Project Name 1Case Studies:Direct Comparison of Cell-Based andCompetitive Ligand Binding Assays toCharacterize Neutralizing Antibody ResponsesBonnie Wu, Ph.D.Senior ScientistImmune Response Assessment and ResearchBiologics Clinical PharmacologyCentocor R&D, Inc.Radnor, PA
Most Biological Therapeutics Can ElicitImmune Responses• Antibodies• Growth Factors/Cytokinesdrug product• Enzymes• Protein Conjugates• Peptidesanti-drug antibodies
Assessment of Immunogenicity of Protein Therapeutics• A critical aspect of drug development and regulatory filing• Requires the availability of reliable bioanalytical methods– Precision and accuracy– Detect all types of anti-drug antibodies (ADAs) toprotein therapeutics• Neutralizing Antibodies—NAb(s) neutralize therapeuticfunction• Non-neutralizing Antibodies—bind the therapeutic, butdo not block function
Tiered Assay Strategy for Immunogenicity AssessmentBiological Fluid SamplesImmunoassaysScreening AssayConfirmatory AssayADA DetectionConfirmed ADA+SamplesTiter Determination, etc.Neutralizing AssayADA Characterization
NAb Detection Assay Formats• Cell-based assays– Typically preferred by regulatory agencies– Functional assays utilizing cell lines responsive to drug treatment– Usually adapted from drug potency assays– NAb activity detected through NAb mediated dose dependentinhibition of cellular responses to drug treatment• Competitive ligand binding assays (CLBA)– Mainly non-cell based formats– Measures binding of drug to target/ligand– NAbs in test samples competitively inhibit the binding of drug totarget/ligand– Practical and technical limitations of cell-based assays largelyabsent
Justification for Using Non-cell Based CompetitiveLigand Binding Assays• Cell-based assays– Susceptible to assay interference derived from• Subtle changes in cell culture condition• Introduction of sample matrix– Lower throughput– Usually longer assay run time– Limited choice of cell lines– Added complexity from working in cellular system• Non-cell based competitive ligand binding assays– Adequate precision and accuracy– Efficient development and validation– Higher throughput– Usually shorter assay run time– Reduced complexity streamlines method transfer, implementation,and maintenance of validated state.
Cell-based Assays Vs. Non-cell Based Competitive LigandBinding Assays• Cell-based assays are the recommended assay format for NAbassessment– Debate ongoing regarding the use of cell-based assays vs. noncellbased competitive ligand binding assays, particularly for NAbsagainst non-MAb drugs• Comparison of the two assay formats requires collection of asufficient body of scientific data– Provide recommendations to regulatory agencies– Facilitate the formation of guidelines on technology platformselection for NAb assays
Comparative Analyses of the Cell-based Assays andthe Non-cell Based Competitive Ligand Binding AssaysCase Study 1: Comparison of the two assay formats for the detection ofNAbs to a therapeutic human mAb that inhibitschemokine XCase Study 2: Comparison of the two assay formats for the detection ofNAbs to a therapeutic human mAb that inhibits IL-12/IL-23
Case Study 1: Therapeutic Human mAb That InhibitsChemokine X• Human mAb drug• An antagonist that blocks the biological function of chemokine X– Soluble drug target• Low-risk of immunogenicity with minor anticipated clinicalconsequence
Outline of Comparative Analysis in Case Study 1• Assay cut point• Assay sensitivity for a panel of anti-drug mAb controls• NAb dose response curve and dynamic range• Assay variability• Assay specificity• Assay selectivity– Matrix interference– Drug tolerance
Case I: Principle of the Cell-based AssayCell-based Assay Flow Chart• Chemokine X stimulates Chemokine Receptorexpressing reporter cells.• Receptor activation and subsequent β-galcomplementation results in a chemiluminescentassay signal.• CNTO X neutralizes chemokine X, reducing theassay signal.• NAb neutralizes CNTO X and restores the assaysignal.• NAb activity is directly proportional to the measuredassay signal.PathHunter TM HEK293Chemokine Receptorβ-arrestin cellsincubation of serumwith CNTO Xincubation of assaymixture with chemokine%Restoration=(RLU_sample – RLU_bkgd)/(RLU_maximal stimulation – RLU_bkgd)*100add the assay mixtureto the cells; chemokine Xactivates the receptorgeneration ofchemiluminescentassay signal
Case I: Principle of the Competitive LigandBinding AssayCompetitive Ligand Binding AssayFlow Chart• Based on Meso Scale Discovery’selectrochemiluminescent (ECL) detectiontechnology platform• Biotin-CNTO X is captured by Streptavidin coatedon the carbon electrode surface.• Biotin-CNTO X is detected by Ruthenium labeledchemokine X (Ru-CX), emitting an ECL assaysignal.• NAb competitively inhibits the binding ofbiotin-CNTO X to Ru-CX, reducing the assay signal.incubation of serumwith Biotin-CNTO Xcapture of biotin-CNTO Xon a Streptavidin coatedassay plate• NAb activity is inversely proportional to the measuredassay signal.%INH = (1-ECL_sample/ECL_Normalization Control)X100biotin-CNTO X is detectedby Ru-CX, emitting anECL signal, whileneutralized biotin-CNTO Xcan not bind to Ru-CX
Assay Cut PointsMeasurement of Assay Cut PointTested Drug Naïve Serum SamplesNormal Human SeraHuman Cancer SeraHuman Autoimmune Disease SeraCell-based AssayCompetitive LigandBinding AssayN=84 N=11224 5230 3030 30Sample Replicate TypeDuplicateTriplicateNAb DeterminantAssay Cut Point%Restoration%Inhibition20.70% 15.01%• The cut point was computed to allow a 1% false positive rate.• For the cell-based assay, the cut point is 20.70% signal restoration.• For the competitive ligand binding assay, the cut point is 15.01% signal inhibition.
Assay SensitivityNAb Assay Sensitivity (ng/ml)Tested NAbs Cell-based Assay Competitive Ligand Binding AssayNAb-1 520.6 146.3NAb-2* 272.7* 109.1*NAb-3 195.9 101.8NAb-4 1247.7 159.7NAb-5 204.7 120.5NAb-6 299.5 132.7NAb-7 287.9 127.6• The assay sensitivity is the lowest concentration of NAb in the neat serum thatgenerates a positive result in the NAb assay.• The competitive ligand binding assay demonstrates greater assay sensitivity toall seven NAbs. Each NAb was detected at a concentration
NAb Dose Response CurvePositive NAb Control in 10% NHS (µg/mL)Positive NAb Control in 10% NHS (µg/mL)• Both NAb dose response curves were generated using the positive NAb control(NAb-2).• The competitive ligand binding assay was more sensitive, capable of detectingNAb control at approximately 2.5 fold lower concentration.• The competitive ligand binding assay generated a larger dynamic range.
%CV of Calculated NAb ConcentrationComparison of Assay VariabilityPositive Control in 10% NHS (µg/mL)• The assay variability was assessed by the %CV of the calculated NAb concentration at thegiven assay response.• %CV = (standard deviation of calculated NAb concentration)/(nominal NAb concentration)X100• The range of quantification falls between the intersection of non-linear regression curveand the 25% CV acceptance limit for assay variation.• The competitive ligand binding assay shows lower %CV and a wider range of quantification.A wider range of quantification generally correlates with lower assay variability.
Comparison of Assay SpecificityAntibody Control in 10% NHS (µg/mL)Antibody Control in 10% NHS (µg/mL)• The assay specificity was assessed by evaluating the positive NAb control and non-NAb control(murine anti-human Fc) in the two assays.• For both assay formats, the positive NAb control generated assay response higher than the cutpoint, whereas no neutralizing activity was detectable from the non-NAb control.• Both assay formats are specific for the detection of drug specific NAbs.
Assay Selectivity: Matrix InterferenceSerumConcentrationAveraged Assay Signal Recovery (%) aCell-based Competitive LigandBiossay Binding Assay• Recovery of NAb activity from different levels of serum matrix was evaluated by testing theserially diluted NAb control in increasing concentrations of human serum in both assay formats.• For both assay formats, the NAb dose response curve was not significantly impacted by thepresence of serum.• The EC50 values and S/N were consistent .EC50 of NAb DoseResponse Curve (ng/ml)Cell-basedBiossayCompetitive LigandBinding AssayCell-basedBioassaySignal to Noise Ratio• The competitive ligand binding assay show better recovery with limited reduction of assaysignal in the presence of serum.• The cell-based assay offered lower recovery from serum matrix. There was significantreduction of assay signal when increasing concentrations of serum were introduced in theassay.Competitive LigandBinding Assay0% N/A N/A 43.5 35.5 4.7 83.25% 43.1 95.6 39.0 38.5 5.4 52.910% 18.6 93.6 37.0 37.5 6.3 61.120% 18.3 93.8 52.0 42.5 6.9 172.2a Averaged assay signal recovery is the mean of signal recovery for serially diluted NAb control tested in thepresence of serum in the NAb assay.
Drug Tolerance (ng/mL)Assay Selectivity: Drug Tolerance• The drug tolerance limit is defined as the minimal concentration of exogenous drug thatproduces a false-negative assay result.• To determine the drug tolerance limit, increasing amount of CNTO X was added to the assaymixture containing 10% serum and 37.5 ng/mL of NAb control in both assays.• The competitive ligand binding assay tolerated approximately five fold greater concentration ofexogenous drug than the cell-based assay.
SummaryComparison of Two Assay Platforms in Case Study 1Assay ParameterCell-based AssayCompetitive LigandBinding AssayMinimal Required Dilution 1:10 1:10Range of Assay SensitivityAssay VariabilityMatrix InterferenceDrug ToleranceAssay Specificity196 to 1248 ng/mLRange of quantification0.024 to 0.082 µg/mLLower assay signalrecovery (18% - 43%)97.5 ng/mLspecific to NAb102 to 160 ng/mLRange of quantification0.008 to 0.178 µg/mLHigher assay signalrecovery (94% to 96%)475.0 ng/mLspecific to NAbHighlighted area indicates superior performance• The competitive ligand binding assay offers superior precision, greatersensitivity, and better assay selectivity (more tolerant to serum matrix andexogenous drug).
Comparative Analysis of the Cell-based Bioassays andthe Non-cell Based Competitive Ligand Binding AssaysCase Study 1: Comparison of the two assay formats for the detection ofNAbs to a therapeutic human mAb that inhibitschemokine XCase Study 2: Comparison of the two assay formats for the detection ofNAbs to a therapeutic human mAb that inhibits IL-12/IL-23
Case Study 2: Therapeutic Human mAb That Inhibits IL-12/IL-23• Human mAb drug product• IL-12/IL-23 antagonist– Soluble drug target• Low-risk of immunogenicity with minor anticipated clinicalconsequences
Case Study 2: Cell-based Assay• Based on the potency assay• Nature killer (NK) cells produces IFN-γ in response to IL-12 treatment.• The assay end point is the level of IFN-γ produced in cell culture supernatant and measured by ELISA.• CNTO Y neutralizes IL-12 and inhibits the IL-12 induced IFN-γ production.• NAb neutralizes CNTO Y and releases IL-12,restoring IFN-γ production.• NAb activity is proportional to the level of IFN-γ in cell culture supernatant.
Case Study 2: Competitive Ligand Binding Assay• Based on Meso Scale Discovery’s ECL detection technology platform• Biotin-CNTO Y is incubated with acid dissociated patient sera.• Biotin-CNTO Y is captured on the Streptavidin-coated MSD assay plate.• After washing, biotin-CNTO Y is detected by Ru-IL-12, emitting ECL assay signal.• NAb competitively inhibits the binding of Ru-IL-12 to biotin-CNTO Y, reducing the assay signal.• NAb activity is inversely proportional to the measured ECL signal.Ru-IL-12Biotin-CNTO YAcid DissociatedPatient Sera(No NAbs)Streptavidin-coatedMSD Assay PlateWash and Detectedby Ru-IL-12ECL SignalBiotin-CNTO YAcid DissociatedPatient Sera(With NAbs)Streptavidin-coatedMSD Assay PlateWash and Detectedby Ru-IL-12No ECL Signal
Measurement of Assay Response• Assay response:- Cell-based assay: percent recovery of IFN-γ response%Recovery of IFN-γ response = (Test Sample – Background Control)/(Maximum IFN-γ Response– Background Control)X100- Competitive ligand binding assay: percent inhibition of assay signalderived from the Normalization Control%INH = (1-ECL_sample/ECL_Normalization Control)X100
Project Name 26Determination of Assay Cut Point• Assay cut point:– Cell-based assay cut point: 36.00% recovery of IFN-γ response• Derived from repeated analysis of 72 drug naïve human serumsamples– Competitive ligand binding assay: 11.96% inhibition of assay signalderived from the Normalization Control• Derived from repeated analysis of 135 drug naïve human serumsamples
Comparison of Critical Assay Parameters Between theTwo Assay PlatformsAssay Parameter Cell-based Assay Competitive Ligand Binding AssayAssay Time 4 Days 1 DaySamples Analyzed per Plate 10 Samples 40 SamplesAssay Sensitivity 720 ng/mL 321 ng/mLDrug Tolerance 1.41 µg/mL 1.50 µg/mLAssay Variability 33.60 %CV 7.77 %CVAssay Robustness Assay and Sample Failure YesHighlighted area indicates superior performance• Compared to the cell-based assay, the competitive ligand binding assay issuperior in assay performance.─ 75% reduction of assay time─ Assay throughput is 4 fold higher.─ Sensitivity is about 2 fold greater (determined by the same polyclonal NAb control).─ Inter-assay precision improved by > 4 folds─ Increased robustness
%Recovery of IFN- Response%InhibitionEvaluation of Clinical Samples from ADA Positive SubjectsUsing the Two Assay Platforms140120Cell-basedAssayr s =0.64 140 Competitive Ligandr s =0.81Binding Assay120100100806040Assay Cut Point8060402020Assay Cut Point01 10 100 1000 10000ADA Titer01 10 100 1000 10000ADA Titer• Twenty-eight clinical samples from ADA positive subjects, including 2 baselinesamples and 26 post-treatment samples, were evaluated by both NAb assays.• The competitive ligand binding assay shows better correlation (determined bySpearman rank order correlation coefficient ) between ADA titer and NAb assayresponse, indicating that CLBA might be more precise in classifying the NAb statusof clinical samples.
Classification of Sample NAb Status: a Comparison*I.E. = inevaluable sample due to matrix interference or sample failure• Two samples inevaluable using the cell-based assay were classified as NAb positivein the competitive ligand binding assay.• Six out of eight post-treatment samples originally classified as NAb negative using thecell-based assay were identified as NAb positive in the competitive ligand bindingassay, while the baseline samples remain negative.• Clinical samples of high ADA titer show matched NAb status in both assays.
SummaryComparison of Two Assay Platforms in Case Study 2• Longer assay procedure in the cell-based assay vs. the competitiveligand binding assay (4 days vs. 6 hours)• The competitive ligand binding assay platform showed superior assaythroughput, sensitivity, precision, and robustness.• For clinical samples of high ADA titer, similar conclusions about NAbstatus were made, but not all NAb positive samples were detected bythe cell-based assay.‣ Eight out of ten clinical samples that were either NAb negative(undetectable) or inevaluable in the cell-based assay were classifiedas NAb positive in the competitive ligand binding assay.
ConclusionsBoth case studies comparing the cell-based assays and the non-cell basedCLBAs for the detection of neutralizing antibodies against human mAb(antagonistic) drugs indicated that:• The competitive ligand binding assay is a viable assay platform for NAbdetection.• Competitive ligand binding assays offer superior assay performancecompared to the cell-based assays.• Competitive ligand binding assays may be better able to correctly classifysamples undetectable or inevaluable using the cell-based assay methods.
AcknowledgementsJamie BaldErik B. CrawfordKenneth GoldbergPersymphonie MillerKrupa RamaniManjula ReddyGeorge R. Gunn, IIIGopi Shankar