Target/drug interference considerations in immunogenicity assessment
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1 Target/drug interference considerations in immunogenicity assessment Eric Wakshull, PhD Senior Scientist March 9, 2016 EMA Workshop on Immunogenicity
2 Outline Page 2 Basic Immunogenicity screening assay Drug Interference o Mitigation strategies Target Interference o Mitigation Strategies Case studies Neutralizing antibody assays (time permitting)
3 Biotin/DIG-Based Homogenous Bridging ELISA Page 3 Color Step 3: Complexes detected with Anti-DIG-HRP DIG-Therapeutic Sample ADA Step 1: The mixture is incubated O/N Biotin-Therapeutic Step 2: Complexes captured on SA plates
4 Drug Interference Page 4 Drug interference: Drug as capture and/or detection reagent in either bridging or sandwich format Drug in sample will compete for ADA binding with labeled (or immobilized) drug False negative Drug tolerance commonly evaluated in assay development and further characterized in validation but we use a surrogate, usually high affinity, positive control may not be representative of a patient immune response 2009, Genentech / Proprietary information Please do not copy, distribute or use without prior written consent.
5 Drug Interference in ADA Assay Page 5 HRP-Mouse anti-dig TMB/H 2 O 2 ADA DIG-therapeutic Color Biotin-therapeutic ADA ADA streptavidin-coated plate Unconjugated drug from samples can interfere in detection of ADA by competition with labeled (or immobilized) drug
6 Drug Interference Page 6 Mitigation Strategies Obtain samples with low/no drug concentrations (washout) Not always feasible Optimize assay for drug tolerance For bridging assays, generally increase sample dilution and/or conjugate concentration: Have achieved >100-fold up to 900- fold molar excess in drug tolerance ([drug]/[ada]} Often a trade-off with assay sensitivity Longer incubation times (allow assay drug to outcompete sample drug) Acid dissociation Basic method (with permutations): Low ph dissociates ICs Provides some increased drug tolerance Unknown effect on patient sample ADAs (low ph denaturation) which may not be apparent using PC to develop optimal conditions May release sol Target from Drug/Target complex interference 2009, Genentech / Proprietary information Please do not copy, distribute or use without prior written consent.
7 Target Interference Page 7 Soluble Target interference: Bridging immunoassays Target may inhibit ADA binding at or near the drug/target interaction domain False negative Multimeric target may form bridge with drug conjugate(s) False positive Sandwich immunoassays Target may inhibit ADA binding at or near the drug/target interaction domain False negative
8 Target Interference: Bridging ELISA Page 8 HRP-Mouse anti-dig TMB/H 2 O 2 DIG-therapeutic Dimeric target =False Pos Biotin-therapeutic Monomeric target =False Neg Anti-Drug Antibodies (ADA) Color Streptavidin-coated plate
9 Mitigation Strategies Considered Page 9 Deplete starget Dissociate drug/starget complexes (if necessary) Affinity capture/remove starget and/or complexes Operationally complex Block drug/starget interaction Murine anti-drug Ab with different CDR Soluble recombinant Receptor Lectin Operationally simple >add to diluent buffer Caveat: Reagent may not be readily available, often needed in large quantitites.
10 starget Blocking Eliminates False Positives (or False Negatives) Page 10 X LIGHT DIG-mAb X starget Biotin-mAb Mu Anti-sTarget mab sol rreceptor lectin Streptavidin coated plate/bead
11 Example 1: Interference from VEGF, a dimeric vascular growth factor VEGF is a dimeric vascular growth factor targeted by Bevacizumab Initial clinical results: 94% ADA+ (??) Page 11 Baseline VEGF levels vary by disease & severity: ~ pg/ml. Post-bevacizumab total VEGF levels can increase 10x, PK effect Cause of high incidence due to VEGF interference?
12 Murine anti-vegf mab blocks VEGF False Positives but not ADA VEGF-induced signals are anti-vegf mumab by Page 12 ADAs are detected in the presence of anti-vegf mumab >250,000 Anti- Bevacizumab CDR ECLU anti-vegf mumab ECLU CP VEGF ug/ml CP VEGF (ug/ml) Clinical results from patient samples tested +/- mu anti-vegf mab: No blocker: 9/18 patients (50%) Ab+ With blocker: 1/18 patients (5.5%) Ab+ Final ADA incidence from 2 piii trials: 1%(6/761; 8/1472)
13 Example 2: Interference from multimeric CA125, Soluble fragment from MUC16 Anti-MUC16 targets membrane bound MUC16 CA125 is a multimeric soluble proteoglycan fragment of membrane MUC16 Biomarker for OvCa Present in serum at levels >11,000 ku/l from clinical samples Page 13 ADA assay signal is proportional to CA125 in samples, potentially producing False Positives CA125-induced signal inhibited by the lectin WGA. Carrasco-Triguero etal 2012
14 WGA Lectin Does Not Impact on the Detection of ADA Page ku/l* CA125 ± ADA were incubated ± WGA in the ADA assay *source: patient-derived ascites Response (ECLU) ATA Detection in Presence of Antigen No WGA WGA 0 CA125 Antigen Antigen CA125 & + ATA ADA ATA ADA Human Serum Pool
15 Drug/Target Interference in NAb Assays: En Brief Page 15 Nab assays: Cell-based and ligand binding-based Significant additional complexities depending on drug MOA and signaling pathway For example: MOA: Drug targets ligand for cell receptor or targets cell receptor, inhibiting ligand/receptor interaction Potential interacting molecules Ligand Assay Cell Receptor Assay Drug Assay NAb Sample Drug Sample Soluble Receptor Sample Ligand Sample Assay Components Derived from Samples Outcomes (True/False, Negative/Positive) depends on the relative concentration and affinity of the interacting molecules
16 Acknowledgments Page 16 John Lowe Mauricio Maia Rebecca Elliott Montse Carrasco-Triguero An Song Patricia Siguenza 2009, Genentech / Proprietary information Please do not copy, distribute or use without prior written consent.
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