swanson, 2005b). In the present study, we found a close correlation to exist between serum levels determined by RIPA and those determined by the double antigen-bridging ELISA (Hoesel et al., 2004). In a previous study, we had shown that the format of the ELISA tests dramatically affects the result (Gross et al., 2006). The present data confirm our previous findings that the double antigen-bridging ELISA using
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biotinylated rhEPO and digoxin labeled rhEPO results in a sensitive, specific and accurate determination of AEAb levels. The relationship between AEAb level and the antibodies' neutralizing activity in patients with rhEPO-induced antibodies are highly variable as shown by follow-up and crosssectional evaluations of the data. In the course of the disease, some patients showed a decreasing neutralizing activity in parallel to decreasing AEAb levels, whereas in other patients the neutralizing activity remained unchanged or even increased despite falling AEAb levels. Cross-sectional analysis has shown that serum samples with a high antibody level (N1000 ng/ml) are associated with a complete neutralizing activity whereas serum samples with lower AEAb levels have differing levels of neutralizing antibodies, with their activity ranging from partially neutralizing to non-neutralizing. On the one hand, these findings might support the coating theory of neutralization assuming that high AEAb levels prevent the binding of the rhEPO molecule to target cell receptors due to steric hindrance (Wilson and McElwain, 2004). On the other hand, the patient-specific time course of the neutralizing activity suggests that antibodies with differing neutralizing activity occur. Most probably, there exists a preferential synthesis of specific neutralizing antibodies with more or less pronounced inhibitory capacities. This assumption is supported by experimental findings. For example, Feldman et al. (1992) produced a series of monoclonal antibodies to amino acid region 99–129 of human erythropoietin that display unique structural features within the putative receptor binding domain of the hormone. Three of the four monoclonal antibodies neutralized the biological activity of EPO in a concentration-dependent fashion in vitro. Similarly, Fibi et al. (1993) produced and characterized recombinant human erythropoietin specific mouse monoclonal antibodies, which were specifically reactive with recombinant human erythropoietin in the enzyme-linked immunosorbent assay. Epitope exclusion studies showed the three distinct epitope regions A, B and C recognized by neutralizing monoclonal antibodies. Non-neutralizing monoclonal antibodies recognized an additional epitope region D. Antibodies defining an epitope region competed with one another for binding sites, but did not compete with antibodies defining a different epitope region. The finding that the apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC50) did not change in the course of the disease and did not correlate to the neutralizing activity of the antibodies confirms previous observations (Casadevall et al., 2002). 5. Conclusion The heterogeneity of the relations between the AEAb levels and the antibodies' neutralizing activity illustrates that the antibodies of the individual patients have an inherent property of being neutralizing or non-neutralizing. This feature might be important in determining the efficiency of therapy or the clinical sequelae (Gupta et al., 2007). Therefore, it is important to decide when and how neutralizing antibodies should be measured in patients treated with rhEPO. A negative result following an ELISA or RIPA test eliminates the need for neutralizing-activity testing, because the binding assay binds all antibodies, both neutralizing and non-neutralizing (Deisenhammer et al., 2004). The present
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study indicates that anti-erythropoietin antibodies above about 1000 ng/ml are most likely to show a complete neutralizing activity, which, in general, makes it unnecessary to determine the antibodies' neutralizing activity. However, in patients with a lower antibody level, where the clinician suspects a loss of clinical efficiency of rhEPO treatment, the determination of neutralizing activities may be helpful when it comes to deciding of whether or not rhEPO therapy should be continued. Therapy with erythropoietin should be discontinued, when neutralizing anti-erythropoietin antibodies are diagnosed. Where non-neutralizing antibodies are diagnosed, continuation of the rhEPO therapy or its restart seem possible as long as the AEA level and the neutralizing activity are monitored, because more rhEPO treatment may induce neutralizing antibodies. Measuring the neutralizing activity of antibodies is no trivial matter (Kawade et al., 2003). There are several methods available for measuring the neutralizing activity of antierythropoietin antibodies (Casadevall et al., 2002; Kelley et al., 2005; Wei et al., 2004). The present study shows that a useful neutralizing test has to be sensitive enough to identify anti-erythropoietin antibody levels between about 10 ng/ml and 1000 ng/ml. Theoretically, human bone marrow cells are best-suited for such tests, but using human bone marrow from healthy donors is neither easily manageable nor ethical. Acknowledgments This work was supported by grants from the University Medicine Charité Berlin (J.G.) and from Roche Diagnostics GmbH (J.G. and R.M.). We are grateful to T. Haselbeck and W. Hoesel (Roche) and to several physicians and nurses for their support and for collecting the serum samples for this study. We thank Prof. Casadevall for performing the RIPA and the neutralization assays and for her cooperation. We wish to thank K.-U. Eckardt for criticism and support. Responsibilities for interpretation of the data and conclusions lies with the authors only. The protocol and the patient information sheet were reviewed and approved by the regulatory authorities of Roche. References Bennett, C.L., Luminari, S., Nissenson, A.R., Tallman, M.S., Klinge, S.A., McWilliams, N., McKoy, J.M., Kim, B., Lyons, E.A., Trifilio, S.M., Raisch, D.W., Evens, A.M., Kuzel, T.M., Schumock, G.T., Belknap, S.M., Locatelli, F., Rossert, J., Casadevall, N., 2004. Pure red-cell aplasia and epoetin therapy. N. Engl. J. Med. 351, 1403. Casadevall, N., Nataf, J., Viron, B., Kolta, A., Kiladjian, J.J., Martin-Dupont, P., Michaud, P., Papo, T., Ugo, V., Teyssandier, I., Varet, B., Mayeux, P., 2002. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N. Engl. J. Med. 346, 469. Casadevall, N., Cournoyer, D., Marsh, J., Messner, H., Pallister, C., ParkerWilliams, J., Rossert, J., 2004. Recommendations on haematological criteria for the diagnosis of epoetin-induced pure red cell aplasia. Eur. J. Haematol. 73, 389. Deisenhammer, F., Schellekens, H., Bertolotto, A., 2004. Measurement of neutralizing antibodies to interferon beta in patients with multiple sclerosis. J. Neurol. 251 (Suppl 2), II31. Feldman, L., Heinzerling, R., Hillam, R.P., Chern, Y.E., Frazier, J.G., Davis, K.L., Sytkowski, A.J., 1992. Four unique monoclonal antibodies to the putative receptor binding domain of erythropoietin inhibit the biological function of the hormone. Exp. Hematol. 20, 64. Fibi, M.R., Aslan, M., Hintz-Obertreis, P., Pauly, J.U., Gerken, M., Luben, G., Lauffer, L., Siebold, B., Stuber, W., Nau, G., 1993. Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization. Blood 81, 670.
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