Technology comparisons for anti-therapeutic antibody and neutralizing antibody assays in the context of an anti-TNF pharmacokinetic study

Journal of Immunological Methods 345 (2009) 17–28

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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

Technology comparisons for anti-therapeutic antibody and neutralizing antibody assays in the context of an anti-TNF pharmacokinetic study Kelly M. Loyet a,⁎, Rong Deng b, Wei-Ching Liang c, Yan Wu c, Henry B. Lowman c, Laura E. DeForge a a b c

Department of Assay & Automation Technology, Genentech, South San Francisco, CA, USA Department of Pharmacokinetic and Pharmacodynamic Sciences, Genentech, South San Francisco, CA, USA Department of Antibody Engineering, Genentech, South San Francisco, CA, USA

a r t i c l e

i n f o

Article history: Received 5 November 2008 Received in revised form 23 February 2009 Accepted 19 March 2009 Available online 2 April 2009 Keywords: Anti-therapeutic antibody assay Neutralizing antibody assay Anti-TNF Electrochemiluminescence Flow cytometry

a b s t r a c t A single-dose cynomolgus monkey pharmacokinetic study was performed comparing two monoclonal anti-TNF antibodies (mAbs), GNExTNFvF and Humira®. Normal pharmacokinetic profiles were observed over the first week of the study, followed by a rapid drop in serum mAb levels after day 8. In order to determine whether an anti-therapeutic antibody (ATA) response led to the abnormal clearance of antibody in this study, ATA assays were developed using two electrochemiluminescent technologies, BioVeris and Meso Scale Discovery (MSD). Characterization of the assays demonstrated that the two platforms gave similar sensitivities and tolerance to the presence of therapeutic antibody. Analysis of the cynomolgus monkey serum samples revealed that all animals developed significant ATA titers with log titer values of 2–4, with the BioVeris and MSD technologies giving very similar results. Immunodepletion studies confirmed the CDR-specificity of the ATA response for the GNExTNFvF-dosed cynos, although the Humira-dosed cynos showed both CDR-specific and human IgG1 frameworkspecific ATAs. To further characterize the ATA response, neutralizing antibody (NAb) assays were developed using two different approaches, flow cytometry and MSD. Flow cytometry and MSD cell-binding assays used Jurkat cells transfected with noncleavable TNF (huTNFNC). Neutralizing activity was assessed by the ability of ATA-positive serum samples to block the binding of biotinylated anti-TNF to huTNFNC Jurkat cells, showing that all but one animal developed neutralizing antibodies. Although both technologies displayed similar trends, the MSD approach showed greater differentiation between samples and could detect a broader range of neutralizing activities. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Tumor necrosis factor (TNF) is a key pro-inflammatory cytokine that up-regulates cytokine and chemokine production as well as adhesion molecule expression (Palladino et al., 2003). Anti-TNF therapies have been demonstrated to be effective in ameliorating deleterious effects of TNF in chronic autoimmune diseases such as rheumatoid arthritis, Crohn's disease, and psoriasis (Palladino et al., 2003; Mousa et al., 2007). Current anti-TNF therapies available on the market ⁎ Corresponding author. Genentech, Inc., MS 98, 1 DNA Way, South San Francisco, CA, 94080, USA. Tel.: +1 650 225 4022; fax: +1 650 225 1770. E-mail address: [email protected] (K.M. Loyet). 0022-1759/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2009.03.012

include: infliximab (Remicade®), a chimeric anti-TNF IgG1 monoclonal antibody (mAb); etanercept (Enbrel®), TNFRIIIgG1 Fc recombinant fusion protein; and adalimumab (Humira®), a humanized anti-TNF IgG1 monoclonal antibody (Shen et al., 2005). The disadvantages of these current therapies include their serum half-lifes that require weekly or biweekly injections and the generation of anti-drug responses. Potential approaches to improving anti-TNF mAb therapy include increasing the binding affinity to TNF as well as improving the pharmacokinetics by increasing the terminal half-life of the anti-TNF antibody. GNExTNFvF is a humanized anti-TNF mAb generated from a parental murine GNExTNF clone (Bringman and Aggarwal, 1987). This antibody was

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humanized (accomplished through swapping of CDR regions into a human framework) and affinity matured. GNExTNFvF and Humira were evaluated in a cynomolgus monkey pharmacokinetic (PK) study. Two different sandwich enzyme linked immunosorbent assays (ELISAs) were developed to quantitate serum concentrations of GNExTNFvF and Humira. While one ELISA was based on binding of the antibody to TNF-coated plates, the other allowed for the specific detection of human IgG (either free or bound to TNF) in cynomolgus monkey serum. During the PK study, a dramatic decline in anti-TNF mAb serum concentrations was observed in both assays for all animals after the day 8 time point suggesting an anti-therapeutic antibody (ATA) response. Therefore, ATA assays were developed to evaluate ATA titers. In order to compare technologies, two different electrochemiluminescence (ECL) assays were evaluated, using either the BioVeris M384 analyzer or the Meso Scale Discovery (MSD) analyzer. Both assays were bridging ECL assays in which ATAs were detected through simultaneous binding to biotinylated-therapeutic and ruthenium-conjugated therapeutic. Upon formation of a complex between anti-therapeutic antibody, biotinylated therapeutic, and rutheniumconjugated therapeutic, light is produced upon application of voltage. This emission of light involves an oxidationreduction reaction with ruthenium metal ions and tripropyl amine (TPA) in which the TPA is in molar excess and the ruthenium can be recycled, resulting in cyclical signal generation and increased sensitivity. Although both the BioVeris and MSD technologies capture the complexes through binding of streptavidin to the biotinylated therapeutic, the BioVeris assay is bead-based, using streptavidincoated paramagnetic beads, while the MSD assay uses streptavidin-coated carbon electrode plates. In addition, signal is detected with the BioVeris technology by photo diodes whereas MSD instruments use a CCD camera or diode array. The two technologies also differ with regard to washing procedure, which may affect the detection of low affinity binding interactions; for the BioVeris platform, there is a single instrument-performed wash, whereas the MSD assay involves multiple washes performed by the user. Therefore, although these technologies are quite similar in many respects, the differences in solid support, signal detection, and washing can potentially impact assay performance. However, given that the BioVeris instrument is no longer supported, comparisons of the technologies are of value in identifying a suitable replacement technology. Finally, two different NAb assays were developed to determine whether ATAs produced in therapeutic-dosed cynomolgus monkeys blocked binding of the anti-TNF mAbs to TNF. Cell-binding assays using either flow cytometry or MSD were developed using Jurkat cells stably transfected with a noncleavable TNF mutant (huTNFNC Jurkat cells). These two different NAb assays were used to test the cynomolgus monkey sera and results were compared. 2. Materials and methods 2.1. Reagents Full-length humanized anti-TNF monoclonal antibody GNExTNFvF was generated at Genentech, Inc. (South San

Francisco, CA) based on a previously described antibody produced by a mouse hybridoma (Bringman and Aggarwal, 1987). Recombinant humanized monoclonal antibodies used as isotype controls and human TNF were also generated at Genentech. Humira was purchased from Abbott (Abbott Park, IL). BSA was purchased from Intergen (Purchase, NY); CHAPS was from Research Organics (Cleveland, OH); bovine gamma globulin (BgG) and fish gelatin were from Sigma Chemical Co. (St. Louis, MO); ProClin 300 was from Supelco (Bellfonte, PA).

2.2. Cyno PK study — animals, study design, and blood sample collection This study was conducted at Shin Nippon Biomedical Laboratories USA, Ltd. (SNBL, Everett, WA), according to their standard operating procedures and in compliance with applicable regulations concerning the use of laboratory animals. Six male and six female cynomolgus monkeys (Macaca fascicularis) were obtained from SNBL stock. Males weighed 2.0–4.5 kg and females weighed 2.5–5.5 kg. All animals used in the study were 1.5–7.9 years old. The monkeys were acclimatized to the study room for a minimum of 14 days before initiation of dosing. Only animals that appeared to be healthy and that were free of obvious abnormalities were used for the study. There were 6 animals (3 male, 3 female) in each of the two anti-TNF antibody groups. These monkeys received a single IV dose of either 5 mg/kg of GNExTNFvF (animals #1 through #6) or Humira (animals #7 through #12). Serum samples were obtained for anti-TNF mAb serum concentration measurements with frequent sampling throughout the first week (predose, and 30 min, 6 h, day 2, day 4, day 6, day 8 postdose), followed by weekly sampling. Blood samples (∼1.5 mL) from restrained, non-sedated animals were collected following overnight food removal by venipuncture from an available peripheral vein into collection tubes without anticoagulant. Serum was harvested then stored at −60 °C or colder prior to analysis for anti-TNF or ATA.

2.3. TNF-binding ELISA ELISA plates (384 well, Nunc, Neptune, NJ) were coated with huTNF in PBS, pH 7.2 at 0.5 µg/mL (25 µL/well) and incubated overnight at 4 °C. Plates were washed three times with Wash Buffer (PBS/0.05% Tween-20), blocked for 1–2 h with Block Buffer (PBS/0.5% BSA, 50 µL/well), followed by a wash step as before, and sample/standard addition (25 µL/ well) for 1.5–2 h. Samples and standards were diluted in PK Assay Buffer (base assay diluent [PBS pH 7.4/0.05% BSA/0.05% Tween-20/10 ppm Proclin 300] supplemented with 0.2% BgG/0.25% CHAPS/5 mM EDTA/0.35 M NaCl). Plates were washed six times followed by addition of goat F(ab')2 fragment anti-human IgG Fc HRP (Jackson ImmunoResearch, West Grove, PA), diluted to 16 ng/mL in PK Assay Buffer (25 µL/well). Plates were incubated for 1–2 h followed by a wash step as before, developed with TMB substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 12–14 min, stopped with 1 M phosphoric acid, and read at 450–620 nm. Separate standards were used for GNExTNFvF and Humira.

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2.4. Human IgG ELISAs

2.7. MSD anti-therapeutic antibody assay

384-well microtiter plates were coated with sheep antihuman IgG (monkey adsorbed, purchased from The Binding Site, San Diego, CA) in 0.05 M sodium carbonate, pH 9.6 at 0.25 µg/mL and incubated overnight at 4 °C. The ELISA was performed as explained above except using sheep antihuman Ig HRP (monkey adsorbed, The Binding Site) at 400 ng/mL as the detection antibody. This assay is based on the generic immunoglobulin pharmacokinetic (GRIP) assay that was recently described (Yang et al., 2008).

Equal concentrations of biotin and ruthenium antibody conjugates were pre-mixed at 250 ng/mL or 500 ng/mL for GNExTNFvF or Humira assays, respectively, and added to polypropylene round-bottom plates (50 µL/well). Samples or controls (25 µL/well) were then added and the plates were allowed to incubate for 1.5–2 h or overnight (Humira) at room temperature with gentle agitation. MSD standard streptavidin plates were blocked with 3% MSD Blocker A for at least 30 min at room temperature or overnight at 4 °C, then washed three times with Wash Buffer. Sample/conjugate mix (50 µL/well) was transferred from the polypropylene plate to the MSD plate and incubated for 1–2 h at room temperature with gentle agitation. Plates were washed six times prior to 1 x Read Buffer T (MSD) addition and read on an MSD machine (Sector Imager 6000 reader).

2.5. Ruthenium and biotin conjugation of antibodies GNExTNFvF and Humira were ruthenium conjugated at a challenge ratio of 8:1 using BV-TAG® (BioVeris, Gaithersburg, MD) or sulfo-TAG (MSD, Gaithersburg, MD) according to the manufacturers’ instructions followed by buffer exchange into formulation buffer (100 mM arginine/10 mM histidine, pH 5.6/0.04% Tween-20). The BV-TAG to protein ratios were 3.5:1 or 2.8:1, and the protein recoveries were 71% or 80% for GNExTNFvF or Humira, respectively. The sulfo-TAG to protein ratios were 6:1 or 4.84:1, and the protein recoveries were 54% or 58% for GNExTNFvF or Humira, respectively. The BV-TAG and sulfo-TAG incorporation ratios were determined by UV absorption as per manufacturer instructions. GNExTNFvF was buffer exchanged into PBS + 0.04% Tween-20 (to aid in solubility) and was biotinylated with EZ-Link SulfoNHS-LC-Biotin (Pierce, Rockford, IL) using a challenge ratio of 8:1. Biotin conjugation of Humira was performed using an EZ-Link NHS-PEO solid phase biotinylation kit pre-packed column as per manufacturer's instructions (Pierce). Biotinylations of both GNExTNFvF and Humira were followed by buffer exchange into formulation buffer. The biotin:protein ratios of GNExTNFvF and Humira were 4.2:1 and 6.3:1, and protein recoveries were 72% and 90%, respectively. The biotin incorporation ratios were determined with Pierce EZ Biotin Quantitation Kit as per manufacturer instructions.

2.6. BioVeris anti-therapeutic antibody assay For both GNExTNFvF and Humira, the biotin and ruthenium antibody conjugates, each at a concentration of 500 ng/ mL, were pre-mixed in ATA Assay Buffer (80 mM HEPES buffer/0.5% fish gelatin/0.25% Tween-20/0.25% CHAPS/0.35 M NaCl/0.05% Proclin 300, pH 7.0) and added to wells of polypropylene round-bottom plates (50 µL/well). Equal volumes of samples or controls diluted in ATA Assay Buffer were then added, and the plates were allowed to incubate at room temperature with gentle agitation for 1.5–2 h or overnight (Humira). Cynomolgus monkey serum positive for anti-human IgG framework was used as a positive control. A suspension containing 0.0667 mg/mL of streptavidincoated Dynabeads M280 (Invitrogen, Carlsbad, CA) was prepared in ATA Assay Buffer and added to the plates (150 µL/well). After a further 1 h incubation, the plate was then transferred to the BioVeris M-series M384 Analyzer for electrochemiluminescence measurement at 620 nm.

2.8. Neutalizing antibody cell-binding assay by flow cytometry The non-cleavable TNF Jurkat cells were prepared as follows. Human TNF was cloned to the pMSCV-GFP (retrovirus) vector (Clontech, Mountainview, CA) and twelve residues (VRSSSRTPSDKP) were deleted by mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Phoenix Amphotropic cells (Dr. G. Nolan laboratory, Stanford, CA) were transfected with this plasmid using the calcium phosphate method and the retrovirus supernatant was used to infect Jurkat cells (ATCC, Manassas, VA) using a spin infection technique. Transfection and infection were performed as previously described (Swift et al., 2001). Jurkat cells were sorted by Fluorescenceactivated Cell Sorting (FACS) for GFP-positive cells and selected for a single cell clone. Clones were stained by antiTNF mAb GNExTNFvF for confirmation. Jurkat cells transfected with retrovirus non-cleavable TNF-IRES-GFP-RV (huTNFNC Jurkat) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1.5 g/L sodium bicarbonate, 10 mM HEPES, 1.0 mM sodium pyruvate, 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM L-glutamine. HuTNFNC Jurkat cells were washed into flow cytometry buffer (0.5% BSA/PBS) and aliquoted into 96-well V-bottom plates, 1 × 106 cells/well, then centrifuged to obtain the cell pellets. Biotinylated GNExTNFvF, Humira, or IgG1 isotype control (500 ng/mL) was combined with a 1/20 dilution of prebleed or day 43 ATA-positive cynomolgus monkey serum in flow cytometry buffer. After 45 min at room temperature with gentle agitation, 100 µL of the mixture was added to the cell pellets for 30 min at room temperature. Washes and all further incubations were done at 4 °C or on ice in flow cytometry buffer. Cells were washed twice with 250 µL buffer, and biotinylated antibody bound to cells was detected with phycoerythrin (PE)-conjugated streptavidin (Becton Dickinson, San Jose, CA) diluted 1:100 (5 µg/mL). Prior to analysis, cells were washed twice. Analysis was performed either immediately or after overnight 4 °C fixation with 1% paraformaldehyde. Only TNF-expressing cells also produced GFP, so GFP-expressing cells were first gated on the FITC channel to detect this population. At least 10,000 events were acquired on a BD FACSCalibur cytometer (Becton Dickinson, San Jose, CA). Neutralizing antibody activity was determined by: (predose

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MFI)/(postdose MFI). Those samples with a neutralizing antibody activity above 2 were considered positive.

3. Results 3.1. Determination of anti-TNF serum concentrations

2.9. Neutralizing antibody cell-binding assay by MSD MSD cell-binding assays were performed as previously described (Lu et al., 2006). In brief, cells were washed into PBS and 100,000 cells per well were seeded onto High Bind MSD plates for 1 h followed by blocking with 30% FBS for 30 min at room temperature with gentle agitation. A dose response curve of biotinylated GNExTNFvF, Humira, or IgG1 isotype control (1–1000 ng/mL) in the presence of a constant concentration of 5% dosed animal serum or control serum (predose pool, n = 28) was prepared in 10% FBS/PBS and incubated for 30 min to 1 h at room temperature. The High Bind MSD plate containing the cells was decanted and blotted thoroughly prior to addition of samples for 1 h at room temperature with gentle agitation. Plates were washed gently three times with PBS before addition of streptavidin-TAG (MSD) at 250 ng/mL. Plates were incubated 1 h at room temperature with gentle agitation, washed as before, followed by addition of surfactant-free read buffer at 1x (MSD) and read on the MSD machine (Sector Imager 6000 reader). Each EC50 value was calculated as the midpoint of the dose response curve using a four-parameter regression curvefitting program (KaleidaGraph, Synergy Software, Reading, PA). For those curves that did not reach saturation, the EC50 was estimated using a curve fit in which the upper asymptote was fixed at the maximal ECL unit (ECLU) value obtained with the predose response curve. For dose-response MSD studies, neutralizing antibody activity was determined by: (postdose EC50)/(predose EC50). For single dose MSD studies, neutralizing antibody activity was determined by: (predose ECLU)/ (postdose ECLU). Those samples with a neutralizing antibody activity above 2 were considered positive.

3.1.1. TNF-binding PK assay parameters and characterization Cynomologus serum samples were analyzed using a quantitative ELISA to measure anti-TNF GNExTNFvF or Humira concentrations. For this purpose, a TNF-binding ELISA was developed in 384-well plates using recombinant human TNF to capture and an HRP-conjugated anti-human IgG Fc antibody to detect the anti-TNF antibodies. Various buffers were tested for their abilities to minimize cynomolgus monkey serum interference in the assay (data

2.10. ATA assay data analysis Cut point Factor Mean + (Zp⁎ x SD) where: Mean Mean signal (untreated population)/mean background (negative control pool) = mean S/B p % false positive rate (5% chosen) p⁎ p − (% outliers) Zp⁎ value obtained from Excel using function NORMINV ((1 − p⁎), 0,1); Zp⁎ = 1.65 for p⁎ = 5% SD standard deviation of mean S/B The Log Titer was determined using the following equation: Log Titer Log [((ECLUHigh − CP)/(ECLUHigh − ECLULow))(DF1− DF2)+ DF2] ECLUHigh ECLU value that is closest to but higher in value than the Cut point ECLULow ECLU value that is closest to but lower in value than the Cut point CP Cut point Dilution Factor of the ECLULow DF1 DF2 Dilution Factor of the ECLUHigh A % titer recovery was calculated as: [(titer obtained in the presence of therapeutic)/(titer in the absence of therapeutic)] x 100

Fig. 1. Evaluation of GNExTNFvF (A) or Humira (B) anti-TNF mAb concentrations using a TNF-binding ELISA (solid line) or huIgG ELISA (dotted line). Both PK assay formats show a rapid decline in anti-TNF mAb concentrations after day 8. The effective lower limit of quantitation was 78 ng/mL. Correlation plot (C) of TNF-binding assay and IgG-binding concentrations (µg/mL) shows that both PK assay formats give similar results (r = 0.99).

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not shown). Negligible interference of 1% serum was observed when a base assay diluent (PBS/0.5% BSA/0.05% Tween-20) supplemented with 0.2% BgG, 0.25% CHAPS, 5 mM EDTA, and 0.35 M NaCl was used as both the standard/sample diluent and the detection antibody diluent. Assay optimization and characterization was performed for the GNExTNFvF and Humira assays concurrently. The assay ranges were 0.78–50 ng/mL and both assays tolerated up to 1% serum. The mean accuracy for both assays was 84– 101% for a range of anti-TNF mAb concentrations tested, and the inter- and intra-assay variability was within ≤15%. No cross reactivity or interference was observed with other recombinant humanized antibodies or other TNF family members (B cell-activating factor belonging to the TNF family (BAFF) and lymphotoxin-alpha). Interference studies showed that detection of the anti-TNF mAbs was not affected by the addition of soluble TNF up to a concentration of 1 ng/ml (GNExTNFvF) or 8 ng/mL (Humira) (data not shown). Endogenously produced soluble TNF would therefore not be expected to interfere in the assay given that it is present in human serum at concentrations less than 1 pg/mL (Saito et al., 1999). In addition, the assay showed good linearity of dilution with percent recoveries from 80–120% for GNExTNFvF and Humira. Finally, low, mid/low, mid, and high analyte concentrations in neat cyno serum were stable for at least three freeze/thaw cycles.

3.1.2. PK profiles in cynomolgus monkeys Both GNExTNFvF and Humira mAbs were well tolerated with no safety signals. The results of PK sampling, as shown in Fig. 1, indicated a rapid decline in anti-TNF mAb concentrations after day 8 and undetectable levels after day 22 for all animals. Due to limited data, PK parameters such as clearance and half-life could not be determined accurately. The rapid drop in anti-TNF mAb concentrations was suggestive of a strong ATA response in the animals. Indeed, it has been reported that in a prior Humira single dose non-human primate study, primate anti-human antibodies developed and affected the pharmacokinetic elimination (http://www.emea.europa.eu/humandocs/PDFs/ EPAR/humira/400803en6.pdf). However, to exclude the possibility that circulating mAb was not being detected in the TNFbinding ELISA either due to degradation of the antigen-binding regions of the mAbs or due to the exceedingly remote possibility of high serum TNF levels, the samples were analyzed in an alternative ELISA format which captured and detected the human IgG portion of the anti-TNF antibodies. The human IgG ELISA format (IgG ELISA) used a sheep polyclonal anti-human IgG antibody that was adsorbed against cyno IgG. This antibody was coated on the plate to capture the anti-TNF mAbs, and then an HRP-conjugated version of the same antibody was used for detection. Like the TNF-binding assay, the human IgG assay range was 0.78– 50 ng/mL, although the human IgG assay could tolerate at least 2% cyno serum (vs. up to 1% for TNF-binding assay). As shown in Fig. 1, the results using the human IgG ELISA were quite similar to the TNF-binding ELISA. Although the TNFbinding assay tended to under-quantitate at days 12 and 22 relative to the IgG-binding assay, the two methods correlated well overall in terms of average concentration (Fig. 1C, r = 0.99).

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3.2. Determination of anti-therapeutic antibody response 3.2.1. ATA assay optimization on BioVeris and MSD platforms For the BioVeris technology, anti-GNExTNFvF and antiHumira antibodies were captured with biotinylated GNExTNFvF or Humira bound to streptavidin-coated paramagnetic beads and detected with BV-TAG conjugated GNExTNFvF or BV-TAG conjugated Humira. For the MSD platform, instead of paramagnetic beads, a standard streptavidin MSD 96-well plate was used. Various concentrations of antibody conjugate pairs (GNExTNFvF or Humira) were tested for optimal ECLU signal/background ratios (S/B) using cynomolgus monkey serum positive for anti-human IgG as a positive control. For both GNExTNFvF and Humira assays, the highest S/B ratio was achieved at conjugate pair concentrations of 500 ng/mL each on the BioVeris platform. For the MSD platform, the Humira conjugate pair had an optimal S/B ratio at concentrations of 500 ng/mL each, while the optimal GNExTNFvF pair concentrations were 250 ng/mL each. Various buffers were tested to determine the optimal assay diluent to be used for samples, controls, antibody conjugates, and paramagnetic bead dilution. ECLU signals were determined for prebleed serum samples from 28 naïve cynomolgus monkeys as well as a negative control serum pool, a positive control serum, and buffer controls using each buffer. Both GNExTNFvF and Humira conjugated antibody pairs were tested to determine which buffer gave the least signal variability between cyno serum samples and showed signals most similar to the negative serum and buffer controls. Buffer containing 80 mM HEPES buffer, 0.5% fish gelatin, 0.25% Tween-20, 0.25% CHAPS, 0.35M NaCl, 0.05% Proclin 300, pH 7.0 gave the lowest signal variability and was chosen for the GNExTNFvF and Humira assays on both platforms (data not shown). The effects of addition of FBS to the final ATA Assay Buffer were evaluated for both the BioVeris and MSD assays. FBS had opposite effects on the BioVeris and MSD assay negative controls. For the BioVeris technology, ATA Assay Buffer supplemented with up to 10% FBS gave the highest S/B for the positive control. However, FBS was found to suppress signal in the negative control serum to below buffer-only control levels and was therefore omitted from the buffer. In contrast to the BioVeris platform, for the MSD platform, negative serum samples had ECLU values below the bufferonly control in the absence of FBS, so 5% FBS was added to the buffer to increase the S/B ratio above 1. The minimum serum dilution was determined for the BioVeris and MSD assays. While there was little difference in S/B between the 1/20 and 1/100 dilutions of the negative control serum pool (S/B ∼ 1.0), the S/B increased to ∼1.2 at a 1/4 dilution. A minimum serum dilution of 1/20 was therefore chosen for the negative control and the samples on both platforms. 3.2.2. BioVeris and MSD assay characterizations The cut point on each assay plate represents the signal above which a sample is considered positive. The cut point was determined by multiplying the mean of the negative control pool replicates (n = 8) by a pre-established, constant value termed the cut point factor. This cut point factor was

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Table 1 Drug tolerance (GNExTNFvF or Humira) of Bioveris (BV) or MSD ATA assays in the presence of either a high (log titer ∼ 4.7) or lower (log titer ∼3.7) positive control. Therapeutic

Titer

Incubation

BV max µg/mL

MSD max µg/mL

GNExTNFvF

Lower

2h Overnight 2h Overnight 2h Overnight 2h Overnight

0.5 ND 2 ND 0.5 1 2 2

0.06 ND b1 ND 0.25 1 1 4

High Humira

Lower High

Assays were performed with a 2-h incubation for all conditions and with an overnight incubation for the Humira assays. The GNExTNFvF assay could not be evaluated with an overnight incubation because GNExTNFvF tended to aggregate in the sample diluent buffer during an overnight incubation.

determined for both anti-TNF mAbs in both the BioVeris and MSD assays by analyzing the individual prebleeds (untreated population) from the study (n = 28). The analysis was performed on at least 3 separate days and the mean cut point factor was obtained (see Materials and methods). For the BioVeris assay, cut point factors for the antiGNExTNFvF and anti-Humira assays were determined to be 1.115 and 1.112, respectively. For the MSD assay, cut point factors for anti-GNExTNFvF and Humira assays were determined to be 1.394 and 1.561, respectively. All assays had expected false positive rates of 5% as recommended (MireSluis et al., 2004). Detection of anti-therapeutic antibodies in the assay may be obscured by interference from circulating GNExTNFvF or Humira in the serum sample. The tolerance of the various assays to therapeutic drug was evaluated, defining tolerance as the highest drug concentration that yielded ≥80% recovery of the titer value. The GNExTNFvF and Humira ATA assays were tested for drug tolerance on both assay platforms and for two different titer samples. Using a high titer positive control (log titer ∼ 4.7) and 2 h incubation, the BioVeris platform could tolerate a slightly higher amount of free therapeutic antibody in the assay (∼ 2 µg/mL for either the GNExTNFvF or Humira assays vs. ≤1 µg/mL or 1 µg/mL, respectively, for the MSD assay). Using a lower titer positive control (log titer ∼ 3.7), the BioVeris platform again tolerated approximately two-fold higher concentrations of free therapeutic than the MSD assay (∼ 0.5 µg/mL for either the GNExTNFvF or Humira assays vs. ∼ 0.25 µg/mL or less for the MSD assay). The ability to improve drug tolerance by increasing the sample incubation time from 1.5–2 h to overnight was investigated. Because GNExTNFvF tended to aggregate in the sample diluent buffer upon overnight incubation, this evaluation was not possible for the GNExTNFvF assays. Using a high titer positive control in the Humira assay, the drug tolerance was not improved with an overnight incubation using the BioVeris platform (∼2 µg/mL), while the tolerance improved to ∼ 4 µg/mL on the MSD platform. With a lower titer positive control, the drug tolerance was improved with an overnight incubation with the Humira assay, tolerating ∼ 1 µg/mL free Humira on either the BioVeris or MSD platforms (Table 1). The presence of endogenous soluble TNF-α may cause false positive signals. Soluble TNF-α exists as homotrimers

and could possibly bridge biotin- and TAG-labeled antibodies in the ECL assay. The potential for TNF-α interference was tested to determine what TNF-α concentration increased the log titer by more than 20% above the minimum log titer of 1.3. For the BioVeris platform (MSD not tested), the GNExTNFvF ATA assay could tolerate TNF-α concentrations up to 2 ng/mL while the Humira ATA assay could tolerate up to 10 ng/mL TNF-α (data not shown). The presence of free serum TNF at 2–10 ng/mL is unlikely given that this study was performed in healthy cynos. It is possible that the TNF bound to the antiTNF therapeutic may reach these levels; however, ATA measurements were largely obtained from samples with below detectable concentrations of therapeutic antibody.

Fig. 2. Evaluation of GNExTNFvF (A) or Humira (B) ATA log titer using BioVeris (solid line) or MSD (dotted line) bridging ECL assay approach. Correlation plot (C) of MSD and BioVeris ATA log titers shows that both approaches give similar results (r = 0.96).

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The sensitivity of an ATA assay is defined as the positive control concentration at the cut point, with an industrysuggested detection limit of 500–1000 ng/mL for preclinical studies (Mire-Sluis et al., 2004). In the present case, the positive control consisted of serum from immunized monkeys, so the concentration of specific anti-human framework antibody is not known, but is estimated to be ∼ 40–55 µg/mL (data not shown). Both the GNExTNFvF and Humira ATA assays had similar positive control titers (log titer ∼4.7) for both the Bioveris and MSD technologies, indicating similar sensitivities for all assays. Accounting for the dilution at the log titer value of 4.7, the sensitivities of the assays are estimated to be in the range of 1–200 ng/mL, conforming to or exceeding the industry standard. It is noteworthy that, although the S/B (ECLU of 1/20 positive control/cut point ECLU) was ∼10 fold higher for the MSD assay (S/B ∼ 800) than the BioVeris assay (S/B ∼ 80), this did not translate to a higher sensitivity on the MSD platform. ANOVA analysis of intra-assay and inter-assay precision for the negative, low positive, and high positive controls was performed for the GNExTNFvF and Humira assays on the BioVeris platform. For GNExTNFvF, all CVs were less than 10%, and for Humira, all CVs were less than 15%. Insufficient data were collected on the MSD platform to perform a comparable analysis.

3.2.3. Results of cyno sera ATA titers were determined on days 15, 22, 29, 36, and 43 for the GNExTNFvF and Humira groups. As shown in Fig. 2A and B, a substantial ATA response was observed for all GNExTNFvF and Humira treated cynos as early as day 15, with the ATA titers increasing by approximately 1 log unit between days 15 and 43. The observed log titer values were somewhat higher for the GNExTNFvF group than the Humira group. The BioVeris and MSD assays gave similar results, and the log titer values determined using the two platforms correlate well (Fig. 2C, r = 0.96). To characterize the immune response further, immunodepletion studies were performed using the BioVeris ATA assay as shown in Fig. 3. A selection of antibodies was spiked at 3 µg/mL into representative day 43 sera from both groups. Test molecules included: GNExTNFvF, Humira, murine parental GNExTNF, GNExTNFvI (an early humanized version), and a recombinant human IgG1 isotype control (rhuIgG1). The specificity of the immune response was measured by the decrease in signal compared to the buffer control (100%), and a signal reduction of greater than 50% as compared to the buffer control was deemed positive. For GNExTNFvF-dosed animals, murine GNExTNF, rhuIgG1, and Humira were incapable of immunodepleting the response (Fig. 3A). GNExTNFvI caused a partial immunodepletion, while GNExTNFvF could strongly inhibit the signal in the assay, indicating that immunoreactivity was the greatest against the complementarity determining regions (CDRs) or changes to GNExTNF specific to its humanization, and not the human IgG framework regions common to rhuIgG1. For Humira-dosed animals, two of three tested sera showed immunodepletion specific to Humira. Animal #12, on the other hand, showed immunodepletion with all test molecules except murine GNExTNF, possibly indicating that anti-human IgG framework antibodies were present (Fig. 3B).

Fig. 3. Immunodepletion of ATA response using 3 µg/mL of test mAbs GNExTNFvF (gray), GNExTNFvI (black), muGNExTNF (diagonal), rhuIgG1 (dotted), and Humira (striped) versus buffer (white) for day 43 ATA-positive GNExTNFvF-dosed (A) animals 2, 3, and 5 and Humira-dosed (B) animals 7, 9, and 12. Effect measured by % ATA response following immunodepletion with buffer control at 100%.

3.3. Neutralizing antibody assays Although immunodepletion studies showed that the ATA responses were, for the most part, specific to the dosed therapeutic, they did not demonstrate whether or not the ATA response was neutralizing. Two types of cell-binding assays were evaluated in neutralizing antibody studies, flow cytometry and MSD. Both cell-based assays used retrovirus infected Jurkat cells expressing membrane-bound non-cleavable TNF mutant (huTNFNC Jurkat cells) as well as green fluorescent protein (GFP). The TNF was modified to eliminate the TNF alpha converting enzyme (TACE) cleavage site so that all TNF present was in a membrane-bound form. The cyno serum samples were tested for neutralizing antibodies by evaluating their ability to block the binding of biotinylated anti-TNF to huTNFNC Jurkat cells. As in the immunodepletion experiments, day 43 serum was used for neutralizing antibody studies. 3.3.1. Flow cytometry cell-binding assay In the flow cytometry based cell-binding assay, the ability of the biotinylated anti-TNF mAbs (GNExTNFvF or Humira) to bind to huTNFNC Jurkat cells in the presence of a predose serum pool was compared to that observed in the presence of

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serum samples with positive ATA titers. A representative predose individual serum sample (prebleed(indiv), Fig. 4) shows the comparability of using a pool instead of individual predose sera. Biotin-GNExTNFvF, biotin-Humira, or biotinIgG1 isotype control at 500 ng/mL was pre-incubated with a 1/20 dilution of predose or day 43 postdose sera prior to addition to the cells. Binding of biotin-mAb to the cells was detected using PE-conjugated streptavidin. With this assay design, a lower signal of the ATA-positive serum compared to the predose serum would indicate the presence of neutralizing anti-therapeutic antibodies. A relative neutralizing antibody activity (predose animal MFI/postdose MFI) over 2 was considered positive. Compared to the biotinylated isotype control mAb, incubation of the huTNFNC Jurkat cells with biotin-GNExTNFvF or biotin-Humira increased the fluorescence from ∼40 to ∼369 mean fluorescence intensity (MFI) units (Figs. 4 and 5).

Fig. 5. Neutralizing antibody analysis by flow cytometry of Biotin-GNExTNFvF (A) or Biotin-Humira (B) binding to huTNFNC Jurkat cells in the presence of ATA-positive (day 43) sera from Humira-dosed cynos 7 (red), 8 (green), 9 (orange), 10 (light blue), 11 (pink) or 12 (brown) versus a prebleed pool (blue). The peak for huTNFNC Jurkat cells treated with a huIgG1 isotype control is shaded gray.

Fig. 4. Neutralizing antibody analysis by flow cytometry of Biotin-GNExTNFvF (A) or Biotin-Humira (B) binding to huTNFNC Jurkat cells in the presence of ATApositive (day 43) sera from GNExTNFvF-dosed cynos 1 (green), 2 (orange), 3 (light blue), 4 (pink), 5 (purple), or 6 (red-brown) versus a prebleed pool (blue) or a representative individual, prebleed(indiv) (red). The peak for huTNFNC Jurkat cells treated with a huIgG1 isotype control is shaded gray.

However, when the cells were incubated with ATA-positive serum from animals dosed with GNExTNFvF (animals 1–6, Fig. 4A), the signal was reduced back to isotype control levels by most sera, indicating the presence of neutralizing antibodies. Although GNExTNFvF-neutralizing antibodies were present in varying degrees in all GNExTNFvF-dosed animals at day 43, the serum from animal #4 showed the lowest neutralizing activity (64 MFI). In contrast, no evidence of Humira-neutralizing antibodies was present in serum from animals dosed with GNExTNFvF, with the exception of a minor effect for animal #4 (237 MFI). This may suggest that the neutralizing antibodies in serum from animal #4 cross-react slightly with a TNF-binding epitope that is similar in both GNExTNFvF and Humira. When the huTNFNC Jurkat cells were incubated with ATApositive sera from animals dosed with Humira (animals 7–12,

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expressed on the surface of the huTNFNC Jurkat cells. However, in contrast to the flow cytometry assay in which the cells are kept in suspension throughout the experiment and analyzed in a flow cell, the MSD assay uses cells immobilized onto high-bind carbon coated 96-well plates. Predose or ATA-positive postdose serum was pre-incubated with biotin-GNExTNFvF or biotin-Humira, and biotin-mAb bound to the huTNFNC Jurkat cells was detected using streptavidin-ruthenium. Relative to flow cytometry, an advantage of the MSD technology includes a higher throughput, making it possible to quickly analyze multiple plates. As shown previously (Lu et al., 2006), in developing an MSD cell-binding assay it is important to first determine the optimal cell density. A range of 12,500–200,000 cells/well was evaluated, and 100,000 huTNFNC Jurkat cells per well proved to be optimal for both GNExTNFvF and Humira assays in terms of providing the highest signal over background for a range of anti-TNF mAb concentrations (data not shown). Binding of a dilution curve (1000–1 ng/mL) of biotinGNExTNFvF or biotin-Humira to huTNFNC Jurkat cells was

Fig. 6. Neutralizing antibody analysis by cell-binding MSD of BiotinGNExTNFvF (A) or Biotin-Humira (B) binding to huTNFNC Jurkat cells in the presence of ATA-positive (day 43) sera from GNExTNFvF-dosed cynos 1 (green, EC50: ∼954 ng/mL (A) or ∼138 ng/mL (B)), 2 (orange, EC50: ∼2059 ng/mL (A) or ∼130 ng/mL (B)), 3 (light blue, EC50: ∼1465 ng/mL (A) or ∼148 ng/mL (B)), 4 (pink, EC50: ∼ 279 ng/mL (A) or ∼298 ng/mL (B)), 5 (purple, EC50: ∼ 531 ng/mL(A) or ∼69 ng/mL (B)), or 6 (red-brown, EC50: ∼1702 ng/mL (A) or ∼133 ng/mL (B)) versus a prebleed pool (red, EC50: ∼62 ng/mL (A) or ∼ 99 ng/mL (B)).

Fig. 5B), the signal was reduced to isotype control levels (40 MFI versus 474 MFI for predose) indicating the presence of neutralizing antibodies. The only exception was animal #9 (355 MFI), which had immunodepleting, although nonneutralizing antibody responses in both the predose as well as the day 43 samples. This indicates that the immunodepletion was due to antibodies that recognize an epitope distinct from the TNF-binding region of the antibody, possibly recognizing a framework epitope that is present in Humira but not other humanized antibodies tested in the immunodepletion studies. No evidence of GNExTNFvF-neutralizing antibodies was present in Humira-dosed animal sera, with the exception of a low amount in animal #12 (87 MFI). Similar to animal #4, although to a greater extent, serum from animal #12 may contain antibodies that cross-react to the TNFbinding regions of both GNExTNFvF and Humira. 3.3.2. MSD cell-binding assay The MSD cell-binding assay has many similarities to the flow cytometry assay in that both are based on the ability of NAbs to inhibit binding of biotinylated anti-TNF mAbs to TNF

Fig. 7. Neutralizing antibody analysis by cell-binding MSD of BiotinGNExTNFvF (A) or Biotin-Humira (B) binding to huTNFNC Jurkat cells in the presence of ATA-positive (day 43) sera from Humira-dosed cynos 7 (red, EC50: ∼42 ng/mL (A) or N 3000 ng/mL (B)), 8 (green, EC50: ∼50 ng/mL (A) or ∼1457 ng/mL (B)), 9 (orange, EC50: ∼76 ng/mL (A) or ∼174 ng/mL (B)), 10 (light blue, EC50: ∼104 ng/mL (A) or N 3000 ng/mL (B)), 11 (pink, EC50: ∼ 84 ng/mL (A) or ∼ 994 ng/mL (B)) or 12 (brown, EC50: 383 ng/mL (A) or ∼795 ng/mL (B)) versus a prebleed pool (blue, EC50: ∼75 ng/mL (A) or ∼61 ng/mL (B)).

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tested in the presence of either predose or ATA-positive postdose animal serum (Figs. 6 and 7). An EC50 value was calculated for each curve, with an increase indicating the

Fig. 8. Neutralizing antibody activity by fixed drug concentration (500 ng/mL ) flow cytometry (A), prebleed MFI/animal MFI; dose response MSD (B), animal EC50/prebleed EC50; or fixed drug concentration (500 ng/mL) MSD (C), prebleed ECLU/animal ECLU. Neutralizing activities of ATA-positive (day 43) GNExTNFvF-dosed animal serum (1–6) or Humira-dosed animal serum (7–12) for biotin-GNExTNFvF are shown in gray and for biotin-Humira are shown in black. The cut point (neutralizing antibody activity at or above which a sample is deemed positive) is shown as a dotted line at neutralizing antibody activity = 2.

presence of neutralizing antibody activity. A relative neutralizing antibody activity (postdose animal EC50/predose EC50) over 2 was considered positive (Fig. 8B). As shown in Fig. 6A, as compared to incubation with predose serum (EC50: ∼ 62 ng/mL), serum from all GNExTNFvF-dosed animals (animals 1–6) increased the EC50 to 279 to ∼ 2000 ng/mL for binding of biotin-GNExTNFvF, indicating that all contain neutralizing antibodies to GNExTNFvF. Similar to the results from the flow cytometry assay, serum from animal #4 (EC50: 279 ng/mL) contained the least neutralizing activity. Fig. 6B shows that the sera from GNExTNFvF-dosed animals have minimal effect on the binding of biotin-Humira to huTNFNC Jurkat cells. The EC50 values ranged from ∼99 ng/mL for predose serum to ∼69 to ∼298 ng/mL for animals 1–6. Again, serum from animal #4 (EC50: ∼ 298 ng/mL) showed a slight ability to neutralize biotin-Humira binding. Analysis of serum samples from animals treated with Humira (Fig. 7) showed a similar pattern of neutralization activity. As compared to incubation with prebleed serum (EC50: ∼ 61 ng/mL), serum from all Humira-dosed animals (animals 7–12) with the exception of animal #9 (EC50: ∼174 ng/mL), increased the EC50 for biotin-Humira binding to ∼383 to N3000 ng/mL, indicating the presence of neutralizing antibodies to Humira (Fig. 7B). With the exception of a low level of GNExTNFvF-neutralizing activity in animal #12 (EC50: ∼ 383 ng/mL), serum from animals dosed with Humira did not affect the binding of biotinylated GNExTNFvF to huTNFNC Jurkat cells, with the predose (EC50: ∼ 75 ng/mL) and ATA-positive postdose serum samples (EC50: ∼ 42– 104 ng/mL) yielding similar EC 50 values for biotinGNExTNFvF binding. The comparative results of the neutralizing antibody analysis performed by flow cytometry and MSD are shown in Fig. 8. In addition to flow cytometry using a 500 ng/mL fixed drug concentration (Fig. 8A) and MSD using a dose response curve (Fig. 8B), also shown is the relative neutralizing antibody activity for a 500 ng/mL fixed drug concentration using the MSD format (predose ECLU/postdose animal ECLU, Fig. 8C). Results are very similar between the dose response curve and fixed drug concentration approaches using the MSD platform with the exception of animal #4, which is no longer positive for either GNExTNFvF or Humira neutralizing antibodies, and animal #12, which is no longer positive for GNExTNFvF neutralizing antibodies with the fixed drug concentration approach. Although the fixed drug concentration approach works well, the dose response approach may be more sensitive for low signal responses. Overall, neutralizing antibody trends were similar for the flow cytometry and MSD formats, although the MSD platform showed more differentiation between samples and the results had a broader range of values for both the dose response curve and fixed drug concentration approaches. It is notable that, for the most part, the neutralizing antibodies were quite specific to the therapeutic with which the animals were dosed, and this specificity was maintained even in the context of two human mAbs targeted against the same antigen. 4. Discussion Administration of recombinant protein therapeutics can result in the formation of ATAs (Frost, 2005). The clinical

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impact of ATAs may include substantial alterations in the PK profile; a reduction or neutralization of the efficacy of the therapeutic; immune system effects such as allergy, anaphylaxis, serum sickness; or neutralization of the endogenous counterpart (Schellekens, 2005). Of particular concern for severe or life-threatening neutralizing antibody responses are enzyme replacement therapies (Wang et al., 2008). Since mAb therapeutics do not have an endogenous equivalent, and patients may also be immunosuppessed at the time of therapy, anti-TNF mAb therapy would represent a lower risk of antibody-related clinical sequelae (Koren et al., 2008). Animal studies, even those conducted in non-human primates, may have limited predictive power for immunogenicity of humanized proteins in humans (Bugelski and Treacy, 2004). This is illustrated by the fact that, despite the strong ATA response seen in the cynomolgus monkeys in the present study, Humira is used clinically with low incidence of ATAs. According to Humira product literature, ∼ 5% of adult rheumatoid arthritis patients receiving Humira developed neutralizing ATAs at least once during treatment. Elsewhere it has been reported that 5–20% of Humira patients develop antibodies against the product (Frost, 2005). Various immunochemical approaches can be used to measure immunogenicity of therapeutic mAbs. These include ELISA, radio immuno-precipitation (RIP), surface plasmon resonance (SPR), and ECL, and each has its own advantages and disadvantages (Mire-Sluis et al., 2004). Anti-Humira antibodies have been previously measured by a commercially available double-capture ELISA (Immundiagnostik AG, Bensheim, Germany) in which F(ab')2 fragments are used for the capture reagent and horseradish peroxidase-conjugated Humira is used as the detection reagent (Bender et al., 2007). This type of ATA ELISA may suffer from masked or altered epitopes due to immobilization on a solid surface, or a reduced ability to detect low affinity antibodies due to loss upon multiple washes. More recently, antiHumira antibodies have been measured with a newly developed radioimmunoassay (Bartelds et al., 2007). Aside from the undesirable feature of using radioactivity, this assay can only detect positives in samples with less than 5 µg/mL of therapeutic. The methods presented here use the ECL method, which has a wide dynamic range (Moxness et al., 2005). It has also been shown that the ECL method is better at detecting both high- and low-affinity antibodies against a human mAb when compared to the ELISA and SPR methods (Liang et al., 2007). In addition, Liang et al. showed a comparison of sensitivities for ATA detection by the BioVeris and MSD technology platforms, indicating similar values for four different antibodies that ranged in affinities from 0.057 to 340 nM. Given that BioVeris reagents and instrumentation are no longer supplied or supported as of the end of 2008, it is necessary to evaluate alternative platforms for ATA analysis, with matching of the performance of the BioVeris assays being highly desirable. As shown in the current study, our results for the BioVeris and MSD ATA assays correlated well. With regard to drug tolerance, the results varied depending on the ATA assay and the incubation time used as discussed above and shown in Table 1. It should be noted that the criteria for assessing drug tolerance was recovery of titer to within +/− 20% of the buffer-only control. Using the maximum amount of drug still

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yielding an ECL signal above the cut point as an alternate definition of drug tolerance, both the BioVeris and MSD assays could tolerate over 500 µg/mL of therapeutic (either GNExTNFvF or Humira). This drug tolerance was more than 100-fold better than the previously used Humira immunogenicity assay, which had a drug tolerance of 5 µg/mL (Bartelds et al., 2007). The MSD assay used half the sample amount as BioVeris (25 µL compared to 50 µL, respectively) per well in 96-well plates. MSD also had a lower background and a higher signal to background ratio, though the sensitivities of the MSD and BioVeris assays were similar. The type of neutralizing antibody assay used depends on the therapeutic antibody's mode of action. It is recommended that a NAb assay consist of “an in vitro assay utilizing cultured cells that interact with or respond to the drug either directly or indirectly in a measurable manner in the presence of the test species matrix for the detection of anti-drug product neutralizing antibodies” (Gupta et al., 2007). Some potential assay endpoints for a NAb assay include: binding to a cell-surface expressed receptor, cell proliferation, intracellular substrate phosphorylation, and protein, mRNA, or reporter gene expression. Possible approaches for determination of neutralizing antibodies against anti-TNF therapeutic mAbs include a bioactivity assay or a cell-binding assay. A potential approach to a bioassay could involve using an appropriate cell line to evaluate cytokine production in response to TNF; inhibition of the response by combining TNF with the anti-TNF therapeutic; and reversal of this inhibition in the presence of NAb-positive serum samples. An example of this type of NAb assay was recently described (Lofgren et al., 2006) using IL-1-stimulated IL-8 production in A549 cells to detect a NAb response to an anti-IL-1R therapeutic mAb. However, advantages of the current cell-binding approach over a bioactivity NAb assay include a quicker read-out in hours instead of days and less possibility of interference from serum factors such as cytokines or growth factors. Although the NAb assay strategy chosen for a clinical program is dependent on a number of factors, for this pre-clinical study, the generation of the huTNFNC Jurkat cells provided an opportunity to evaluate the feasibility of using them as a basis for a NAb assay. Since TNF can be cleaved from the cell surface to produce a soluble form, it was imperative to use a non-cleavable mutant of TNF for this approach to work. Moreover, the display of native conformation, noncleavable TNF on the surface of the cells provided a more physiologically relevant means of immobilizing TNF than other potential methods. The NAb assays described here generalize to other mAbs that bind to cell surface proteins. As shown in the current study, both MSD and flow cytometry cell-binding approaches show similar NAb activity trends. The flow cytometry approach was used with a fixed drug concentration while the MSD approach was used with both fixed drug concentration and dose response formats. With a few exceptions, the two formats yielded quite similar results, with the dose response format better able to detect low signal responses. The MSD NAb assay approach allows for use of 96- or 384-well plates, enabling a high-throughput approach to quickly test various doses of biotinylated antiTNF binding to huTNFNC Jurkat cells, thus obtaining EC50 values for binding of the biotinylated antibodies. The flow cytometry approach is not as conducive to high-throughput analysis, making it less feasible to run dilution curves.

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In summary, ATA assays for anti-TNF therapeutics were developed using the BioVeris and MSD platforms. The results demonstrated that the assays compared favorably overall and gave titer values that correlated well (r = 0.96). The NAb cellbinding assay on either the flow cytometry or MSD platforms showed data with similar trends, although the MSD NAb assay enabled a higher throughput and allowed better differentiation between samples. Acknowledgements The authors thank Jiabing Ding for cloning non-cleavable TNF construct, and Qinglin Ou and Qian Gong for providing non-cleavable TNF infected Jurkat cells. We would also like to thank Mercedesz Balazs, Mark Nagel, Phil Hass, Suhas Iyer, and Ly Nguyen Kawaguchi. Thanks also to James Araujo and Cecilia Leddy for helpful ATA assay advice and to Ms. Leddy for providing the ATA assay positive control; thanks to Saileta Prabhu and Wai Lee Wong and her team for review of the manuscript; and thanks to Greg Spaniolo and Dan Coleman for cut point factor determination advice. References Bartelds, G.M., Wijbrandts, C.A., Nurmohamed, M.T., Stapel, S., Lems, W.F., Aarden, L., Dijkmans, B.A., Tak, P.P., Wolbink, G.J., 2007. Clinical response to adalimumab: relationship to anti-adalimumab antibodies and serum adalimumab concentrations in rheumatoid arthritis. Ann. Rheum. Dis. 66, 921. Bender, N.K., Heilig, C.E., Droll, B., Wohlgemuth, J., Armbruster, F.P., Heilig, B., 2007. Immunogenicity, efficacy and adverse events of adalimumab in RA patients. Rheumatol. Int. 27, 269. Bringman, T.S., Aggarwal, B.B., 1987. Monoclonal antibodies to human tumor necrosis factors alpha and beta: application for affinity purification, immunoassays, and as structural probes. Hybridoma 6, 489. Bugelski, P.J., Treacy, G., 2004. Predictive power of preclinical studies in animals for the immunogenicity of recombinant therapeutic proteins in humans. Curr. Opin. Mol. Ther. 6, 10. Frost, H., 2005. Antibody-mediated side effects of recombinant proteins. Toxicology 209, 155. Gupta, S., Indelicato, S.R., Jethwa, V., Kawabata, T., Kelley, M., Mire-Sluis, A.R., Richards, S.M., Rup, B., Shores, E., Swanson, S.J., Wakshull, E., 2007. Recommendations for the design, optimization, and qualification of cellbased assays used for the detection of neutralizing antibody responses elicited to biological therapeutics. J. Immunol. Methods 321, 1.

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